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Periplasmic binding protein FhuD of Escherichia coli K-12:
Overexpression in Bacillus subtilis, purification, and
renaturation of the recombinant FhuD
BY
Francisco C. Ferreira
Department of Microbiology and Immunology
McGill University
Montreal, Canada
March, 1998
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment
of the requirements of the degree of Master of Science
@Francisco Ferreira, 1998
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TABLE OF CONTENTS
TABLE OF CONTENTS
ABSTRACT
RESUME
LIST OF ABREVIATIONS
LIST OF FIGURE3
ACKNOWLEDGEMENTS
LITERATUW REVIEW
A) Structure and composition of the outer membrane
ems B) Periplasrnic-binding protein dependent transport syst
1 ) Generalities
2) The periplasmic binding proteins
i ) the affinity of binding
ii ) the conformational change
iii) the crystal structure
iv) the sequence
v ) the bctional regions of the binding proteins
3) The membrane-bound components
i ) the integral cytoplasmic membrane proteins
ii ) the ATP-binding cassette (ABC) subunits
PAGE
1
iv
vi
tiii
ix
i i
4) Model for the mechanism of cytoplasmic membrane translocation of substrate
in binding protein dependent-ABC transport systems
5) High-affinity iron uptake in Escherichia coli
i ) iron
ii ) siderophores
iii) outer membrane receptor for ferric siderophores
iv) TonB protein
v ) fenichrome-iron uptake
vi) regulation of iron transport
C) Overexpression of recombinant proteins in Bacillus
1) Secretion of heterologous proteins in Bacillus
2) Production of heterologous proteins intracelluiarly in
Bacillus subtilis
D) Rationale for thesis
MATEFUALS AND METHODS
Bacterial strains and plasmids
Media, reagents and enzymes
Molecular cloning of the maturejhuD sequences
Isolation of inclusion bodies (IBs)
Purification of urea-solubilized IBs containing BacFhuD-His,
Preparation of antibodies
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 4 1
Immuno blotting
Renaturation of purified urea-solubilized BacFhuD-His,
Purificatiodrenaturation of GuanidineHCI-solubilized IBs-containing
BacFhuD-His,
Detection assay for secreted BacFhuD (secBacFhuD)
Protease protection assay for BacFhuD-His,
Binding activity assay for BacFhuD-His, and FhuA
RESULTS
Expression of recombinant BacFhuD and BacFhdhHis,
Purification of urea-denatured BacFhuD-His,
FhuD specific anti-peptide antibodies
Production of the secreted form of FhuD
Renaturation of urea-solubilized BacFhuD-His,
Purification/renaturation of GnHCl-solubilized BacFhuD-His,
Binding activity of FhuA and recombinant FhuD
DISCUSSION
REFERENCES
TABLES
FIGURES
ABSTRACT
FhuD (266 amino acids; M, 29,160) is found in the periplasrn of Escherichia coli
K-12 and it is a binding protein that is involved in the transport of Fe3+-ferrichrome across
the bacterial cell envelope (Coulton et al., 1987). FhuD is a component of the ferric
hyiroxamate gtake (Fhu) system which also includes a high afinity outer membrane
receptor (FhuA) and two cytoplasmic membrane-bound proteins (FhuWFhuC) [Coulton
el al. , 19871. FhuD is detected in very low concentrations in the E. coli K- 12 periplasm.
To generate large amounts of FhuD, thefhuD gene of E. coli was cloned into a plasmid
vector (pKTH288) and expressed in Bacillus subtilis. This construct was designed so that
the recombinant FhuD would accumulate intracellularly. Alternatively, thefiuD gene of
E. coli was also cloned into a secretion plasmid @KTH132) and expressed in Bacillus
subtilis. In this construct it was expected that the recombinant FhuD be secreted into the
exterior of the cell. The pKTH288--uD construct yielded recombinant FhuD that was
produced at about 180 rng/mI in B. subfilis and which aggregated into intracellular
inclusion bodies. Polyclonal anti-FhuD peptide] sera were generated in rabbits and
irnrnunoblotting confinned the identity of FhuD. Histidine-tagged recombinant FhuD
(BacFhuD-His,), solubilized from inclusion bodies in 8 M urea or 6 M Guanidine-HCl
was purified by N?'-NTA chromatography under denaturing conditions. In order to
obtain protein in a native conformation it was necessary to remove the denaturant (urea or
guanidine-HC1). Three different methods were attempted to remove the denatwant and
hence refold the purified BacFhuD-His, into a biologically active form. The first method
involved stepwise dialysis of 8 M urea to 0 M urea. The second renaturation protocol
consisted of a slow, FPLC-mediated linear decrease in urea or guanidine-HC1. The third
renaturation method consisted of FPLC-mediated fast removal of the denaturant
guanidine-HCI. In the last two methods BacFhuD-His, was immobilized on a N~"-NTA
v column prior to removal of the denaturing agent. All methods led to different amounts of
BacFhuD-His, remaining in solution and stability of the soluble protein varied. To test
the biological activity of the renatured, soluble protein, two assays were used: (i) a
protease protection assay of BacFhuD-His, in the absence and presence of ferricrocin
(femchome analogue); (ii) equilibrium dialysis experiments in which radiolabelled
ferricrocin was dialyzed against a small volume of protein.
FhuD (266 acides amines; 29,160 Da) est une proteine se trouvant dans le
Hriplasme d' Escherichia coli K-12 et qui est impliqube dam le transport du femchrome
~ e ~ + a travers la membrane cellulaire (Coulton et a\., 1987). FhuD est une composante du
systeme de 'Terric hydroxamate uptake" (Fhu) qui se compose Cgalement d'un recepteur
extra-membranaire ii haute affinitt (FhuA) et de dew protbines liCs a la membrane
cytoplasmique (FhuBFhuC) [Couiton et al., 19871. FhuD se trouve H de tres faibles
concentrations dam le pkriplasme d'E. coli K-12. Afm d'obtenir de grandes quantites de
FhuD, Ie genefhuD fut clod dam un plasmid @KTH288) et exprime dam BocilIus
subtilis. Ce vecteur fut con~u de telle sorte que la proteine FhuD recombinante
s'accurnule dam le milieu intracellulaire. Le genefhu. d'E. coli fbt igalernent clonkdans
un plasmide de sicr6tion (pKTH132) et exprime dans Bocil1u.s subtiiis. Ce vecteur devait
en principe excreter la prothe FhuD recombinante a I'extirieur de la cellule. Le
plasmide pKTH288-jhuD donna la protdine FhuD recombinante a un concentration
autour de 180 rngM dam B a c i h subtilis qui s'aggregea en "corps d'inclusion". Des
sera polyclonaw anti-(peptide FhuD) k n t gene& dans des lapins et I'identite de FhuD
fut c o n h e e par imrnuno-"blotting". La protbine recombinante FhuD (BacFhud-Hiss),
solubiliztk a partir des "corps d'inclusion" dans de P'de 8M ou du HCl-guanidine, fut
purifide par chromatographie N~~+-NTA sou conditions dhahuantes. Il fut necessaire
d'enlever I'agent denaturant ( d e ou HCI-guanidine) afin d'obtenir la protbine dam une
conformation native. Tmis protocoles de renaturation h n t essay& a cette fin. Le
premier consistait en me dialyse progressive d ' d e 8M jusqu'a OM. Le second protocole
vii
de renaturation consistait en un diminution lineaire de I'ur6e ou du HCl-guanidine i
l'aide de FPLC. En fin, le troisihne protocole consistait en l'dlimination rpide par FPLC
de l'agent dhahuant. Dam les dew demikres mkthodes, la protCine BacFhud-Hiss fut
immobilMe sur me colome N~~'-NTA avant I'elimination de l'agent denaturant. Les
trois methodes varierent quaat a la quantitti de BacFhud-His6 restant en solution et la
stabilitb de la pro the soluble. Dew moyens h e a t employes a h de tester l'activitd
biologique de la proteine soluble et renatur6e: (i) un test de protection de BacFhud-His6 A
la protbase en absence et en pdsence de ferricrocine (analogue de femchrome); (ii) des
expiriences de "equilibrium dialysis" oh de la fenocricine radioactive fbt dialysC contre
un faible volume de proteine.
LIST OF ABREVIATIONS
bp: base pairs
kDa: kilodalton
PAGE: polyacrylamide gel electrophoresis
Gn-HCI: Guanidine HCl
SDS: sodium dodecy l sulfate
2.uLb15: 2x Luria broth with 25 pg/ml kanamycin
L ~ p l ~ ~ : Luria broth with 125 pg/ml ampicillin.
X-Gal: 5-bromo-4-chloro-3-indoy1-b-D-galactoside
IPTG: Isopropyl-b-D-thiopyrogalactopyranoside
PMSF: Phenylmethyl sulfonyl fluoride
BacFhuD: Recombinant FhuD
BacFhuD-His,:Recombinant FhuD possessing a 6-histidine C-terminal tag
Ni2'-NTA: ~i"-nitrilotriacetic acid
P-ME: 5-P-mercaptoethanol
TBS: Tris buffer saline
EDTA: ethylenediaminetetraacetic acid
CBB: Coomassie brilliant blue
IB: inclusion body
Abs: antibodies
LDAO: Laury 1-dimethy lamine-N-oxide
BPD: Binding proteindependent
LIST OF FIGURES
Figure 1. Schematic representation of the cell wall of Gram-positive and Gram-negative
bacteria. 87
Figure 2. Diagrammatic representation of a periplasmic binding protein. 88
Figure 3. Diagrammatic representation of the structural organization of a typical ATP
transporter. 89
Figure 4. Model of transport mechanism across the cytoplasmic membrane involving the
CM complex and a periplasmic binding protein. 90
Figure 5. Strategy for overexpression and purification of BacFhuD-His, 91
Figure 6. Inclusion bodies from B. subtilis IH6 140 strains overexpressing BacFhuD and
BacFhuD-His,. 92
Figure 7. Purification of BacFhuD-His, fiom urea-solubilized inclusion bodies by ~ i " -
NTA chromatography employing a Biologic FPLC (Biorad). 93
Figure 8. Identification of BacFhuD-His, by immunoblotting. 94
Figure 9. Detection of secreted BacFhuD (secBacFhuD) by irnrnunoblotting. 95
Figure 10. Analysis of renatured BacFhud-His,. 96
Figure 1 1. FPLC-mediated linear gradient GnHCl removal experiment profile (Biologic,
B iorad). 97
Figure 12. Analysis of renatured BacFhuD-His, obtained by FPLC-mediated linear
gradient removal of GnHCl. 98
Fi p re 13. FPLC-mediated fast (one-step) batch-method GnHCl removal experiment
profile (Biologic, Biorad). 99
Figure 14. Analysis of BacFhuD-His, removed by FPLC-mediated, fast (one-step)
removal of GnHC 1. 100
Figure 15. Analysis of BacFhuD-His, renatured in a large scale,"batch"-binding, FPLC-
mediated one-step removal of GnHCI. 101
Figure 16. Analysis of digestion of BacFhuD-His, using proteinase K. 102
ACKNOWLEDGEMENTS
I would like to express my gratitude to my research supervisor, Dr. James Coulton
for his support, encouragement and patience. I also wish to thank all the great friends that
were or still are members ofthe laboratory and whose help and friendship made the time
spent here an enjoyable experience (Andrew, Mark, Alpesh.. .). I would especially like to
express my thanks to the former lab members Greg Moeck. R Srikurnar and David
Dahan for aii their guidance and for introducing me into science. Finally, I would like to
thank my family for their encouragement in the hardest times. The monetary help
(NSERC scholarship) provided by the government of Canada is also very appreciated.
LITERATURE REMEW
Bacteria can be divided into Gram-positive and Gram-negative organisms,
according to their reaction to a staining protocol (Nikaido and Saier, 1992). In Gram-
positive bacteria the cytoplasmic membrane (CM) is surrounded solely by a mechanically
rigid and rather porous peptidoglycan cell wall (Figure 1). On the other hand, in Gram-
negative bacteria such as Escherichia coli, the plasma membrane is surrounded by a
second membrane, the outer membrane (OM), which is located external to an aqueous
region, the periplasm (Figure 1). In addition to a thin peptidoglycan layer, the periplasm
also contains processing enzymes such as phosphatases and nucleases, anionic
oiigosaccharides and transport proteins termed peripiasrnic binding proteins (Nikaido,
1993). These components make the periplasm an important region for processing and
vectorial trafEc of' various substances (Ames et al., 1990)
A) STRUCTURE AND COMPOSITION OF THE OM
The OM of Gnun-negative bacteria is an asymmetric biiayer. The inner
monolayer of the OM is composed of several types of lipids, including the phospholipids
phosphatidylethanolamine and phosphatidylglycerol (the lipid composition of the inner
monolayer is similar to that of the CM bilayer) pikaido and Saier, 19921, whereas the
outer monolayer contains in addition to phospholipids the unique Lipid species
lipopolysaccharide (LPS) [Nikaido, 19931. The l l l y asymmetric distribution and the
chemical characteristics of LPS are responsible for many of the unique barrier properties
of the OM [Ghuysen and Hakenbeck, 19941. LPS is an anionic lipid, which self-
associates through divalent cation cross-bridging and as such it plays an important role as
a penetration barrier against many hydrophobic compounds and other large molecules
(Nikaido, 1993; Ghuysen and Hakenbeck, 1994). The OM bilayer is approximately 50%
covered with protein (Nikaido, 1993). However, despite this abundance of proteins,
relatively few protein species are present. The OM contains less than ten major proteins
which are very abundant and about 50 minor ones (Nikaido and Saier, 1992; Nikaido,
1993). The major OM proteins (OMPs) of E. coli can be expressed in more than 100,000
copies per cell and combined they comprise 80% of the OM protein content (Nikaido and
Saier, 1992). In E. coii the major OM proteins are OmpF and OmpC, which produce
non-specific trirneric transmembrane diffision channels, OmpA which constitutes a
monomeric slow-rate diffusion channel and lipoproteins which can be found bound to the
petidoglycan layer and are therefore thought to play structural roles (Lugtenberg and van
Alphen, 1983; Nikaido, 1993; 1994). Furthermore, other E. coii OM proteins such as
LamB and PhoE are induced to become major proteins under certain growth conditions;
LamB by the presence of rnaltodextrins and PhoE by phosphate starvation (Benz, 1988;
Nikaido, 1993). Minor proteins o f the OM of E. coli include Fe3+-chelator high-affSty
receptors, such as FhuA, which is the OM component of the ferric bdroxamate &take
(Fhu) system. The Fhu system will be described in more detail below.
Because of its asymmetry and protein content the OM functions as a molecular
sieve that prevents most environmental noxious substances (hydrophobic and
hydrophilic) tiom entering the bacterial cell, while allowing the selective uptake of some
essential nutrients (Hancock, 1987; Nikaido and Saier, 1992). In particular, OMS exclude
a variety of proteins, including proteases, lipases, phospholipases, peptidases, saccharide
hydrolases, deoxyribonucleases, and channel-forming toxins, all of which are capable of
damaging bacterial constituents (Nikaido, 1993). Most hydrophilic molecules above a
certain size limit are excluded from transversing the OM of Gram-negative bacteria
(Hancock. 1 987; Nikaido, 1 993). Furthermore, most Gram-negative bacteria also exclude
hydrophobic and amphiphilic molecules, as indicated by the resistance of these bacteria
to hydrophobic agents such as the antibiotics fusidic acid and erythromycin and the
detergent deoxycholate (Hancock and Bell, 1988; Nikaido and Saier, 1992).
The OM contains three major classes of channel-like proteins that make this
membrane selectively permeable to various hydrophilic compounds:
(i) Pore-forming proteins known as porins (such as OmpF of E. coli) consist of
large. open water-filled channels that allow for the non-specific diffusion through the OM
of hydrophilic molecules below the exclusion limit of about 600 Da (Nikaido, 1992;
1993; 1994). Porins are P-barrel proteins that have monomer molecular weights that
range between 28,000 and 48,000 Da and that are present in the membrane usually as
trimers that tend to be resistant to sodium dodecyi sulfate (SDS) denaturation (Nikaido
and Saier. 1992; Nikaido, 1993).
(ii) Porin-like proteins that contain specific binding sites carry out the facilitated
diffusion of some nutrients that would penetrate only slowly through porin channels.
These are designated as specific channels and include the L a d protein of E. coli
(Nikaido and Saier, 1992).
(iii) Several "minor" P-barrel OM proteins that are involved in the specific
active transport of large substrates across the OM against large concentration gradients
(Nikaido, 1993). These proteins have their expression in the OM increased if there is an
environrnental stimulus. The expression of the high-affity hydroxamate OM receptor
protein FhuA increases in the low-iron media. Furthermore, these proteins are
components of active tmspon systems which require in addition to the specific high
affinity OM protein receptor, the TonB protein, whic!. IS anchored to the CM and couples
solute translocation to energy input (Klebba et a[., 1993; Killmann et af., 1993). These
OM high affinity protein receptors have been implicated in ~ e ~ k h e l a t o r transport and
vitamin BI2 uptake (Nikaido, 1993; Ghuysen and Hakenbeck, 1994).
The research described in this thesis is related to the high&finity periplasmic
binding protein-dependent transport of ~e"-chelator complexes in E. coli. Therefore, the
remainder of this review will deal primarily with key components of periplasmic binding
protein-dependent bacterial transport systems.
B) PEIUPLASMIC BINDING PROTEIN-DEPENDENT (BPD) TRANSPORT SYSTEMS
1) Generalities
Periplasmic BPD transport systems are used by Gramnegative bacteria to
transport inorganic ions, amino acids and sugars into the cells (Ames, 1986; Higgins,
1992). These transport systems are characterized by the presence of an OM protein
component which can be: (i) a passive diffusion pore (i.e. OmpF) of little affinity but
high rate of difhion, (ii) a specific diffusion channel of low affinity (i.e. LamB) or, (iii)
a high affinity receptor with a low rate of transport (i.e. FhuA) [Nikaido, 1992; 19941.
BPD transport systems also possess a periplasmic substrate binding protein that is
thought to shuttle the ligand between the OM receptor and the CM components of the
vansport system. The CM components of BPD transporters typically consist of four
membrane-associated domains which will be described in more detail below.
2) The periplasmic binding proteins
Periplasmic binding proteins are the major characteristic of BPD dependent
transport systems. Periplasmic binding proteins seem to provide directionality to BPD
transport systems since this type of transporter is only involved in nutrient uptake and not
export (Higgins, 1992). Recently proteins similar in structure and hct ion to periplasmic
binding proteins have been found in Gram-positive organisms (Schneider and Hantke.
1993); these proteins are anchored to the external side of the CM by a lipid tail
(Schneider and Hantke, 1993). The significance of periplasrnic binding proteins was
initially discovered by a procedu-e involving plasmoiysis of cells at room temperature
followed by osmotic shock. This procedure releases most periplasmic proteins (and some
cellular proteins) into the medium without significant loss in viability (Ames, 1994). The
fmt periplasmic binding proteins to be isolated using this technique were those specific
for Ieucine, sulfate, galactose or histidine (Ames, 1986; 1994). The main argument for
their involvement in transport was: - (i) The close similarity of their substrate binding KD
with the Ks of the corresponding transport system in whole cells, (ii) their periplasm
localization, and (iii) the specific reduction in transport activity in whole cells when the
binding proteins were removed by the osmotic shock procedure (Ames and Lever, 1972;
Ames 1986; Ames et a/., 1990; Ames 1994).
Approximately two dozen periplasmic binding proteins have been identified
(Ames et ai., 1990; Higgins, 1992; 1995; Nowalk el a[., 1994). These proteins are
monomeric and they have molecular weights ranging between 25,000 Da (histidine) and
56.000 Da (oligopeptide) [Ames. 1986: Higgins. 19951. Furthermore. periplasmic
binding proteins bind their substrates with high affinity while undergoing a
conformational change upon the binding of the ligand; they possess close structural
resemblance but surprisingly limited amino acid sequence similarity; and they possess
two functionally and genetically separable active domains (Ames et al., 1990). These
later five features will be discussed in more detail below.
(0 The afFinity of binding
Periplasmic binding proteins demonstrate a very high affinity of binding to their
substrates (K,) in the order of 1 pM or lower (Silhavy er of., 1975; Miller et a/., 1983;
Ames et a/., 1990). They have also been shown to have a single binding site per
polypeptide chain (Miller et al., 1983; Ames et al., 1990). The binding affinity (and
stoichometry of binding) of these proteins is usually measured by equilibrium dialysis
wherein the protein is dialyzed against a large volume of buffer containing varying
concentrations of the radioactively labeled substrate. After dialysis a sample of protein is
counted in an appropriate scintillation fluid. Blank levels are measured (and subtracted)
by assaying the radioactivity present in the external fluid and inside dialysis bags not
containing proteins (Lever, 1972; Ames, 1994). Binding affinity can also be measured by
following fluorescence changes upon addition of substrate. This technique can be used
for those binding proteins in which the loaded form shows a different intrinsic tryptophan
fluorescence than the free form so that the on and off rates of substrate can be measured
and the K, determined (Boos et al., 1972; Miller et a/. , 1983).
Aside fiom the role of the binding protein in the initiation of translocation of
substrate, their functioa due to the inherent property of high affinity binding proteins in
substrate retention (Ames et al.. 1990: Higgins. 1992). is to act as a "trap" for their
substrate preventing it fiom leaving through the OM.
(ii) The conformationat change
Periplasmic binding proteins have been shown to undergo a significant
conformational change upon substrate binding. Initially substrate-dependent
conformational changes were seen with the galactose-binding protein. This protein was
observed to have an altered effective protein charge upon binding of ligand, as indicated
by altered mobility on non-denaturing polyacrylamide gels (Boos et al., 1972). A second
parameter suggesting conformational change was the alteration in the intrinsic protein
fluorescence upon substrate binding. This was initially recognized in the galactose-
binding protein (Boos et al., 1972). The alteration in fluorescence was interpreted as
being due to the alteration in the environment around tryptophan residues since
differential absorption spectroscopy in the presence and absence of substrate coincided
with the absorption spectrum of a tryptophan model compound (McGowan et al., 1974).
Nuclear magnetic resonance (NMR) was also utilized to detect the conformational change
by incorporating '%-labeled amino acids and 5-fluorotryptophan in histidine and
g lutarnine-binding proteins (Cedel et al., 1 984; Ames, 1 986). Protease protection
experiments have also been used as indicators of conformational change (Koster and
Braun, 1990; Rohrbach et al., 1995a). In these essays it was demonstrated that the E. coli
periplasmic binding protein FhuD, when bound to its ligands (i.e. femchrome), shows
greater resistance to digestion by proteinase K and trypsin. Conformational change has
also been tested using other techniques, such as scanning calorimetry, immunology
(Ames. 1986). The phenomenon of conformational modification was further confirmed
by the solving of the structure of several periplasmic binding proteins by X-ray
crystallography (Ames, 1986; Adam and Oxender, 1989; Bnrns er al., 1997).
(iii) The crystal structure
Many periplasmic binding proteins have been crystallized and their structure
determined by X-ray crystallography. This work has been done mostly by F. A. Quiocho
and his collaborators (Ames, 1986; Higgins. 1992). Recently the structure of an iron
binding periplasmic protein has been solved by Bruns er al. (1997). This protein is a
ferric iron-binding protein found in Haemophilus infuemae (hFBP). The hFBP, unlike
the siderophore-binding proteins of E. coli (which are inferred to bind iron in the context
of their specific metal chelate-iron complex), was shown to bind the free form of iron
(Fe33 within the periplasmic space (Adhikari el al., 1995; Bruns et al., 1997). The
prominent feature emerging from the crystal structure analysis is that all binding proteins
adopt a similar structural motif. (Quiocho, 1986; Adam and Oxender, 1989; Tam and
Saier. 1993; Bruns et al., 1997). The periplasmic binding proteins are arranged in two
similar folded globular domain or lobes (the N-lobe and the C-lobe as they contain the
respective ends of the polypeptide chain) which form a cleft that is connected by a hinge
region is made up of three polypeptide chains that are far away from each other in
primary sequence (refer to Figure 2). Although both lobes are made up of non-
contiguous polypeptide segments both have a similar secondary structure (largely of a P-
structure ~~4th connecting loops rich in a-helices). The polypeptides that constitute the
hinge region make the base of the deep cleft that is formed between the two globular
domains. The substrate binds within the cleft mainly through hydrogen bonding,
although hydrophobic and steric components of binding are also important (Ames, 1986;
Quiocho, 1986). It has also been shown, in the case of H influenzae hFBP that this
periplasmic binding protein utilizes two exogenous components, water and a phosphate
ion, as iron ligands (Bruns et al., 1997). Periplasmic binding proteins are flexible so that
the cleft closes down on the substrate upon binding, thus trapping the ligand deeply
within the protein ("Venus flytrap" model, [Mao ef al., 1982; Ames et al. 19901).
Finally, there is a high level of similarity between the structures of the eukaryotic iron-
binding proteins transferrin and lactoferrin and the structures of the periplasmic binding
proteins specific for arabinose, galactose, leucine/isoleucine/valine, and sulfate (Anderson
et al.. 1987). The periplasmic ferric iron-binding (FBP) proteins of H influenzae and
Neisseria meningitidis have been also recently shown to be structurally similar to
transferrin (Nowalk et ai., 1994; Bruns et al., 1997). Furthermore, the coordination of
iron by these FBPs is also homologous to the manner by which transferrins bind iron
(Nowalk et al., 1994; Adhikari et al., 1995). Each of these periplasmic proteins is similar
in size two a single lobe of lactoferrin (or transfenin) [-300 residues]. Transferrin and
lacto ferrin also possess ellipsoidal two-domain structures in which the substrate binds
between the domains (Anderson et ai., 1987; Mason et al., 1993). Furthermore these
eukaryotic iron-binding proteins have a folding pattern and distribution of P-strands, a-
strands and helices that is similar to that of periplasmic binding proteins (Anderson et al.,
1987; Nowalk et al., 1994; Bruns et al., 1997).
(iv) The sequence
As indicated above, periplasmic binding proteins all have similar three-
dimensional structural features (as seen in their X-ray structures) but limited primary
sequence homology (Adams and Oxender, 1989; Tam and Saier, 1993). However close
analysis of these sequences has revealed "signature sequences" which suggest the
divergence of binding proteins from a common origin (Tarn and Saier. 1993).
(v) The functional regions of the binding protein
There is genetic and biochemical evidence that at least some periplasmic binding
proteins possess regions responsible for protein-protein interactions with the CM
components of BPD transport systems (distinct from their substrate binding region).
Much of this data has been obtained both for the histidine- and n~altose-binding proteins
(Kustu and Ames, 1974; Treptow and Shwnan. 1988). In the case of the histidine-
binding protein. a protein mutant has been obtained which is capable of binding to its
wild-type substrate (histidine) and yet it cannot function in transport (Kustu and Ames,
1974; Ames et at., 1990). Therefore, a region in this mutant protein (distinct fkom the
ligand binding region) was postulated to be involved in the interaction with the
membrane complex (Ames et al., 1990). In fact, several suppressor mutations have been
found in the HisP protein (part of the CM protein complex, see below) which overcome
the mutation of the binding protein H id (Ames and Spudich, 1976; Ames et al., 1990),
fUrther suggesting the interaction between these two components (Hid and HisP). The
close similarity between the histidine-binding protein and the closely related 1 ysine-
arginine-ornithine-binding protein which interact with the same membrane complex also
supported the hypothesis that a domain of the binding proteins interacts with the
membrane complex. In fact, these proteins share some regions of very high homology
which are much superior to the overall homology between them. Furthermore, the Hid
mutation was found to be located within that region. Similar genetic evidence was also
obtained for the interaction between the maltose-binding protein and the membrane
complex components of the maltose BPD transport system (Treptow and Shuman, 1988).
The direct interaction between binding proteins and the membrane complex has also been
demonstrated biochemically (both in vivo and in v i m ) by employing crosslinking
methods (Prossnitz et a!.. 1988; 1989; Ames et al., 1990; Rohrbach et a/., 1995b). By
using the water-soluble crosslinker formaldehyde (in vivo) [Prossnitz et al., 19881 and
Su1fosuccinimidy1-6-(4'-azido-2'-nitrophen1ino)hexoate (Sulfo-SANPAH), a
photoactivatable cross-linking reagent (in vitro) [Prossnitz et al., 1988; 19891 it was
demonstrated that protein-protein interactions occur between the periplasmic binding
protein H i d and a component of its corresponding CM complex (HisQ) both in vivo and
in vztro. Recently, W e r evidence for the interaction between periplasmic binding
proteins and the CM complex has been found (Mademidis et al., 1997). This group
demonstrated that, the periplasmic binding protein FhuD, of the ferric hydroxamate
uptake system interacts with regions of two loops of the CM complex protein FhuB, by
using synthetic FhuD peptides in a modified enzyme-linked immunosorbent assay
(ELISA) wademidis et al. 19971. FhuD was suggested to interact with a region
contained within a loop of FhuB (predicted to be located in the penplasm) and also within
another region predicted to include a transmembrane region and a cytoplasmic loop
(Mademidis et at., 1997). Binding of FhuD to the transmembrane region and cytoplasmic
loop have led to the suggestion that periplasmic binding proteins may bind within a pore
formed by the CM complex (Mademidis et a!., 1997). Interaction of FhuD with FhuB
has also been shown by chemical cross-linking of FhuB to His-tagged FhuD added to
spheroplasts (Rohrbach et al.. 1995b) and by inhibition of proteolytic degradation (by
trypsin and pmteinase K) of FhuB by FhuD (Rohzbach et ol., 1995a; 1995b)
3) The membrane-bound components
In addition to the periplasrnic binding proteins and a specific or non-specific OM
channel, BPD transport systems have CM-associated complexes which are thought to
form a channel required for the active transport of substrates across the CM (Higgins,
1992; 1995). These complexes are generally composed of four membrane-associated
domains (Figure 3). Two of these domains are highly hydrophobic and are therefore
predicted to span the CM, constituting the translocation channel. The remaining two
domains have an amino acid sequence which is not recognizably hydrophobic (Arnes et
at., 1990; Higgins, 1992; 1993, despite the fact that they are also CM-associated. These
relatively hydrophilic membrane-associated domains have an extensive region of high
sequence homology among different bacterial and eukaryotic transporter proteins. This
region constitutes an ATP binding motif. All transporters that possess such an ATP
binding motif (i.e. BPD transport systems) belong to the larger family of transporters of
the ATP Binding cassette (ABC) [Higgins et a[., 1986; Higgins, 1992; 19951. These four
core domains appear to provide sufficient machinery to mediate the transmembrane
translocation of solutes (Higgins, 1992). The major characteristics of the transmembrane
and ATP-binding domains will be reviewed in more detail below.
(i) The integral CM proteins
Computer-aided analysis of the primary structure of several membrane proteins
revealed that these possess several uncharged, highly hydrophobic sequence stretches that
are predicted to form a-helices and span the membrane bilayer (Traxler et at., 1993). The
majority of BPD transporter inner membrane domains analyzed so far (Higgins, 1992;
1995) are predicted to comprise two domains of six transmembrane helices ("two-times-
six") helix model joined by hydrophilic sequence stretches that form three periplasrnic
and two cytoplasmic loops (Higgins, 1992). These two domains can either be expressed
as separate polypeptides, such as in ribose and histidine transport systems; or as single
multifUnctiona1 two-domain polypeptide (i.e. Fhd3 of the iron-hydroxamate transport
system) [Higgins, 19921. The C- and N-termini of these proteins are located at the
cytoplasmic face of the membrane. The topology of seven membrane components of
BPD transport systems kom E. coli (MalF, MalG and Prow) and S. typhimurium (HisQ,
HisM. OppB and OppC) has been determined by biochemical and genetic analysis
(Higgins et al., 1982; 1992). Such analysis indicated that there are exceptions to
computer-predicted six-membrane spanning organization, such as in the case of the
integral membrane protein MalF, which is predicted to be comprised of eight membrane-
spanning segments (Froshauer el al., 1988), and HisQ and HisM which possess only five
such membrane spanners (Kerppola and Ames, 1992).
Comparison of amino acid sequences of transmembrane proteins of different
transporters reveals, in most cases, Little or no similarity between these proteins (Higgins,
1 W ) , despite a high degree of conservation of their secondary structure (Higgins, 1992;
Kerppola and Ames, 1992). This has been explained by the fact that hydrophobic a-
helical structures can be satisfied by many alternative amino acid combinations (Higgins,
1992). However, one highly conserved sequence feature can be found in the inner-
membrane proteins of BPD transport systems. This sequence (EAA--G---I-LP) is
located at the C-terminus of each of these proteins, within the last cytoplasmic loop
("EAA" loop) [Dassa and Hofnung, 1985; Higgins, 19921. The high degree of
conservation of the "EAA" loop is suggestive of its importance in the h c t i o n of
transmembrane proteins. This idea is supported by deletion experiments of amino acids
within the EAA loop of different proteins (i.e. MalG and FhuB), which abolished
transport completely (Dassa, 1990; Koster and B o b , 1992). There is also evidence that
a region corresponding to the "EAA" loop of FhuS interacts with the periplasmic protein
FhuD, and that this interaction is essential to femchrome-iron transport (Mademidis et
al.. 1997). Mademidis et al. (1997) demonstrated that FhuB artificial peptides
encompassing the EAA loop region bound to purified FhuD (Mademidis et al., 1997).
The exact function of the "EAA" loop is not yet clear, however its location in a
cytoplasmic loop suggests that it may interact with the conserved membrane-associated
ATP-binding subunit (see below) [Koster and Bohm, 1992; Kerppola and Ames, 1992;
Mademidis et al-, 19971. In fact, the "EAA" loop maybe involved in signal transduction
between the ATP-binding subunit and the integral membrane components of the transport
systems (Kerppola and Ames, 1992; Mademidis et al., 1997).
Because of their hydrophobic nature, their membrane-spanning characteristics,
and their structural relatedness to transport protein of the poton motive force (PMF) type
[Henderson, 19931 the function of the integral membrane proteins of BPD transporters is
as the actual translocators of the system (with the periplasmic binding proteins as
"transport trigger" and the An-binding component as the energy module [see below])
[Higgins. 1 9921.
(ii) The ATP binding cassette subunits
In addition to the periplasmic binding proteins and the integral membrane
proteins, each BPD transport system possesses two energy-transducing domains
contained within two polypeptides (i.e. FhuCIFhuC homodimer) or a single
multifunctional polypeptide (i.e. RbsA) that are essential lor BPD transport. These
domains of BPD transport systems were shown to have striking homology among several
bacterial and eukaryotic transport proteins that extends over a region of 200 amino acids
and includes two short Walker nucleotide binding fold motifs (Walker et al., 1982;
Higgins, 1992; 1995). Due to the presence of this conserved nucleotide binding domain,
BPD transport systems have been grouped together into the ABC transporter or trafXc
ATPase superfamily (Ames, 1986; h e s et d, 1990; Higgins, 1992). Examples of ABC
transporters of eukaryotes include the multiple drug resistance protein (MDR) and the
cystic fibrosis protein (CFTR), both of which exist in humans (Nikaido and Saier, 1992;
Higgins. 1992; 1995).
Biochemical evidence (mostly from studies performed on the HisP component of
the histidine transport system) shows that the ATP-binding proteins are tightly associated
with the inner membrane (Kerppola et al., 1 Wl), even though their amino acid sequence
does not include hydrophobic stretches long enough to transverse the lipid bilayer
(Higgins, 1992). Furthermore, studies of the energy transducing subunit of BPD
transport systems (namely HisP) also indicates that the ATP-binding subunits form a
complex with the inner membrane components of the transport complex (Kerppola el al.,
1991). Studies on HisP also suggest that ATP-binding proteins are found on the
cytoplasmic face of the membrane (Kerppola er al., 1991), even though a region of these
proteins may extend through the inner membrane components into the periplasm
(Kerppola el al., 1991; Baichwal el al., 1993). Structural models of ATP-binding
domains predict sequences extending from the core ATP-binding structure which could
potentially filfill such a role (loop 2/3 region; Higgins, 1992).
The predicted nucleotide binding fold has been shown to bind both ATP and ATP
analogues (i.e. 8-azido-ATP) [Hobson el af., 1984; Ames, 19861, and to hydrolyze ATP
(and GTP) [Ames and Joshi, 1490; Davidson and Nikaido, 199 I], strongly suggesting
that ATP is in fact the source of energy for ABC type transporters, such as the BPD
transport systems. In fact it has been found that both binding, and hydrolysis of ATP (or
GTP) are required for transport of substrates across the CM (Hobson el al., 1984; Bishop
et al., 1989). The stoichometry of ATP hydrolysis and substrate transport has also been
analyzed in different in vivo and in vitro systems (Ames et al., 1990; Higgins, 1992).
Numbers obtained generally point to a value of around two molecules of ATP hydrolyzed
per substrate molecule transported. This number would be in accordance with the fact
that two ATP binding domains are present in all BPD transport complexes (Ames et al.,
1990; Higgins, 1992).
Due to the absence of experimental data on the structure of the domains of the
ATP-binding subunits, models for the structure of these subunits have been derived based
on the known tertiary structure of the ATP-binding proteins adenylate kinase (Hyde et al.,
1990) and rasp21 (Higgins, 1992). HisP, the ATP hydrolyzing component of the
histidine uptake system of Salmonella enterica serovar typhimirium was used as the basis
for the model. Analysis of the information obtained indicates that ATP-binding domains
consist of a central domain of five hydrophobic P-sheets connected by a-helices or turns.
Three of these P-sheets form a Rmsman nucleotide binding fold and Walker motif A
(Rossman et af.. 1975; Walker et aL, 1982). Protruding from the core structure, between
the P-sheets 2 and 3 there is a large predominantly a-helical loop (loop 2 3 ) that extends
from the core structure and is not involved in binding ATP. A possible function of this
loop might be the coupling of ATP hydrolysis and transport (Hyde et al., 1990). A model
consistent with the data currently available predicts that regions corresponding to the
helical domain (loop 2 3 ) undergo a conformational change upon nucleotide binding and
hydrolysis [Hyde et aL, 19901. Therefore, the helical domain of the BPD transporter ATP
binding subunit might serve as a "lever" to transduce a structural change caused by ATP
hydrolysis to a conformational change of the membrane bound complex.
4) Model for the mechanism of CM translocation of substrate in BPD-ABC
transport systems
Various models for the mechanism of BPD transport systems have been proposed
(i.e. Shuman, 1982; Arnes, 1986; Davidson et al., 1992). All of these models suggest that
substrate loaded binding protein interacts with the membrane bound component (see
above). The following model proposed by Davidson et al. (1992) [Figure 41 is most
widely accepted:
During transport, the membrane components, driven by ATP hydrolysis, proceed
through a series of different states. (i) State I is fully energized but incapable of
tnnslocating the free substrate. It can however, bind the closed form of the substrate-
loaded periplasmic protein. (ii) Binding of this form triggers the conversion of the
membrane complex to state I1 which exhibits a higher affinity for the form of the
periplasmic binding protein that opens its substrate-binding site to the membrane
complex. The membrane complex then reaches its lowest state of energy (state HI) as the
substrate is discharged into the cytoplasm and the binding protein leaves the complex.
ATP hydrolysis by the ABC subunit is then thought to restore the membrane complex
back to state I.
The research presented in this thesis deals with the periplasrnic binding protein
component of the BPD-high fiinity Fhu system of E. coli. Therefore, the remainder of
this review will deal with high affmity iron uptake in E. coli, in general and then finally
with the Fhu iron uptake system itself. The function, structural features and regulation of
the Fhu system will be analyzed.
5) High affinity iron uptake in E. coli
( iron
Iron is an essential element to all cells. Its roles in microbial physiology are
numerous. Iron is a main constituent of heme enzymes, which include cytochromes and
hydroperoxidases. Ribonucleotide reductase also contains iron, and non-iron
nitrogenases require an iron protein in a complex for their activity (Briat, 1992). Iron is
the fourth most-represented element in the earth's crust. Being so abundant it should not
be a limiting growth factor for bacterial growth. However oxygen is even more common
in the environment, so that at neutral pH iron is particularly insoluble and tends to
precipitate as ferric hydroxides (Briat, 1992). In humans most iron is found bound to
eukaryotic iron-binding carriers such as tranferrin in serum. to lactoferrin in secretory
fluids and phagocytes, and to intracellular femtin (Anderson et al., 1987; Mason el al..
1993). Therefore in humans most iron is unavailable to support bacterial growth within
host.
(ii) siderophores
Iron starvation leads to the synthesis of native siderophores by bacterial and
fungal organisms that live in aerobic conditions, and by microbial pathogens that live in
an animal host in the presence of the eukaryotic iron-carriers mentioned above.
Siderophores are low molecular weight molecular carriers (400 to 1000 Da) pankford,
19731, characterized by a very high affmity for femc iron (Neilands, 1981). These
molecular carriers are secreted into the environment where they bind the insoluble ferric
iron ( ~ e ~ ' ) . Siderophores have divergent structures that can be classified into three main
groups: the hydroxamate type, the phenolate type and the organic acids (such as citrate).
The structures of the hydmxamates femchrome, aerobactin, coprogen and rhodotorulic
acid as well as the phenolate enterobactin have been previously described (Neilands,
1981). E. coli normally produces and secretes the phenolate type siderophore
enterobactin. Its synthesis is regulated by the internal iron supply (Neilands, 1981). Of
all the hydroxarnate type siderophores above, only aerobactin is synthesized and secreted
by certain strains of E. coli. Coprogen, ferrichrome and rhodotoluric acid are produced
by fungi of the Genera Penicillia, Neurospora and Ustilago (Muller er al. , 1984). E. coli
uses high affinity OM proteins which specifically recognize the fungi-synthesized
siderophores.
(iii) OM receptors for ferric siderophores
Most siderophores are either too large to cross the OM through porins (which
have a channel exclusion limit of about 600 Da) or have a difision rate through porins
that is too slow to provide growth. Furthermore siderophores are very scarce in the
environment so that iron transport systems require high affmity OM receptors to increase
their effective concentration on the bacterial OM surface.
There are three known high af'finity iron uptake systems in E. coli and one high
affinity B,? uptake system and these are served by eight different OM receptor proteins.
These are the Fep (Elkins and Earhart, 1989), the Fhu (Coulton er al., 1987), the Fec
(Staudenmaier er al., 1989) and the Btu (Friedrich el al., 1986) transport systems. The
Fhu transporter system utilizes the OM receptor FhuE to transport coprogen, FhuA for
femchrome and IutA for aerobactin. The Fep system transports enterobactin using the
FepA OM receptor (Braun, 1985), and dihydroxylbenzoylserine as well as
dihydroxybenzoic acid using the Fiu and Cir receptors (Maddock et al., 1993). The Fec
transport system uses FecA to bind dicitrate (Braun, 1985). Vitamin B,, is h-ansported
into the cell by the BtuB receptor (deVeaux et al., 1986; Friedrich et al. , 1986). The Btu
transport system is structurally and hctionally similar to the femc-siderophore transport
systems (deVeaux et al., 1986). All these active transporters depend upon the energy-
coupling protein TonB to catalyze ferric-siderophore or vitamin B,, transport (below)
[Skare et a/.. 19931.
The receptor proteins for fenic-siderophores (and vitamin B,J are minor proteins
of the OM (Lugtenberg and van Alphen, 1983). The receptor proteins may be detected by
the analysis of OM proteins of E. coli by SDS-polyacrylamide gel electrophoresis (i.e.
C m e l and Coulton, 1991). The protein bands corresponding to the OM receptors are
evident upon comparison of OM proteins from cells grown in iron-depleted media versus
cells grown in excess iron (Neilands, 1994). OM receptor proteins have monomeric
molecular weights that range between 60 kDa and 80 kDa (Neilands, 1982). These
receptors are thought to be iknctionally independent monomers even though there is
evidence that FepA and FhuA may form trimen in vivo (Skare et a[., 1993; Moeck et al.,
1997). High affinity OM receptors are essential components of their respective transport
systems: without them transport activity is abolished. Furthermore, while in classic BPD
transport systems such as the histidine BPD transport system the location of substrate
recognition is the periplasmic binding protein, in the high iron uptake transport
systems the location at which major substrate recognition takes place is the OM receptor
(Koster and Braun, 1990). Consistent with this conclusion is the observation that the
binding protein associated with the Fhu transport system (FhuD) binds all the
siderophores with less affinity than the individual OM receptors (KBster and Braun,
1990).
Analysis of amino acid sequences of OM receptors shows that they possess a
larger number of hydrophilic amino acids than would be expected for a macromolecule
that resides in a membrane. In fact these OM proteins have few regions of sufficient
hydrophobicity to span the bilayer (Klebba et al., 1990, Klebba et al., 1993).
Crystallographic studies on the OM non-specific channels OmpF and PhoE of E. coli
have shown that these porins are composed of sixteen amphipathic transmembrane P-
strands separated by loops oriented toward the cell surface and turns located in the
periplasm [Cowan et al., 19921. Computer modeling (Klebba ei al., 1993) of OM high
atlinity receptors based on their amino acid distribution and the crystallographic studies
on porins (Cowm el a/., 1992), as well as, immunochemical monoclonal mapping
(Klebba r i al., 1990; Moeck et a/., 1995) and biophysical (Moeck et al., 1996) studies of
the OM receptor proteins FhuA and FepA have led to the prediction that these OM
receptors are also made up of several P-strands arranged an arnphiphilic P-barrel
interspersed by hydrophilic loops and turns. Deletion of some externally exposed loops
of FhuA (Killmann et al., 1996) and FepA (Rutz er al., 1992) turned these proteins into
non-specific diffusion channels, suggesting that OM high flinity receptors possess
certain surface located loops that confer ligand specificity and cover an underlying
channel. Furthermore, evidence of confonnational changes of OM high affinity receptors
upon ligand binding has been seen for both FepA (Liu et ai., 1994) and FhuA (Moeck et
ol., 1996; Moeck et ai., 1997). In the case of the femchrome receptor FhuA the
conformational changes have been suggested to involve a periplasmically located loop
that could interact with the cytoplasmic energy-transducing protein TonB (Moeck et a&.,
1996).
(iv) TonB protein
The energy requirements for translocation of all ironcomplexes and vitamin B,?
across the OM requires the product coded by the tonB gene of E. coli (Plastow and
Holland, 1979). The TonB protein has been shown to be anchored in the cytoplasmic
membrane and to extend into the periplasmic space between the OM and CM (Plastow
and Holland, 1979; Postle, 1993). Furthermore, TonB has been found to be associated
with the CM proteins ExbB and ExbD. Together, these three proteins are necessary for
coupling of PMF with active transport at the OM (Postle. 1990; 1993; Larsen et al.,
1994). The need for TonB to function as a energy-transducing element was inferred from
observations that transport across the OM is driven by PMF, although a proton gradient
cannot exist across the OM due to its porosity. In fact, ferric-siderophore and vitamin B,2
uptake have been shown to be inhibited by protonophores, which dissipate PMF, and by
arsenate which depletes nucleoside triphosphate pools (Bradbeer and Woodrow, 1976).
Therefore it was suggested that TonB couples metabolic energy with the OM receptor
proteins (Hantke and Braun, 1978). Without TonB the OM receptors are still capable of
binding to their ligand but do not carry active transport (Hantke and Braun, 1978). TonB
(and ExbBD) has been found to have homologues in many other Gram-negative bacteria
such as Pseudomonas aeruginosa, Yersinia enterocolitica and Haemophilus influewe
(Poole et al., 1996; Moeck et a&. , 1 997).
To& is composed of an uncharacteristically high content of prolyl residues
(16%) [Klebba et al., 19931 and it contains few recognizable p-turns (Wilmot and
Thornton, 1988). The N-terminal domain of TonB is predicted to span the membrane
bilayer as a hydrophobic helix (Hannavy et al., 1990; Klebba et al., 1993; Postle, 1993).
TonB also includes two rigid, highly charged internal domains whose hctions are not
known: a negatively-charged Glu-Pro repeat followed by a positively-charged Lys-Pro
repeat. The region between these charged regions has been shown to possess certain
conformational flexibility (Brewer et a/.. 1990). The C-terminal domain of TonB is
predicted to consist of three arnphiphilic structures (P-strand-a-helix-P-strand) separated
by p-turns. The terminal amphiphilic P-strand is essential for TonS h c t i o n (Klebba et
a/., 1993). It has characteristics that suggest it may span the OM bilayer (Klebba, et a[.,
1993; Postle. 1993). The charged regions of TonB are predicted to be located in the
periplasm (Hannavy el al., 1990; Klebba et ul., 1993) while the C-terminal region of this
protein exists either in the periplasm or OM (Klebba et al., 1993).
TonB is thought to interact with periplasmically-located loops or transmembrane
strands OM high affinity receptors, inducing them to release their substrate to periplasm
in binding proteins. The following three findings lend credibility to this hypothesis:
(i) All known high-affinity OM receptors have homologous eight-amino acid
region, characterized by invariant Thr and Val residues and termed the TonB box (Koch
et al., 1987; Postle, 1993). The fact that this domain is highly conserved suggests that
this amino acid stretch has been evolutionarily preserved to interact with another specific
macromolecule.
(ii) Suppressor mutations have been observed between TonB and the OM receptor
proteins that compensate for mutational deficiencies (Heller et al., 1 988; Bell et al., 1 990;
Braun et a!., 1991). For example, Heller et al. (1988) isolated extragenic supressor
mutations that partly restored the defect in vitamin B,, utilization in a btuB mutant strain
(Leu-8 in the middle of TonB box was converted to proline) carrying the tonB gene on a
rnulticopy plasmid. Suppressor mutations were identified in the plasmid-encoded t m B
gene, and they converted Gln-165 to Leu or Lys, which suggested that direct interaction
occurs between TonB and the TonB-box region of BtuB.
(iii) There is available in vivo and in vitro evidence for the physical interaction
between the OM receptors FhuA and FepA and TonB (Hannavy et al., 1990; Skare et a/.,
1993; Moeck et a!., 1997). The in vivo studies were performed by employing
formaldehyde crosslinking techniques using whole cells (Skare et a/., 1993; Moeck er al.,
1997). Moeck er al. (1997) have shown that TonB favorably crosslinks with ligand-
loaded forms of the OM receptor FhuA, suggesting that the ligand-loaded FhuA has a
conformation which differs from ligand-free FhuA. Furthermore. purified TonB was
shown to be retained on a Ni2'-NTA agarose resin containing histidine-tagged FhuA
(Moeck ei aL. 1997). The retention of TonB was enhanced when ligand-bound histidine-
tagged FhuA was applied to the column (Moeck et al., 1997). This in vino assay further
suggests that OM receptors (such as FhuA) and TonB interact favorably in the presence
of ligand.
(v) Ferrichromeiron uptake
Femchrome is a cyclic hexapeptide siderophore of the hydroxamate type. It has a
molecular weight of 740.54 Da and it was first isolated from the smut h g u s Ustiiago
sphaerogena (Neilands, 1952). The high affinity uptake of femchrome is dependent on
the fiu operon. This operon consists of four genes in the following order: fiw4 jhuC
fiuDfhuB (Fecker and Braun, 1983; Coulton et al. , 1986). Transcription of these genes
proceeds fromjhu. tofhuB (Fecker and Braun, 1983; Coulton et al., 1986).
TheJhuA gene encodes the protein FhuA (M, 78,992; 714 amino acids) which is
the hydroxamate femchrome receptor of E. coli. The FhuA protein is a minor, inducible
component of the outer membrane of E. coli and is found in about 10) copies per cell
(Hantke. 1981). In response to iron limitation or to constitutive synthesis of iron
transport proteins in a regulation-deficient fir mutant (Hantke, 198 1 ), the copy number of
FhuA can be increased to about lo4 copies per cell. [n addition to binding femchrome,
FhuA acts as the receptor for the phages $80, TI, TS and UC-I and the bactericidal
proteins colicin M, rnicrocin 25 and albomycin (Neilands, 198 1; Coulton er aL, 1987).
FhuA like all other known OM receptors contains a consensus TonB-box near its N-
terminus (Coulton et al., 1986; Postle. 1993). Furthermore FhuA has been shown to
interact with the TonB energy-transducing protein both in vivo and in vitro (Hannavy et
a!. , 1990; Moeck et al., 1997). TonB has been suggested to couple the PMF across the
CM with the energy necessary for the release of ferrichrome into the periplasm (above).
Similar to all other BPD iron-uptake systems, the overall Y, (0.06 pM) of ferrichrome
transport is determined by its OM receptor, in this case FhuA. If cells are treated with
protease which renders the OM permeable, independent of FhuA, the Y, increases by 10-
fold (0.7 pM) [Wookey et of., 19811.
The fhuB, fhuC and fiuD genes encode proteins which catalyze the transport of
ferrichrome and other ferric hydroxamate compounds (femc aerobactin, ferric coprogen)
from the periplasm through the CM into the periplasm (Coulton et al., 1987).
FhuB and FhuC are the CM components of the Fhu transport system. ThefiuC
sequence revealed a fairly hydrophilic protein with some hydrophobic stretches, and
possessed two regions which displayed strong homology to ATP-binding proteins
(Burkhardt and Braun, 1987; Coulton et al., 1987). Such proteins are found in transport
systems for sugars, peptides, amino acids and other ferric siderophores (above; Ames,
1986; Higgins. 1992) that require a periplasmic binding protein. Initially this was taken
as evidence that transport of ferrichrome across the OM follows a periplasmic BPB
transport mechanism. When point mutations were introduced into the two domains of
FhuC presumed to contain the ATP-binding sites (Becker et a/., 1990) the resulting FhuC
derivatives were no longer able to transport fenichrome and albomycin. This result
helped confirm that the two domains that represent consensus sequences among all CM
proteins of BPD transporters are involved in substrate transport. The ATP-binding
cassette subunit of FhuC (Coulton et a/., 1987) places the Fhu transport system in the
BPD ABC superfamily of transporters (above). The fhuB gene encoded a very
hydrophobic protein of 659 amino acids with a predicted molecular weight of 70,329.
The molecular weight of FhuB deduced from the nucleotide sequence was twice as large
as usually found for hydrophobic membrane proteins of the BPD ABC transporter family
(above; Higgins, 1992). The first 20 amino acids have the characteristic features of a
signal peptide. It was concluded that FhuB is a membrane protein since it migrated
anomalously during SDS-polyacrylamide gel electrophoresis and was similar to other
hydrophobic proteins (Koster and Braun, 1986). Furthermore, when thejhuB gene was
cloned downstream of the strong phage T7 promoter the very hydrophobic FhuB protein
was found to localize to the CM (Kiister and Braun, 1989). These results together with
the homology of FhuB with other CM proteins (Higgins, 1992) of the BPD ABC family
have led to the prediction that FhuB possesses two domains, each one made up of a-
helices that span the membrane six times (Higgins, 1992). Most bacterial AE3C
transporters encode for two proteins of this class (Higgins, 1992; 1995). Therefore it is
predicted thatfhuB arose by duplication and fusion of a primordial gene (Higgins, 1992,
Tam and Saier. 1993). FhuB and FhuC are thought to interact and form a channel
through which ~ e ~ + or ~e)'-femchrorne is pumped across the CM.
The FhuD protein is the only candidate for a periplasmic protein in the fhu
operon. FhuD displays some homology to the periplasmic binding proteins BtuE, FecB,
FatJ3 and FepB (Staudenmaier er al., 1989; Tam and Saier, 1993). All these proteins are
thought to have arisen from a common ancestor (Higgins, 1992; Tam and Saier, 1993).
The sequence of theFuD gene has been determined (Coulton er al., 1987). ThefhuD
gene encodes a protein which contains a typical N-terminal thirty-amino acid signal
sequence essential for its translocation across the CM, and a mature protein of 266 amino
acids (M, 29,610). Similar to other periplasmic binding proteins and some eukaryotic
iron-binding proteins, such as transfemn and lactoferrin (Anderson er al., 1987; Tam and
Saier, 1993), FhuD is thought to be a two-domain protein, which undergoes a
conformational change upon ligand binding (above; Ames, 1986; Higgins, 1992). The
molecular details of this mechanism remain to be elucidated with FhuD. Rohrbach el al.
(1995a) have been able to overexpress a form of FhuD in E. coli in which the signal
sequence was replaced by a histidine tag. This group was to able to obtain significant
amounts of histidine tagged FhuD which accumulated in the cytoplasm of E. coli in an
apparently native form. Subsequently, in vivo reconstitution studies and in v i m protease
protection assays performed on the purified his-tagged FhuD seemed to indicate that this
protein is capable of binding and transporting ferric-siderophores across the CM
(Rohrbach et al., 1995a). Recently, the his-tagged FhuD has also been shown to interact
with the integral membrane protein FhuB (Mademidis et a!., 1997)
(vi) Regulation of the iron transport
Greater than thirty genes in E. coli have been found to be involved in siderophore
synthesis and ferric-siderophore transport. These genes are derepressed by iron-limiting
conditions. One gene termed@ gerric &take regulation) plays an important role in iron
regulation (Hantke 198 1 ; 1982; Litwin and Caiderwood, 1993). In fir mutants it has
been found that iron-related genes are derepressed (Hantke 198 1; 1982; Fleming et al.,
1983). The nucleotide sequence of an 868 bp hgrnent containing the fiv gene was
determined (SchafTer er al., 1985) and the amino acid sequence derived from the
nucleotide sequence offir comprised 148 amino acids corresponding to a polypeptide of
16,795 Da. Since the Fur protein represses the expression of all iron-related genes so far
tested (which include thejhu andfec operons) in iron-rich media, it has been suggested to
act as a repressor when cells are grown in an environment containing iron or iron-related
compounds. Therefore the Fur protein was suggested to be a DNA-binding protein. Fur
was found to contain a sequence from amino acids 1 14-1 29 that exhibits homology with
the consensus pattern found in other DNA-binding proteins (de Lorenzo et al., 1987).
DNA-footprinting experiments of the iron-regulated gene iucA of E. coli, using purified
Fur showed that a region in the vicinity of the iucA promoter was protected (de Lorenzo
er al., 1987). This region was also shown to contain a dyad symmetry element.
Additional work has revealed that the dimeric form of Fur, complexed with ferrous iron,
Mn2' or cd2+ binds to the promoter region of the aerobactin promoter [iucA is the fist
gene of the aerobactin promoter] (Bagg and Neilands, 1987). Furthermore, in the
presence of either Mn" or ~ e " , the addition of purified Fur to cell extracts prepared from
a fur mutant was found to repress in vitro transcription of a DNA template containing an
iucA-lac2 fusion (Bagg and Neilands. 1987). The results above suggest that Fur acts as a
repressor of transcription in the aerobactin operon, preventing RNA polymerase from
binding or utilizing the aerobactin promoter in vivo as long as the intracellular iron supply
remains above some critical level,
Therefore all these results indicate the Fur element is probably a repressor DNA-
binding protein, whose trigger to bind DNA is its binding to inorganic ions (i.e. Fe2'). In
the absence of inorganic ions (such as in iron-deplete media) the Fur repressor can no
longer bind the DNA operator regions genes involved in iron-metabolism, so that RNA
polymerase can bind the promoter DNA regions and transcribe the genes involved in the
high affinity uptake of iron (Litwin and Calderwood 1993).
C) OVEREXPRESSION OF RECOMBINANT PROTEINS M BACILLUS
1) Secretion of Heterologous proteins in Baciillus
The overall pattern of secretion in the Gram-positive bacteria of B a c i h Genus,
which has been studied mostly in B. subtilis, foIIows the principle established for the
secretion of proteins across the cytoplasmic membrane in E. coli. Most components of
the protein export apparatus of E. coli (such as SecA, SecB, SecY and SecE) share
homology with proteins of B. subtilis also shown to be involved in the secretion pathway
(Pugsley 1993; Simonen and Palva, 1993). Furthermore, secreted proteins of B. subtilis,
as those of E. coli, must possess a signal sequence (leader peptide) that is cleaved as the
protein transverses the CM. The presence of a functional signal sequence is an absolute
requirement for secretion to take place. It contributes to the folding of the nascent
polypeptide chain, it is the target of the binding and recognition by the translocation
machinery, and it interacts with the components of the translocator in the initiation of the
translocation of the polypeptide across the membrane (Wickner ei a!., 1991). On the
other hand, the mature sequence of a secreted protein is thought to have no active
h c t i o n in the process of secretion (Chan et al.. 198 1 ; Sarvas, 1995). The considerations
above led to the prediction that the hsion of a signal sequence functional in B. subtilis to
the DNA sequence encoding a protein of interest (preferably a naturally secreted protein)
would result in secretion from the cell, as long as there were also functional expression
signals for transcription and translation (Sarvas, 1995). The expression signals and signal
sequences of a-amylases and proteases that are secreted to high levels in many species of
Bacillus were considered a logical choice for this purpose. Initially, sequences encoding
for a-amylase were cloned into the high-copy plasmid pUB110 by Palva (1982) so that
amyE was put under the control of a strong bacillar promoter. This resulted in up to 1 gfl
of a-amylase being secreted into the media (Palva, 1982). This is 2500-fold more than
the endogenous level of a-amylase secretion in B. subiilis. Since then several more
bacterial and eukaryotic proteins have been cloned into various bacillar expression
vectors leading to different levels of expression and secretion (Simonen and Palva, 1993;
Sarvas, 1995). For instance, when the E. coli periplasmic protein TEM P-lactamase was
cloned into different B. subtilis expression vectors levels of secretion ranging between 20
mg/l and 150 mg/l were obtained (Simonen and Palva, 1993; Sarvas 1995). The level of
secretion depended mostly on the strain used (i.e. whether it was exoprotease deficient)
but it was also related to the promoter, the signal peptide and the type of growth media
used (Nakarnura et a!. 1985: Simonen and Palva 1993: Sarvas. 1995). Some subunits of
Pertussis toxin have also been secreted at up to 500 mg/ml under optimal conditions
(Sarvas, 1995). Most eukaryotic protein have been secreted to only low levels in Bacillus
(0 - 10 mg/ml) [Simonen and Palva, 1993; Sarvas, 19951. This was suggested to be due
to the differences in protein processing pathway between eukaryotic and prokaryotic
systems (Sarvas, 1995). Most limitations in the secretion of protein to high levels are
related to the secretion of eight major exoproteases and several minor exoproteases by
most Bacillus species (Nakamura et a!., 1985; Simonen and Palva, 1993; Sarvas, 1995).
These exoproteases (produced mostly in the stationary phase) can reduce secreted
heterologous protein yields by several fold in strains that are not exoprotease-deficient
andfor in the absence of significant amounts of protease inhibitors.
2) Production of heterologous proteins intracellularly in B. subtilis
Most efforts to overexpress recombinant proteins in B. subrilis have concentrated
on the extracellular mode of production. However, Bacillus species can be used even if
synthesis of protein takes place intracellularly. The main problems limiting the high level
production of secreted proteins (i.e. degradation by exoproteases, arrest at the cell wall)
can be circumvented if the proteins remain intracellular (Sarvas, 1995). By slightly
modifying the secretion vectors it becomes possible to achieve high levels of intracellular
accumulation. For example, cloning of a sequence encoding only for the partial signal
sequence of a-amylase into a Baciilus expression vector has led to high level expression
and intracellular accumulation of several heterologous proteins whose sequences were
inserted downstream to the partial leader peptide (Puohiniemi et n l , 1992; Sirnonen and
Palva, 1993). Some of these proteins, such as OmpF and OmpA of E. coli have been
produced intracellularly to high levels in B. subtilis (10 - 40 mg/l OmpA; 40 - 50 rngA
OmpF) [Puohiniemi el al., 19921. However, both OmpF and OmpA were found in an
insoluble form in B. stibtilis. They were located in the fast sedirnenting fraction after
breakage of the cells which is consistent with the presence of inclusion bodies (IBs)
[Puohiniemi et al., 1 992; Sarvas 1 9951. IBs are intracellular globules of electron-dense
material in which proteins predominantly exist in a denatured state, being partially or
completely refolded (Sarvas, 1995). The intracellular production of heterologous proteins
as an IB provides the advantage of an easy early purification step, using simple
techniques cellular breakage and low-speed centrifigation (Srikumar, el al., 1993).
Moreover, the protein recovered from the IBs is often highly enriched in the protein of
interest (Srikumar, el al., 1993; Sarvas, 1995). Because the proteins contained within IBs
are highly insoluble they have to be treated with a strong denaturant (such as urea or
guanidine-HCl) in order to be solubilized. This process results in the complete unfolding
of the protein. In order to renature denaturant-solubilized protein into a native
(biologically active) form it is necessary to remove the denaturing agent. Reduction of
the denaturant concentration can be done by dilution, dialysis or any other method that
removes the chaotrope. Unfortunately the conditions to be used in this process are highly
dependent upon the specific protein being refolded and must be determined in an
empirical. case-by-case manner. An example of a protein that has been successllly
renatured is the eukaryotic plasminogen activator protein (TPA). IBs containing TPA
were soluhilized in guanidine-HCI, was subsequently diluted to allow refolding.
Purification by afinity chromatography produced a protein with the native enzymatic
activity of plasminogen activator (Wang et al., 1989). The Haemophilus influenzae type
B porin (Hib) has also been successfilly refolded by a similar process (Dahan et al.
1 996).
D) RATIONALE FOR THESIS
FhuD is the only candidate for periplasmic binding protein within thefhu operon.
Until recently, no studies on E. coli FhuD had been possible since this protein cannot be
expressed to high levels in its host system (E. coli K-12). The work presented in this
thesis deals with the overexpression of the fhuD gene in a BaciNus subtiiis expression
system and purification and refolding of the recombinant jhuD gene product. The
recombinant FhuD was modified to contain a C-terminal hexahistidine tag (BacFhuD-
His,) to enable for purification and refolding on a Ni2+-NT~ matrix. Furthermore,
overexpression and secretion of recombinant FhuD in a Bacillus secretion system was
also attempted. Finally, studies on the binding Smity of FhuD as compared to that of
the high affinity OM receptor FhuA (whose wild type phenotype has previously been
demonstrated) were also performed. These results could indicate whether the
recombinant FhuD obtained in this study has a wild type phenotype as far as the binding
to one of its natural ligands (i.e. ~e"-femchrome) is concerned.
MATERIALS AND METHODS
Bacterial strains and plasmids. Bacterial strains and plasmids that were used
are described in Table 1.
Media, reagents and enzymes. All liquid and solid media have previously been
described (Silhavy et d, 1984; Sambrook et aL, 1989; Srikumar et al., 1993). For
plasmid selection, the antibiotics ampicillin (125 pg/ml) and kanarnycin (25 pglml) were
added to the media. B. subtilis transformants containing either pFCFO1, pFCFO2 or
pFCF03 were grown in 2xLw, (no NaCI). On agar plates containing this medium it is
possible to differentiate clones containing pFCFO1, pFCF02 and pFCF03, because
overexpression of recombinant proteins leads to difference in colony size and
morphology as compared to wild type. Restriction endonucleases were purchased from
Amersharn Ltd.. Boehringer Mannheim Canada and New England Bio Labs. The T4
ligase was purchased from Bethesda Research laboratories. Shrimp alkaline phosphatase
was obtained from United States Biochemicals (USB). Growth condition protocols
described by Silhavy et al. (1984) were followed throughout. For digestions with
restriction endonucleases and separation of hgments by agarose gel electrophoresis,
standard techniques described by Sambrook et al. (1989) were used. The protein
denatminting agents Guanidine(Gn)-KC1 and ultra-pure urea were purchased from ICN
Biomedicals, Inc. The protease inhibitors, proteinase K and trypsin were obtained from
Boehringer- Canada. When commercially obtained kits were employed, the protocols
suggested by the companies were followed.
Molecular cloning of the mature fkuD sequences. Two previously PCR-
amplified fragments (Ferreira, 1995) BacjhuD and BacfhuD(CAQ6 encoding,
respectively, the mature sequence of FhuD and FhuD-His, were trimmed with Hind11
and subcloned into Hind11 restricted pBS. Sub-cloning was required to increase the yield
of the PCR-generated fragments. Both Fragments BacfiuDIHindIII and BacfiuD-
(CA?J6/HindIII were ligated at two-fold molar excess to pBS which had been Hind11
restricted and dephosphorylated to decrease the background of re-ligated plasmid. E. coli
DHSa cells were made competent, transformed with 345 ng of postligation species and
selected on L,,,,, plates onto which %Gal (20 mg/ml) and IPTG (120 mg/ml) had been
spread. Both blue (containing the re-ligated plasmid) and white colonies were observed.
White colonies were selected as candidates because they did not hydrolyse X-Gal,
suggesting that the P-galactosidase gene was interrupted by the presence of the inserts.
The selected candidate colonies were patched on L,,,?, Analysis of the plasmids from
the patches suggested that most candidates contained an insert. The plasmids from the
clones of interest were purified by using a mini-prep plasmid purification kit obtained
from QIAGEN. To confirm the identity of the inserts the candidate plasmids were cut
with Hind11 and PHI. Then, 5 pg of two plasmids containing respectively BacfhuD and
BacjhuD-(CAg6 inserts were restricted with H i n d u and run on a preparative 0.7 %
agarose gel. After electrophoresis for 2 hours at constant voltage (60 mV) the bands
corresponding to the inserts were cut fiom the gel and purified using the GENECLEAN
kit fiom Bio 101.
The fragments [B acfhuDIHindIII and BacjhuD- (CA 7J dHindIII] were then ligated
at hvo-told molar excess into the Bacillus expression vector pKTH288 that had also been
restricted with HindIII, but not dephosphorylated. The vectors were not dephoshorylated
because when attempts were made to ligate the fragments with previously
dephosphorylated Baci2lzi.s vectors no candidate recombinant colonies were obtained
upon transformation of B. subtiIis cells. The B. sztbtilis IH6140 cells were made
competent and transformed [by the method of Gryczan et al., (1978)l with 700 ng of
postligation species, and selected on plates of Luria broth containing kmamycin (25
pg/ml) [2*xLm,]. The transformation frequency was 1000 colonies per pg input of DNA
[CFUI(pg DNA)]. Candidate recombinant colonies were easily identified because of
their different morphology. Candidates were patched onto 2xL,, plates and screened
for the presence of the plasmid of the predicted size, 5.4 kbp. Plasmids from candidates
were restricted with Hind11 to confirm the presence of the inserts. Glycerol fiozen stocks
were prepared for clones with plasmids corresponding to the size of interest and placed at
-70°C.
The fragments BacjhuDllfindIII and BacfhuD-(CAT)6 /Hind11 were also ligated
onto the B. subtilis secretion vector pKTH132, which had been previously cut with
Hind11 but not dephosphorylated. The inserts were added to the ligation mix at 10-fold
excess of vector. The total ligation mix contained 1,800 ng of post-ligation species. The
transformation efficiency was 750 CFU/(pg DNA). Candidate colonies were selected on
the basis of their pinpoint size. The candidate recombinant colonies were patched onto
ZxL,, plates and screened for the presence of a plasmid of the predicted size, 4.55 kbp.
Plasmids from the candidates were restricted with Hind11 to c o n f i r the presence of the
insert, and with PstI and k'bai to confirm the orientation of the hgment. Glycerol frozen
stocks were prepared for clones with plasmids corresponding to the size of interest and
placed at -70 C.
Isolation of inclusion bodies. The following large-scale protocol based on a
method devised by Srikumar er al. (1993) was used to isolate inclusion bodies (IBs)
which contained BacFhuD and BacFhuD-His,. Recombinant bacteria (200 ml - 1.0 liter)
were grown at 37* C overnight in 2xLuria (L) broth containing kanarnycin (25 pg/ml).
Cells were harvested by centrifugation, washed three times with distilled water,
resuspended in 0.2 volumes of 10 mM Tns-HCI (pH 8.0) and converted to protoplasts by
treatment with lysozyme (0.5 mg/ml) for one hour, at 37OC. The addition of DNase (10
mg/rnl) and RNase (20 mg/ml) reduced the viscosity of the lysed protoplast suspension;
phenylmethyl sulfonyl fluoride [PMSF] (10 rnM) was added to minimize proteolysis of
BacFhuD and BacFhuD-Hiq. The material was centrifuged (12.000 x g) to recover
membranes plus IBs. To solubilize the membranes the pellet was extracted three times at
room temperature for 20 min each with buffer containing a nonionic detergent and high
salt: 40 m M Tris-HCI (pH 8.0), 1% Nonidet P-40 (NP-40), 1.0 M NaCI, 10 rnM PMSF
and 5mM EDTA.
Purification of urea-solubilued IBs containing BacFhuD-His,. Purification of
BacFhuD-His, was performed using ~i"-nitrilotriacetic acid (Ni2+-NTA) matrix and
columns purchased from QIAGEN. The IBs obtained from a clone overexpressing
BacFhuD-His, were solubilized in the urea-containing buffer B (8 M urea, 100 m M
NaH,PO,, 10 mM Tris-HC1 pH 7.9). A total of 10 mg of IB protein in 3.3 ml buffer B
were mixed for 45 min in batch format with 2 mi of Ni"-NTA which had been previously
equilibrated in the same buffer. To pellet the matrix the mixture was centrifuged at 500 x
g for L min. The supernatant (flow-through) was collected and stored at - 20°C for
M e r analysis. The matrix containing the bound BacFhuD-His, was poured into a 2 ml
Biorad column, which was connected to an FPLC East Protein Liquid Chromatography)
device (BioLogic, BioRad). In an initial washing step 10 column volumes (20 ml) of
buffer B were passed through the column at a constant flow rate of 1 ml/min. In a second
washing step, 10 column volumes (20ml, 1 ml/min) of buffer C (8 M urea, 100 mM
NaH,PO,, 10 mM Tris-HC1 pH 7.9, 20 m M imidazole ) were used. Ten fractions ( 1 ml
each) were collected and stored at -20°C. To elute the protein bound to the matrix , 5
column volumes (1 0 mi) of buffer EB (8 M urea, 100 mM Na H,PO,, 10 m M Tris-HCI
pH 7.9, 250 mM imidazole) were applied to the column at a flow rate of L ml/min. The
(1 ml) eluted fractions were collected and stored at -20°C for analysis. In an alternative
method. 3.3 ml IB protein was mixed with 6.7 ml buffer B, and applied directly (by
injection) to a pre-mounted 2 ml N?'-NTA matrix column. The washing and elution
steps were the same as above.
Preparation of antibodies. A synthetic peptide (FhuD 207/222) corresponding
to amino acids 207 to 222 of mature FhuD sequence (Coulton et al., 1987) was
synthesized and conjugated to keyhole limpet hemocyanin [KLH] (Sheldon
Biotechnology Center, McGill University). A polyclonal anti-FhuD antiserum (PAb 993)
was raised by immunizing New Zealand White rabbits with FhuD207R22-KLH as
follows: each animal received a primary (sub-cutaneous) immunization at five different
sites with 100 mg antigen per site. Four weeks later the rabbits were boosted with 500
mg of antigen at five different sites. Ten days after the secondary immunization blood
was collected fiom the rabbits, and immune serum was separated by centrifbgation.
Immune serum was stored at -20" C. The protocols for raising Abs were those of Harlow
and Lane (1988). Our practices comply with those prescribed by the Canadian Council
on Animal Care.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Before electrophoresis all protein samples were solubilized by boiling for 5 min in
electrophoresis sample buffer (2% SDS) with or without 5% P-mercaptoethanol. For
analysis of fractions containing 8 M urea and 6 M GnHCl, proteins were first precipitated
by dilution with distilled water and addition of two volumes of ice-cold absolute ethanol
or ice-cold acetone. Proteins were resolved on either 8%, 10% or 12 % polyacrylamide
gels with a vertical slab apparatus (BioRad) according to the method described by
Laemmli (1970). Gels were either stained with Coomassie brilliant blue (CBB), by the
silver staining method, or used for immunoblotting.
Immunoblotting. SDS-solubilized inclusion body or ~i"-NTA purified proteins
were resolved by SDS-PAGE and transferred electrophoretically to nitrocellulose
(Schleicher & Schuell) overnight at 4 O C , at constant voltage (25 V). All subsequent
washes were done at room temperature with shaking. Nitrocellulose membranes were
blocked for one hour with 1% bovine serum albumin in Tris-buffered saline (TBS 10
mM Tris-HCI pH 7.4, 150 m M NaCl). The blocking solution was drained, then a
solution of anti-FhuD 993 polyclonal antibody (PAb 993) in TBS (1 : 1000 dilution) was
added and incubated for one hour. The primary PAb 993 was drained and a series of four
five-minute washes was done; the first and fourth with 40 ml TBS and the second and
third with 40 ml TBS plus 0.5% NP-40. Goat anti-rabbit polyclonal secondary antibody
conjugated with alkaline phosphatase, diluted in TBS (1 2000 dilution) was added. After
one hour the solution was drained and the four washes were repeated. The blots were
developed with nitroblue-tetrazoliurn and bromo-chloro-indoly 1 phosphate (NBT and
BCIP; BRL) at 150 and 300 rng/ml, respectively, in carbonate buffer (100 rnM NaHCO,,
1 mM MgCl,). The cotour reaction was stopped by washing the membrane in water.
Renaturation of purified urea-solubilized BacFhuD-His,. Since the
purification of BacFhuD-His, by Ni2+-NTA was performed under denaturing conditions
(8 M urea), two strategies were attempted to refold the purified protein into its native
conformation.
The fint renanuaton protocol to be attempted (stepwise dialysis) was adapted
fiom that of Miller et al. (1979): ~i~+-NTA-purified BacFhuD-His, was diluted to 100
pg/ml in buffer B (8 M urea, 100 m M NaH,PO,, 10 mM Tris-HCI pH 7.9). The
recombinant FhuD-His, was introduced into a low ~olecular weight cut-off dialysis bag
(mwco 3,500; Spectrapor), and dialyzed against 250 volumes of 10 mM Tris-HCl pH 7.9,
5 mM EDTA and 5 mM P-mercaptoethanol containing 6,4,2 , 1,0.5, 0.25 and 0 M urea,
respectively. Each step of the dialysis protocol had a duration of 2.5 h. The refolding
protocol was carried out at 4OC. with constant stirring of each dialysis solution. Mer
dialysis the contents of the dialysis bags were transferred to an Eppendorf tube and
centrifuged (20,000 x g, 5 min) to collect protein precipitates (i.e. aggregated, misfolded
protein).
The second method to renature urea-solubilized BacFhuD-His, (linear gradient
refolding on a column) was based on a protocol by Jaenicke and Rudolph (1990). It is
suggested that by immobilizing one end of the protein during renaturation the formation
of misfolded aggregates can be prevented. This protocol has been used to successfilly
refold several proteins that could not be renatured or could only be obtained at low yields
by stepwise dialysis (Jaenicke and Rudolph, 1990). In this method, different amounts of
~ i " - ~ ~ ~ - ~ u . i f i e d BacFhuD-His, (500 pg, 1.0 rng and 1.5 mg) were re-bound to ~ i " -
NTA matrix (after overnight dialysis to remove the irnidazole), either by the batch or
direct inject methods (described above). Renaturation was attempted by using a linear 8
M to 1 M urea gradient in 100 m M NaH2P0,, 10 mM Tris-HC1 pH 7.9, 5 rnM P-
mercaptoethanol and either 20 % glycerol (buffer 21) or no glycerol (buffer 22). The
gradient was FPLC-mediated to ensure linearity, and the renaturation was allowed to
proceed for 2 h, 3 h or 5 h at a constant flow rate of 1 mVmin. After the gxadient, the
protein was eluted with 10 column volumes (10 ml, 1 rnl/rnin) buffer X (100 m M Na
H,PO,, 10 mM Tris-HC1 pH 7.9, 5 m M P-mercaptoethanol, 250 rnM imidazole).
Generally, ten 1 mi fractions were collected, transferred to Eppendorf tubes and
centrifuged (20,000 x g, 5 rnin) to remove unwanted protein aggregates. The eluted
fractions were either stored at 4" C or -20 * C. After the elution step, the column was
washed with buffer Z1 + imidazole (8 M urea 100 rnM NaH,PO,. 10 m M Tris-HCl pH
7.9, 5 mM P-mercaptoethanol, 250 mM imidazole) to remove any protein that might have
precipitated on the column. The protein concentration of the eluted fraction was
determined using the BioRad assay, and by CBB staining of 15 p1 samples applied to
SDS-PAGE gels.
Purificatiodrenaturation of Guanidine(Gn)HCl-solubilbed IBs containing
BacFhuD-His,. As an alternative for solubilizing BacFhuD-His, IBs, 6 M GaHCl was
utilized as the denaturing agent instead of urea. Two different methods were used to
attempt refolding of GnHCl-solubilized BacFhuD-His,. The main difference between the
two methods is that the denaturing agent (GnHCl) was either removed slowly (by linear
gradient) or quickly (one-step 6 M - 0 M GnHCI).
The fiat method is similar to linear gradient refolding of urea-solubilized
BacFhuD-His, described above, except that the non-ionic detergent Lauryl-
D imethy lamine-N-oxide (LD AO, Fluka) was added to the all buffers. Different amounts
(750 pg, 1.0 mg, 1.5 mg, 2.0 mg) of IBs containing BacFhuD-His, were solubilized in
buffer A (6 M GnE-ICl, 40 rnM TrisHCl pH 7.8, 250 mM NaCI, 2 rnM CaCl, 0.05 %
LDAO, 5 m M P-mercaptoethanol). The protein was subsequently applied to 1 ml or 2 ml
of N~"-NTA matrix pre-equilibrated in buffer A (10 column volumes, I drnin) , by
FPLC-mediated direct sample injection [5 ml, 0.5 ml/min] (described above). The
renatwation was attempted by using a linear gradient of 6 M to 1 M GnHCl. The
gradient was FPLC (BioLogic, BioRad) mediated. Buffer B (40 mM TrisHCl pH 7.8,
250 mM NaCl. 0.01 % LDAO. 2 m M CaCl?. 5 mM PMSF and 5 mM P-mercaptoethanol)
was used as the gradient buffer. The gradient was extended over a period of 3.5 or 4 h
(0.5 mVrnin flow rate). Afler renaturation, proteins were eluted either by the addition of
250 mM imidazole or 50 mM EDTA (I0 column volumes, 1 ml/min) buffer E: 40 mM
TrisHCl pH 7.8,250 m M NaCl, 0.01 % LDAO, 2 mM CaCI,, 5 m M PMSF and 5 rnM P-
mercaptoethanol, 250 mM imidazole or 50 mM EDTA). PMSF (5 mM) was also added
to the eluted fractions to prevent proteasemediated degradation of the solubilized protein.
Generally, 10 x 1.0 ml fractions were collected. After elution, the different fractions were
transferred to eppendorf tubes and centrifbged at 20,000 x g for 5 min to eliminate
undesirable protein aggregates. The elution Fractions were stored either at 4OC or -20°C
for fiuttler analysis. After the elution step the column was cleaned with buffer 1 plus
imidazole (or EDTA) to remove protein that that could have precipitated on the column.
The concentration of the proteins eluted in the different fractions was estimated by the
BioRad assay method and by CBB analysis of SDS-PAGE gels containing 15 pl samples
from each of the 1.0 ml elution hctions. The purity of the eluted protein samples was
determined by CBB and silver staining of SDS-PAGE gels and immunoblotting.
Imrnunoblotting (with PAb 993) was also used to confirm the identity of the band
corresponding to BacFhuD-His,.
The second method that was used to attempt to renaturate GnHCl-solubilized
BacFhuD-His, was based on a protocol developed by Holzinger et al. (1996). In this
method the renaturation of the GnHCl-solubilized BacFhuD-His, involved one-step FPLC-
mediated fast removal of the denaturing agent, rather than slow linear gradient removal
(above). Several proteins possessing histidine tags either at their N- or C-termini have been
refolded with high efficiency at relatively high concentrations (750 pg/ml- 1.5 mghl) using
this simple technique (see Holzinger er al. 1996). Different amounts of BacFhuD-His,-
containing IBs (1.0 mg, 1.5 mg, 2.0 mg) were solubilized in buffer 1 (denaturaturation
buffer; 6 M GnHCI, 20 rnM Tris-HCl pH 7.9.500 mM NaCl [or 250 rnM NaCl], 0.01 %
LDAO. 5 mM P-ME). The GnHCl-solubilized IBs were applied to 2 mi N?+-NTA
matrix pre-equilibrated in buffer 1 (12.5 column volumes) either by the batch method
(described above) or direct injection (5 mi, 0.33 mllmin) into a ~i"-NTA matrix-packed
2 ml (Pharrnacia) column. The direct injection of the protein sample and all subsequent
steps of this method were FPLC-mediated (as before). Following the binding of
BacFhuD-His, onto the Ni-NTA matrix, buffer 2 (wash buffer; 6 M urea, 20 m M Tris-
HCl pH 7.9, 500 m M NaCl [or 250 mM NaCl], 0.01 % LDAO, 5 m M P-ME, 30 m M
imidazole, 'Complete cocktail' protease inhibitor (Boehringer-Mannheim Canada) was
applied to the buffer (12.5 column volumes, 1 m h i n ) . The low concentration of
imidazole (30 mM) in buffer 2 helped to reduce non-specific binding to the N?'-NTA
matrix. Subsequently, 1 2.5 column-volumes (1 ml/min) of buffer 3 (solubilization
buffer; 20 rnM Tris-HCI pH 7.9, 500 m M NaCL [or 250 mM NaCI], 0.01 % LDAO, 5
m M P-ME, 'Complete cocktail' protease inhibitor) were applied to the ~i"-NTA matrix
to remove the denaturant (urea) and renature BacFhuD-His,. Finally, the Ni2+-NTA
matrix-bound protein was eluted from the column using 12.5 column volumes (1 mlhin)
of buffer 4 (elution buffer, 20 mM Tris-HCI pH 7.9, 500 mM NaCl [or 250 mM NaCL],
0.01 % LDAO? 5 MM P-ME. 'Complete cocktail' protease inhibitor. and 50 rnM EDTA
[or 400 m M irnidazole]). Five fractions were collected (1.5 rnl each). These Fractions
were transferred to Eppendod tubes and centrifuged at 20,000 x g to remove insoluble
protein aggregates. Fractions were either stored at 4" C or -20 C. The concentration of
the proteins eluted in the different fractions was estimated by the BioRad assay method
and by CBB staining analysis of SDS-PAGE gels containing 15 p1 samples from each of
the 1.5 ml elution fractions. The purity of the eluted protein samples was determined by
CBB and silver staining of SDS-PAGE gels and by immunoblotting. fmmunoblotting
(using PAb 993, see above) was also used to confirm the identity of the band
corresponding to BacFhuD-His,.
The method above was also attempted at a larger scale. In this variation 18 mg of
IBs containing BacFhuD-His, were bound to 25 ml Ni2'-NTA matrix by the batch method
(above). Afier batch binding of BacFhuD-His, the Nil'-NTA matrix was packed into a 25
ml (BioRad) column. The same buffers (1 - 4) and steps (denaturation, washing,
solubilization, elution) as above wen used. However, in each step 8 column volumes of
each of the buffers were applied to the column at a flow rate of 2.5 d m i n (instead of
12.5 column volumes at 1 ml/min). Moreover, the volume of fractions collected was 3
ml instead of 1.5 ml. Further modifications of the small-scale method included: (i) use
of no detergent (LDAO) in any of the buffers, (ii) use of lower detergent concentration in
all buffers (0.0025, 0.005 or 0.0075 % LDAO), (iii) lack of protease inhibitors, (iv) use
of lower salt concentration or no salt in all buffers (0 - 150 m M NaCl), and (v) utilization
of a different buffer (10 mM ammonium acetate pH 7.9 instead of 20 mM TrisHC1 pH
7.9). Different pH ranges were also attempted (pH 7.5 - pH 7.9).
Detection assay for secreted BacFhuD (secBacFhuD). Candidates for secreted
BacFhuD (secBacFhuD) were grown overnight and the growth media (2xLM5) was
screened for the presence of secreted BacFhuD. The candidate cells were inoculated into
50 ml 2xL,,, containing the cocktail protease inhibitor "Complete" and incubated at
37 O C ovemight. The 50 ml growth media was subsequently concentrated into 2 ml using
a TCF 10 ultrafiltration system (Amicon), and then Curther to 300 p1 using a centricon40
concentrator (Amicon). For each candidate, 100 p1 of concentrated growth media were
transferred to wells of a 96-well titer plate along with 100 pl of 8 M urea. To one control
well. 100 pl sterile 2xLw, were added to 100 p1 urea. In a second control well 100 pls
of 8 M urea were mixed with 100 p1 IB protein (BacFhuD in Tris-EDTA). Subsequently
the titer plate was incubated for 1 hour with mixing to ensure denaturation of proteins
present in the growth media (2xL,,). Then, the contents of each well were transferred
from the titer plate to nitmcellulose membranes, using a milliblot apparatus (BioRad).
The spots in the nitrocellulose membrane corresponding to each well were washed with
200 11 TBS. M e r the washing steps the nitrocellulose membrane was removed from the
milliblot apparatus and screened with PAb 993 for the presence of secBacFhuD. The
immunoblotting protocol was the same as described above. The presence of secreted
BacFhuD in media was also assayed by SDS-PAGE of samples followed by CBB and
silver staining.
Protease protection assay for BacFhuD-His,. To test for the correct folding of
renatured BacFhuD-His, a protocol based on a method developed by Koster and Braun
(1990) was used. Kaster and Braun showed that native FhuD exhibited an increased
resistance to digestion by trypsin and proteinase K when pre-incubated with one of its
ligands (i.e. ferrichrome). Therefore, in our method BacFhuD-His, was digested with the
protease proteinase K in the presence or absence of ferricrocin (a ferrichrome homologue)
or with trypsin (in the absence of ferricrocin).
For the proteinase K digestion protocol, 5 pg of purified BacFhuD-His, in buffer
4B (20 mM Tris-HCI pH 7.9,250 m M NaC1,0.01 % LDAO, 5 mM P-ME, 5 m M CaClJ
were digested for different amounts of time (2, 5, 10 min) at 37OC either in the presence
or absence of 30 pM ferricrocin. One microgram (or 500 ng) of proteinase K was utilized
in the digestion (3 7 C). These amount correspond to a 1 :5 or 1 : 10 ratio of proteinase K
to protein. The concentration of the proteinase K used in the digestion mix was 50 pg/ml.
The proteinase K digestions were stopped by the addition of 5 rnM PMSF. After the
digestion reactions were completed, sample buffer was added to the digestion mix and the
samples were run on SDS-PAGE gels and stained with CBB. As controls, 5 yg of
BacFhuD-His, (no proteinase K) and 1 pg (or 500 ng) of proteinase K were also run on
the SDS-PAGE gels. Both samples were kept at 37" C for the maximum length of the
digestion (1 0 min), prior to loading.
In the trypsin digestion protocol, 5 pg of purified BacFhuD-His, in buffer 4C (20
m M Tris-HCI pH 7.9, 250 m M NaCI, 0.01 % LDAO, 5 m M P-ME) were used. The
digestions were done for 1, 3 h or overnight at 37" C, either in the absence of femcrocin
(30 pM). The trypsin digestions were stopped by the addition of 5 mM PMSF. AAer the
digestion reactions were completed, sample buffer was added to the digestion mix and the
samples were analyzed on SDS-PAGE gels by CBB staining. As controls, 5 pg of
BacFhuD-His, (no trypsin) and 100 ng of txypsin were ran on the SDS-PAGE gels. Both
samples were kept at 37 C for 3 h or overnight, prior to loading.
Binding activity assay for BacFbuD-His, and FhuA. To determine whether the
purified BacFhuD-His, was refolded into its native conformation a protocol initially
suggested by Lever (1972) was employed. This method is based on the premise that
periplasmic binding proteins bind their substrates with very high affinity. Therefore
dialysis of a small volume of protein solution against large buffer volumes containing
radiolabeled substrate should lead (after equilibration) to an increase of ligand specific
activity within the dialysis bag. In our experiments, both purified BacFhuD-His, and
purified FhuA (OM receptor for ~e"-femchrome), which has previously been shown to
be biologically active (i.e. Killmann et al., 1996; Moeck et al., 1996; 1997), were tested
for their ligand binding activity.
To radioactively label the defem form of femcrocin (dFCo), an aliquot of dFCo
(in ddH20) was incubated with equimolar amounts of "~eC1, (Amersham Buchler; 1.01
rnCi/ml, 2 10 pg/ml) for 1 hour at room temperature (Coulton et al., 1979). After the
reaction was completed the yellow "Fe-ferricrocin solution was stored at - Z O O C.
In the equilibrium dialysis experiment using purified BacFhuD-His,, two different
amounts of protein (6.75 pg and 3.375 pg) were introduced into a low mwco dialysis bag
(mwco 3,500; Spectrapor). The protein was diluted in buffer 4X (20 rnM Tris-HCI pH
7.9, 250 m M NaCI, 0.05 % LDAO, 5 mM P-ME) to a total volume of 3.0 mI in the
dialysis bag. The protein was subsequently dialyzed against 1.5 1 of buffer 4X (20 mM
Tris-HCI pH 7.9, 250 m M NaCI, 0.05 % LDAO, 5 mM P-ME) containing 1 nM " ~ e -
femcrocin. The dialysis was done at 4" C with gentle stirring for 16 - 20 h. A 250 p1
sample was withdrawn, added to 5 ml liquid scintillation fluid (EcoLite, ICN
Biornedicals) and counted on an LKB liquid scintillation counter. Blanks levels were
measured (and subtracted) by assaying the radioactivity present in the external fluid and
inside bags that did not contain protein. All tubes (containing samples plus liquid
scintillation fluid) were counted for five minutes, and the counting was repeated three
times. The equilibrium dialysis protocol for purified FhuA is identical to the one
described above. The amounts of FhuA tested were: 18 pg, 8 pg, and 4.5 pg.
RESULTS
Expression of recombinant BacFhuD and BacFhuD-His,. Two recombinant
fhuD gene constructs were expressed in Bacillus subtilis IH6 140 to yield the recombinant
proteins BacFhuD and BacFhuD-His,. The B. subtilis vector used was pKTH288, in which
expression of the inserts was under the control of the promoter for a-amylase of Bacillus
amyloliquefaciee (Palva el al., 198 1). By PCR amplification of thefhuD gene, the mature
form of FhuD was obtained as a Hindi11 hgrnent (Ferreira, 1995). As an alternate
approach, the 3' end of thefiuD gene was modified using a primer containing 6 contiguous
histidine codons ~ u P ( C . ~ 6 ] and two terminal Hind111 sites. The rationale of this
approach (refer to Figure 5) was to generate a recombinant FhuD with a C-terminal hexa-
histidine extension (BacFhuD-His&; this extension was used as an affinity tag for metal
chelate chromatography. Both the BUD and fhuo-(CA g6 HindIII fragment. were ligated
to pKTH288 restricted with HindIII so that the codons for the first 7 amino acids of the a-
mylase signal sequence plus four linker amino acids were fused in-frame to codons for the
266 amino acids of FhuD or to codons for the 272 amino acids of FhuD-His,. The ligated
plasmid species @FCFO I , pFCF02) were used to transform BuciIIus subtilis M6 140 and
selected on plates containing kanamycin. Differences in colony morphology allowed for
identification o f the desired transformants fiom a background of kanamycin-resistant
colonies. The differences in colony morphology between colonies derived fiom protein-
producing clones and those colonies producing no recombinant protein has been used
previously as a basis to initially identify recombinants (i.e. Dahan et al., 1996).
Candidate plasmids @FCFO1 and pFCF02) were confirmed to harbour the desired inserts in
pKTH288 fiuD or jhuD-(CAv6) by their size and by their restriction endonuclease
patterns.
The proteins produced by these fusions (FhuD, 277 amino acids and FhuD-His6,
283 amino acids) were predicted to have the following amino acid sequence at their N-
termini: Met-l Ile Gln Lys Arg Lys Arg Asn Ser Lys Leu M a 4 2 Ala- 13 Ile- 14 Asp- 15 Pro-
16 Asn- 1 7 Arg- 1 8. Ala-12 to Arg- 1 8 correspond to the N-terminus of the mature fom of
FhuD. The recombinant protein FhuD-His, was also predicted to harbour the amino acids
Ile-273 Gly-274 Gly-275 Lys-276 Ala-277 (His),, at its carboxy terminus. Ile-273 to Ala-
277 correspond to the C-terminal amino acids of FhuD. Both BacFhuD-His, and BacFhuD
were expressed to high levels, as indicated by the formation of inclusion bodies within the
cells. To isolate the inclusion bodies, the desired transformants were converted to
protoplasts, followed by treatment of the collected inclusion bodies with a b e e r containing
a non-ionic detergent plus high salt concentration. This buffer had previously been shown
to solubilize membrane proteins (Srikurnar et aL, 1993). Because inclusion bodies are
refkctory to this treatment, they were collected and resuspended in 10 mM Tris-HCI (pH
8.0), 1 mM EDTA. The concentration of protein in the inclusion bodies was approximately
5 rng/ml.
Identification by SDS-PAGE of the proteins at each step of the above protocol
showed almost no loss of inclusion body proteins by detergent extraction (data not shown).
Furthermore, the inclusion bodies were enriched for the recombinant proteins (BacFhuD or
BacFhuD-His6) Fig. 6, lanes 1 to 81. When electrophoresis sample buffer with no P-
mercaptoethanol was used to solubilize the inclusion bodies, three proteins with relative
mobilities of approximately 14 kDa, 30 kDa and 66 kDa were observed. When sample
buffer containing P-mercaptoethanol was used, the band corresponding to 66 kDa
disappeared and the intensity of the band corresponding to the 30 kDa protein became more
prominent (Fig. 6, lanes 3 - 4 and lanes 7 - 8). However, the 14 kDa band persisted. This
band has been observed in other instances in which the Bacillz~~ expression system was
employed (Srikumar et at., 1993; Dahan et at., 1996). Several other minor protein species
of both higher and lower molecular mass than BacFhuD or BacFhuD-His, were also
consistently observed in the preparations of inclusion bodies. The calculated molecular
mass of 3 1,011 Da for BacFhuD (277 amino acids) and of 3 1,834 Da for BacFhuD-His,
(283 amino acids) matched the expected migration of the proteins on SDS-PAGE, using
standard proteins as reference (Fig. 6, lane M). Since FhuD has a single cysteine residue, it
is possible that dimea form by disulfide bond formation, resulting in the appearance of the
66 kDa protein in the absence of P-mercaptoethanol. The migration of the 66 kDa protein
in SDS-polyacrylarnide gels is close to the calculated values of 63,668 for BacFhuD-His,
dimers or 62,022 Da for BacFhuD dimea (Fig. 6, lanes 1 - 2 and lanes 5 - 6).
Purification of urea-denatured BacFhuD-His,. In this protocol recombinant
FhuD-His, from inclusion bodies was purified by FPLC-mediated N~''-NTA
chromatography under denaturing conditions. The IBs were solubilized using the
chaotropic agent urea (8 M urea); no pellet was observed after centrifbgation of the urea-
solubilized inclusion bodies. The chaotrope-solubilized [Bs were then applied to a N2'-
NTA column. The recombinant FhuD-His, was retained by the ~i"-NTA matrix (Fig. 7,
lanes 4 - 6); only a small amount of BacFhuD-His, was detected in the flow-through (Fig. 7,
lane 3). Contaminating proteins were eluted by using urea-containing wash buffer with 20
mM imidazole (almost no protein washed off the matrix upon application of the imidazole).
hnidazole has a structure similar to that of histidine, so that at very high concentrations (100
mM - 500 mM) imidazole competes with. and displaces the His-taged protein from the
Ni2'-NTA matrix. At low concentration (20 mM -30 mM) imidazole displaces
contaminants that are weakly bound to the resin. BacFhuD-His, was eluted using urea-
containing buffer with 250 mM imidazole (Fig. 7, lanes 4 - 6). After ~i"-NTA affmity
chromatography, the dirneric form of FhuD could still be observed by Coomassie brilliant
blue (CBB) staining of the gels (Fig. 7, lane 4). As seen with IBs, this species was
converted to the monomeric form after treatment with sample buffer containing P-
mercaptoethanol(ME) (Fig. 7, lanes 5 and 6). The yield of purified (denatured) BacFhuD-
His, from one litre of culture was 180 mg. By this affinity chromatography purification
protocol the protein was shown to be about 95 % pure.
FhuD specific adpeptide antibodies. BacFhuD was confirmed by
immunoblotting, to be the recombinant protein of interest. The primary polyclonal
antibody (PAb 993) used in the Western blot was obtained fiom immunization of a rabbit
with a 16-amino acid synthetic peptide corresponding to a region of FhuD encompassing
amino acids 207 to 222. Using the software Predict7 v1.2 (Carmenes et aL, 1989) this
region was identified to be the most hydrophilic and therefore probably the most antigenic
in the FhuD sequence. The antibody was shown to react strongly to the protein expected to
be BacFhuD-His, (Fig. 8, lanes 1 - 2 and lanes 4 - 6). A strong reaction of PAb 993 with
the band migrating near 66 kDa was also observed suggesting that it represents a FhuD-His,
dimer. The antibody was shown to be slightly cross reactive with the outer membrane
protein FhuA (data not shown). Pre-bleed serum, collected from the rabbit before
immunization showed no anti-FhuD reactivity (data not shown).
Production of the secreted form of FhuD. Two recombinant fhuD gene
constructs were expressed in B a c i h subtifis to attempt expression of a recombinant form
of FhuD (or FhuD-His,) that instead of accumulating intracellularly as aggregated inclusion
bodies. could be secreted into the growth media (secBacFhuD). The B. ~ h t i l i s vector used
was the secretion vector pKTH 132, in which expression of the inserts was under the control
of the promoter for a-amylase of Bacillus amyloliquefociens (Palva et al., 198 1; 1982).
Unlike the intracellular vector pKTH288 which encodes in addition to the a-amylase
promoter only seven amino acids of the a-amylase signal sequence, the pKTH132 vector
encodes the full signal sequence of the a-amylase of B. amyloliquefaciens. The mature
jhuD/ and jhuD-(CA dHindIII hgments were ligated to pKTH 1 3 2 restricted with HindIII
so that the codons for the 30 amino acids of the a-amylase signal sequence plus two linker
amino acids were b e d i n - h e to the codons for the 266 amino acids of FhuD (or 272
amino acids of Fhd9His6). The ligated plasmid species (pKTH132-fluD [pFCF03] and
pKTH L 32-fhuD-(CAg6) were used to d o r m Bacillus subtilis M6 140 and selected on
plates containing kanamycin. Attempts to ligate restricted pKTH 132 with thefhuD-(CA Z'J 6
insert were not successll as no recombinant candidate colonies were ever observed upon
transformation of competent B. subtilis cells. Several attempts to clone thejhuD insert into
pKTH132 yielded a single clone harbouring the pKTH 132-jhuD [pFCF03] plasmid. The
candidate plasmid (pFCF03) was confirmed to harbour the desired insert (j7m.D) in
pKTH132 by its size and by its restriction endonuclease patterns. Pin-point colony
morphology allowed for identification of the desired transformant &om a background of
kanamycin-resistant colonies. Furthermore, the clone harbouring pFCF03 had a slower
growth rate than the wild-type (untransformed) B. subrilis IH6 140 (in liquid media or solid
media). During the process of protein expressionlexport, when the ss-FhuD fusion protein
is translocated across the B. subfilis cell wall it is expected that the signal sequence be
cleaved OK Based on this premise, the secreted recombinant FhuD (secBacFhuD) would
then possess the two linker amino acids (Gly Lys) fused to the mature sequence of FhuD.
Detection of secBacFhuD (from IH6 140lpFCF03) was attempted by growing
candidates expected to express and secrete secBacFhuD. Subsequentiy the growth media
from these candidates was transferred to nitrocellulose membranes using a milliblot
apparatus. Blotting was carried out using the polyclonal antibody PAb 993. By this assay
secBacFhuD-His, was not detected in the growth media (Fig. 9) for any of the clones tested.
The clone possessing the plasmid pFCF03 was placed in wells l a through Id in Figure 9.
All other wells contained cells that do not possess the plasmid of interest. CBB and silver
staining analysis SDS-PAGE gels showed no protein in the growth media with the
predicted molecular weight of secBacFhuD (data not shown). Furthermore, as predicted, no
secBacFhuD was shown to accumulate as IBs following the IB purification protocol
(Materials and Methods). The IB purification protocol also showed, no detectable
secBacFhuD was arrested in the B. subtilis cell wall (data not shown).
Renaturntion of urea-solubilized BacFhuD-His,. The purification of FhuD-His,
was performed under denaturing conditions. To achieve the native confoniiation of the
protein. it was necessary to remove the urea The urea removal was carried out by using
two different protocols. The fim method employed stepwise dialysis of the purified, urea-
solubilized BacFhuD-His, against Tris-HCl buffer containing decreasing amounts of urea.
AAer dialysis, the sample was centrihged, and the soluble proteins were analyzed by SDS-
PAGE. Soluble BacFhuD-His, was detected in the supernatant, and analyzed by CBB of an
SDS-PAGE gel (Fig. 10, lanes 2 - 3 and 4 - 5). Recovery of urea-solubilized BacFhuD-
His6, as soluble BacFhuD-His, was determined to be approximately 30 % by CBB analysis
of SDS-PAGE gels and by the BioRad protein estimation assay (-30 pg/ml). Attempts to
dialyse out any of the components of the buffer (i.e. EDTA or P-ME) containing the
purified. presumably refolded protein led to BacFhuD-His, precipitation Eom solution.
Attempts to concentrate the protein using Centricon40 units also resulted in precipitation.
Due to the low concentration of the recovered protein and the precipitation problems, this
protein was not utilized in the binding activity assays (see below).
The second method to refold the (purified) urea-solubilized BacFhuD-His6, was
carried out by FPLC-mediated linear gradient decrease in the concentration of the
denaturant urea. CBB and silver staining of SDS-PAGE gels to which samples of the
eiuted hctions were applied did not indicate recovery of any BacFhuD-His, (data not
shown). Therefore, under the experimental conditions attempted (Materials and Methods)
no soluble BacFhuD-His, was obtained by FPLC-mediated linear gradient urea removal.
Purif~cation/renatumtion of GnHCI-soh bilized BacFhuD-His, The denaturing
agent GnHCl was also used to denature BacFhuD-His6-containing IBs. IBs were easily
denatured using this chaotrope and after high-speed centrifugation no pellet was observed.
To obtain a native form of the protein it was necessary to remove GnHCl. Two different
methods were employed to purifyhenature BacFhuD-His, solubilized in GnHCI.
In the first purificationlrenaturation method GnHCI-solubilized BacFhuD-His, was
fim applied to a ~i"-NTA column. BacFhuD-His, was then purified and renatured by
FPLC-mediated linear gradient removal of the denaturant GnHCI. Figure L 1 represents the
FPLC-mediated linear gradient denaturant-removal profile for one such experiment. The
(left) Y-axis represents a 280 nrn OD reading and it allows for the protein to be tracked for
the duration of the protocol (flow-through, wash, linear gradient, and elution stages). The
eluted protein fractions obtained from this purificatiodrenaturation protocol were
cenaihged to remove aggregated protein and contarninants and analyzed by CBB staining
and silver staining of SDS-PAGE gels. Soluble, purified BacFhuD-His, was detected in
two elution fraftions both before and after centrihgation [20, 000 x g 5 min] (Fig. 12;
hction 23, lanes 3; hction 24, lane 4). The starting material (GnHCl-solubilized 1%) and
flow-through (material that did not bind to the matrix) were loaded in lanes 1 and 2,
respectively. The elution fractions were also shown to be greatly enriched for the protein of
interest (BacFhuD-His& by silver staining analysis of an SDS-P AGE. Western blotting
confirmed the identity of the major band migrating at about 30 kDa (Fig. 12) as being FhuD
(data not shown). The experiment above (represented in Figure 1 I), whose eluted protein
was analyzed by CBB of an SDS-PAGE represents the highest level of purity/recovery of
soluble BacFhuD-His, obtained using the linear gradient GnHCl removal protocol. The
parameters/experhental conditions used were the following (refer to Materials and
Me hods): (i) 1.5 mg of BacFhuD-His, were applied (by FPLC-mediated direct injection)
to 2 ml ~i"-NTA matrix (750 pg/ml matrix); (ii) the solubilization and gradient buffers
used (A and B) were the same as those described in Materials and Methods; (iii) the GnHCl
concentration was slowly decreased over a 3.5 hour period (6 M - 1 M); at the elution step
(using buffer E containing 250 mM imidazole) most protein was collected in fractions 23
and 24 (see OD peak in Figure 11); (iv) 5 rnM PMSF was added to the two fractions of
interest; and (v) samples were stored at -20°C for subsequent analysis. CBB analysis of
SDS-PAGE gels and the BioRad protein estimation assay suggest that approximately 350
pg of BacFhuD-His, were recovered in fractions 23 (-150 pg/ml) and 24 (-200 pg/ml),
corresponding to 23 % recovery of protein from the starting GnHCl-solubilized IBs. Since
IBs consist of approximately 75 % BacFhuD-His, the actual protein recovery was actually
about 30 %. Little BacFhuD-His, can be detected in the flow-through of FPLC-mediated
linear gradient refolding experiment [when material is first applied to the N?*-NTA matrix
(Fig. 12; lane 2). Little protein is lost during GnHCl removal (before application of
imidazole; Figure 11). After elution of the soluble BacFhuD-His, fiom the matrix, most
lost protein can be recovered as a white precipitate after washing of the N?'-NTA matrix
with 6 M GnHCl plus 250 mM acetic acid (data not shown). This indicates that under these
experimental conditions most protein (- 70 %) aggregates and precipitates on the column
during the linear gradient removal of the denaturing agent (GnHCl). Attempts to
concentrate the protein using Centricon40 concentrator units resulted in total protein loss
(by precipitation). Furthermore, removal of detergent or salt by dialysis also resulted in
protein precipitation.
The second method to attempt renatwation of GnHCl-solubilized BacFhuD-His,
involves one-step fast removal of the denaturing agent. This method was also FPLC-
mediated. Figure 13 represents the BioLogic (FPLC) run report for such protocol, for one
of the experiments with the highest recovery of soluble BacFhuD-His,. As above, the (left)
Y-axis represents a 280 nm OD reading and it allows for the protein to be tracked for the
duration of the protocol (tlow-through, wash and elution stages; see arrows). Figure 13
represents a "batch"-type binding experiment (Materials and Methods). In this experiment
1.7 mg of BacFhuD-His, (in GnHCl) were bound to 2 ml of N?'-NTA resin by the batch
method (850 p1 proteinlng resin) [see Materials and Methods] to ensure an uniform
distribution of protein within the matrix. Soluble, purified BacFhuD-His, was detected in
two fractions after high-speed centrifbgation by CBB staining of a 10 % SDS-PAGE gel
(Fig. 14; tiaction 13, lanes 1 ; hction 14, lane 4). Lanes 2 and 3 (Fig. 14) of the SDS-
PAGE were loaded with samples of the starting material and flow-through respectively.
The band corresponding to BacFhuD-His, can be seen only faintly in the flow-through
(after wash with buffer 2; below) attesting to the efficiency of the binding. A western blot
confirmed the identity of the BacFhuD-His, band, and confirmed the purity of the protein
recovered (data not shown). The buffers used in this experiment were described in
Materials and Methods. After elution the samples ( b t i o n s 13 aud 14) were stored at -
20" C. CBB analysis of SDS-PAGE gels and the BioRad protein estimation assay indicated
that about 400 pg of BacFhuD-His, were recovered in kctiom 13 (-250 pg/rnl) and 14
(-150 pg/mi), corresponding to 24 % recovery of protein from the starting GnHCl-
solubilized IBs. Since IBs consist of approximately 75 % BacFhuD-His, the actual protein
recovery was actually about 32 %. A white precipitate could be observed after washing the
column with 6 M GnHCl and acetic acid, presumably corresponding to protein that
aggregated and precipitated on the ~i"-NTA matrix. Recovery of protein in batch binding
experiments did not prove to be much higher on average than recovery of protein in which
experiments in which BacFhuD-His, was injected onto the matrix on the column [30 % vs.
27 %] (see Materials and Methods). However the purity of soluble BacFhuD-His, obtained
from batch binding experiments was slightly better. As in the linear gradient renaturation
protocol, concentration of protein (using Centricon- 10 units) led to protein precipitation.
Dialysing away detergent or salt also led to protein loss (data not shown). However, in
several experiments, it was possible to dialyse the P-ME and imidazole, a requirement for
some activity assays, without significant protein loss (see below).
A variation of the batch binding experiment described above was also performed at
a larger scale (see Materials and Methods). In this experiment 18.75 mg of GnHCl-
solubilized BacFhuD-His, were bound to 25 rnl Ni2'-NTA resin (750 pg/rnl). Soluble,
purified BacFhuD-His, was detected in two 3 ml elution fi.actions after centrihgation (Fig.
15, lanes 1 and 3). The buffers used in this experiment were the same as described above.
By CBB analysis of the SDS-PAGE gel and the BioRad protein concentration estimation
assay it was calculated that the BacFhuD-His, concentration in the elution W o n
corresponding to lane 1 was 600 pg/ml and in the fraction corresponding to lane 2 it was
120 pg/ml. In total, approximately 2.2 mg of protein were obtained in this experiment
corresponding to a recovery of only 18 % of input BacFhuD-His6. Lanes 2 and 4 (Fig. 15)
contain the purified protein recovered after dialyzing out of the P-ME and imidazole. No
significant loss of BacFhuD-His, was observed after dialysis. The protein samples obtained
were then used in the binding affinity assays (see below).
Binding activity of FhuA and recombinant FhuD. Two different assays were
attempted to test whether the purified, refolded recombinant BacFhuD-His, had been
successfidly refolded into a biologically active conformation.
In the first assay samples of BacFhuD-His, were digested with the protease
proteinase K (500 ng) for different time intervals either in the presence and absence of
ferricrocin (Materials and Methods). The digested BacFhuD-His, was analyzed by CBB of
an SDS-PAGE. (Fig.16). When BacFhuD-His, (5 pg) was incubated at 37°C (10 min) in
the absence of femcrocin it degraded suggesting that the protein has low stability (Fig.16,
lane I). It can also be observed (Fig. 16, lanes 3 - 8) that the recombinant protein
BacFhD-Hk, was completely digested both in the presence or absence of ligand
(ferric rocin) under these experimental conditions. S irnilar experiments, performed using
trypsin instead of proteinase K, also indicated that the recombinant protein (BacFhuD-
His,) was sensitive to the protease treatment regardless of the presence or absence of
ligand.
In the second biological activity assay, preliminary experiments were canied out in
which small amounts of protein (BacFhuD-His, and FhuA) were dialyzed against large
volumes of radiolabeled femcrocin. If the periplasmic binding protein bound to its ligand it
was expected that the radiolabeled femcrocin accumulate in the dialysis bags containing the
pmtein. FhuA has been shown to bind femcrocin and therefore it was used as a positive
control. Table 2 shows the results obtained fiom the preliminary dialysis experiments.
After dialyzing 18 pg (12 pg were tested - see Materials and Methods) of FhuA against
radiolabeled femcrocin the concentration of diolabeied femcrocin was observed to be
1260 counts per minute (CPM). When 8 pg and 4.5 pg of FhuA were dialyzed against the
same amount of radiolabeled ferricrocin the concentration of the radiolabeled ligand inside
the dialysis bags was found to be 539 and 290 CPM, respectively. The concentration of
radiolabeled fenicrocin in dialysis bags containing no protein and in the dialysis buffer was
154 and 174 CPM respectively (for the same experiment). Therefore in these preliminary
experiments involving FhuA an increase in the concentration of radiolabeled ligand within
the dialysis bags containing the protein was observed (over background) for three different
amounts of pmtein that were tested. Two different amounts of purified BacFhuD-His, were
tested (4.5 pg and 2.25 pg). It was observed, in both instances the concentration of
radiolabeled ferricrocin (1 80 and 1 70 CPMs, respectively) in the protein samples did not
increase after dialysis with respect to the background concentration of femcrocin (dialysis
buffer and dialysis bags with no protein; Table 2).
DISCUSSION
The objective of this project was to attain high-level expression of FhuD, a
periplasmic binding protein of Escherichia coli K-12, necessary for the transport of
ferrichrome iron (Coulton et al., 1987). Previous attempts in our laboratory to overexpress
FhuD in E. coli have been unsuccessfid. We therefore investigated two novel strategies for
obtaining high level expression of FhuD in the Gram-positive bacterium Bacillus subtilis.
For the first strategy, we hypothesized that the B. subtilis a-amylase signal sequence
fused to the mature form of E. coli FhuD would allow for the secretion of recombinant
FhuD by B. subtilis cells. This assumption was based on previous reports that other
similarly modified periplasmic proteins of E. coli could be produced and secreted to high
levels by B. subtilis (Sarvas, 1995). Accordingly, the gene sequence encoding mature FhuD
was cloned into a secretion expression vector (pKTH132) so that FhuD could be
overexpressed in IH6 140, an exoprotease deficient strain of B. subtilis. A single clone was
obtained harbouring the vector (pFCF03) with the modified fhuD sequence, inserted in the
right orientation. This clone had a slower growth rate than the wild-type (untransformed) B.
subtilis IH6140 (in liquid media and solid media). On agar plates, the colonies harbouring
this construct (pFCF03) formed pin-point colonies. We are not sure of what accounts for
the pin-point colony size and slow growth of the clone. We found no reports in literature
involving this Bacillus secretion expression system of similar phenotypes. We have
hypothesized that the growth of the clone was slow and that the colonies were very small
because the expression of this particular foreign protein (FhuD) somehow interfered with
the production of other essential cellular proteins, hence inhibiting normal growth..
Attempts to detect secreted FhuD in the growth media using our polyclonai anti-FhuD
antibody (PAb 993) proved dhitfbl. Furthennore, no FhuD was detected either within
the cells or arrested at the cell wall using the same polyclonal antibody.
B. mbtilis produces seven major exoproteases (Sarvas, 1995) which have been
shown to be very efficient in degrading extracellular protein, especially when bacteria
remain at the stationary phase for any length of time (Sarvas, 1995). In our experiments a
cocktail protease inhibitor and PMSF were used throughout growth and attempts were made
to collect the bacteria prior to the stationary phase. However it is possible that the protease
inhibitors did not totally efficiently eliminate exoprotease activity. Furthermore, it is
known that a residual Bacillar exoprotease activity is sufficient to quickly degrade secreted
proteins (Simonen and Palva, 1993). Moreover, in some instances the recombinant
proteins are degraded intracellularly immediately following translation (Sarvas, 1995).
Th is could explain why no recombinant FhuD was detected (by blotting) within the
bacteria or arrested in their cell wall. Unexpectedly, not even hgmented forms of
recombinant FhuD were found within the bacteria or in the growth media. To explain
this peculiarity, I propose that perhaps a truncated form of the protein that does not
include the polyclonal antibody recognition sites was being expressed instead of the full
size protein. Since only a single clone was obtained which possessed the modifiedfhuD
gene in the correct orientation, it is possible that expression of the recombinant protein is
lethal to the cells. Therefore the single done could possess a mutation within the inserted
BUD gene which truncated the protein and allowed the cells to grow. This hypothesis
could be tested by sequencing thefhuD insert.
The second strategy for overexpressing FhuD, involved cloning the sequences
encoding mature FhuD @ustidine tagged or untagged) into a Bacillus expression vector
(pKTH288). Histidine-tagged FhuD (BacFhuD-His6) and untagged FhuD were
overexpressed in this B. subtilis IH6 140. This Bacillus expression system has successllly
been used for the overexpression of many outer membrane proteins (Nurrninern et a/. , 1992;
Srikurnar et a/., 1993: Dahan et a/.. 1996 1. The Coulton laboratory has previously used this
expression system to express two proteins: FhuA of E. coli (Moeck el 01.. 1995) and p i n
of Haemophilus influeme type b (Srikumar et at., 1993). Overexpression of BacFhuD-
His, led to its intracellular accumulation in the form of insoluble inclusion bodies.
Inclusion bodies are aggregates of insoluble proteins that are usually formed when a protein
is produced at very high-levels. Inclusion bodies probably correspond to attempts at partial
(mis)folding and exclusion of water by aggregation of hydrophobic patches. In the
construct (pKTH288) the modified jhuD gene is b e d the partial signal sequence (seven
amino acids) of a-amylase. The presence of the partial signal sequence is necessary for
protein expression (Sarvas, 1995) and it may also be directing inclusion body formation.
Inclusion bodies containing recombinant protein are often formed when using a Bacillus
expression system (Nurrninem et al., 1992; Srikumar et al., 1993). M y results indicate that
the Bacillus expression system can also be used to achieve high-level expression of
recombinant periplasmic binding proteins (1 80 mg inclusion body p r o t e a culture). In the
absence of the reducing agent P-mercaptoethanol, bands corresponding to dimers of
BacFhuD and BacFhuD-His, were observed by SDS-PAGE. FhuD contains a single
cysteine so that it has the potential to form a disulfide bridge with another molecule of
FhuD (and hence a dimer). However, there is evidence that the dimerization of
recombinant FhuD is an artefact of the high-level expression and aggregation of FhuD into
Bacillus inclusion bodies. Disulfide bridge formation has been observed in other instances
in which a BaciZZus expression vector was used to overexpress recombinant proteins
(Nurminem et a!., 1992; Sarvas, 1995). The inclusion bodies containing BacFhuD-His,
were solubilized in a seong denaturant (either urea or GnHCl) and purified by nickel
chelate chromatography. Finally, three different strategies were used in an attempt to
renature the purified BacFhuD-His, into a biologically active conformation.
In the furt strategy the denaturant (urea) was removed gradually by stepwise
dialysis. This is a traditional empirical refolding technique that has been used successfully
on many proteins (i.e. Miller et al., 1979). By using this technique I was able to obtain
small amounts of soluble, recombinant FhuD. However, recombinant FhuD refolded in this
manner was very unstable; it quickly precipitated from solution when the reducing agent
was removed by dialysis or when the protein was concentrated. Funhermore, storage of the
protein at low temperature (either 4 "C or -20 "C) led to its degradation and precipitation
From solution. This instability is unusual as periplasmic binding proteins are very resistant
and tend to maintain their activity even after such harsh treatment as boiling (Ames et a/.,
1990). Another disadvantage of the stepwise dialysis approach for refolding is that this
technique is not sufficiently cost and time efficient to produce the large amounts of purified
protein (100 mg amounts) required for X-ray crystallography trials; the refolding
concentration has to be very low and large buffer volumes are required. We therefore
explored the possibility of refolding FhuD while it is immobilized to a N?+-NTA column.
This technique allows for higher protein concentrations and requires less buffer and time.
With BacFhuD-His, protein bound to the Ni2+-NT~ matrix the denaturant
concentration was slowly lowered using an FPLC device to ensure linearity. The protein
was treated with protease inhibitors throughout the experiment to decrease degradation.
Using this technique soluble BacFhuD-His, was obtained, which was at a five-fold higher
concentration than the protein from the stepwise dialysis experiment. However, this soluble
BacFhuD-His6 was not very stable. Attempts to concentrate the protein or to dialyze away
the irnidazole. NaCl or P-mercaptoethanol led to BacFhuD-His, precipitation fiom solution.
In the third strategy the denaturant (GnHCl) was removed quickly (in one-step) with
the BacFhuD-His, bound to N~'+-NTA matrix. This technique was also FPLC-mediated.
Soluble BacFhuD-His, was recovered in solution with an efficiency similar to that observed
when the denaturant concentration was slowly lowered. Although it wasn't possible to
concentrate the protein, BacFhuD-His, refolded according to this third strategy remained
soluble following the removal by dialysis of irnidazole or P-mercaptoethanol yielded a
protein that remained in solution. The correct folding of the recombinant FhuD was
assessed by analyzing its ferrichrome binding activity.
Soluble recombinant FhuD was treated with the proteases proteinase K and trypsin.
A properly folded form of recombinant FhuD should show some resistance to protease
digestion in the presence of ferricrocin, a fenichrome homologue, and a Iigand of FhuD.
Recombinant FhuD was rapidly degraded by the proteases both in the presence or absence
of fenicrocin, suggesting that recombinant BacFhuD-His, was not properly refolded.
Furthermore, in the absence of added proteases BacFhuD-His, degmdation was observed
following a simple ten minute incubation at 37 "C. These data suggest low stability and
indicate that the protein was probably misfolded. Equilibrium dialysis assays have been
used for sevenl years to demonstrate binding of periplasmic binding proteins to their
substrate (i.e. Ames, 1986). Preliminary evidence from equilibrium dialysis experiments
also seems to indicate that the soluble BacFhuD-His, has no femcrocin binding activity and
is therefore not folded properly. This same equilibrium dialysis assay, seemed to confirm
that the high-affinity femchrome OM receptor FhuA is capable of binding femcrocin.
FhuA is the OM receptor for fernchrome, and it has been shown to bind ferricrocin by
different methods (i.e. Moeck et al., 1996). Finally, the entire refolding process had to be
performed in the presence of a low concentration (0.01 %) of a non-ionic detergent
(LDAO). This requirement of detergent is unusual as E. cofi FhuD is thought to be a
soluble periplasmic protein. In fact Rohrbach et al. (1995) used an experimental approach
to overexpress FhuD that did not require the presence of detergent for FhuD purification or
storage. Other methods that examine the secondary structure of proteins could also be used
to assess correct folding of a stable recombinant FhuD. The crystal structures of several
periplasmic binding proteins have been determined, so that the average percentages of
alpha-helical, beta-sheet and random coil structures for the periplasrnic binding protein
group have been calculated (Adam and Oxender, 1989; Bruns et a[., 1997). Therefore,
the folding of soluble recombinant FhuD could also be partly assessed by employing
techniques such as circular dichroism (CD), Fourier transform i&ed spectroscopy
(FTIR) or tryptophan fluorescence spectroscopy which estimate the secondary
composition of a protein. Unfortunately, the low stability of the soluble BacFhuD-His,
prevented us tiom using these techniques.
I suggest three possibilities as to why recombinant FhuD did not renature into a
biologically active conformation. The recombinant FhuD-His, differs from the wild-type E.
coli FhuD in which it has an 11 amino acid N-terminal extension that is thought to be
involved in the inclusion body formation (an artefact of the Bociflus expression system).
The recombinant protein also possesses a 6-histidine C-tennind extension (the affinity tag).
Both the N-terminal extension or the C-terminal hexahistidine tag may interfere with the
proper folding of recombinant FhuD. However, other proteins which also have the 11
amino acid N-terminus extension have been refolded successfuliy (i.e. Hib porin; Dahan
et a!.. 1996). Still, there are no reports in the literature of the successful refolding of
periplasrnic binding proteins possessing a C-terminal affmity tag. It is also possible that
the protein is properly folded but that the N-terminal extension or C-terminal
hexahistidine tag (or both) interfere with ligand binding. Finally, FhuD may bind the
free form of iron (Fe") rather than the siderophore-cornpiexed form of iron. There is
crystal structure (Bruns el a/. , 1 997) and biochemical data (i.e. Adhikari et al., 1 995) that
indicate that the periplasmic binding proteins of Neisseria and Haemophilus species bind
to free Fe3+. In fact, there is no crystal structure evidence and there is little biochemical
data (Koster and Braun, 1990) to directly support the assumption that the E. coli
peri p lasmic binding proteins bind the siderop hore-complexed form of iron. However
these last two possibilities do not explain the low stability of the soluble BacFhuD-His,.
To test the hypothesis that the C-tenninal hexahistidine tag interferes with folding
recombinant FhuD lacking the C-terminal extension could be refolded by stepwise
dialysis. To find out whether the actual ligand of E. coli FhuD is free iron rather than
siderophore-iron a variation of the equilibrium dialysis binding activity assay could be
camed out using radiolabelled Fe3+. The same equilibrium dialysis assay could be used
to determine the Kd and Km of ligand binding for FhuA and FhuD, and to determine the
stoichometry of substrate binding for both proteins.
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85
Table 1 - Escherichia coli and Bacillus subtilis strains and plasmids used in this study.
E. coii strains
MC4 100 FaraD A(argF-lac) Ul69 rspL [hi reLl flbB deoC pstF rbsR
DHSa supE44 AlacU 169(+801acZAM15) hsdRl7 recA 1 endA 1 gyrA96 thi- 1 relA 1
E. coli plasmids
pPM18
B. subtilis plasmids I
blajhzul pBR322 replicon, 13.9 kb
pBS I1 KS (H-) B, szrbtilis strains
I * Bacillus subtilis strain IH6140 is a prototrophic, sporulation defic
Phagemid bla+, 2.96 kb
Source
Sarnbrook et al. 1989
Coulton at al., 1983
S tratagene
Palva et al., 1981
Palva et al., 1981
Palva, et a[., 1982
This study
This study
This study
-
ent derivative of Margurb strain 1A298 from the Bacillus genetic stock center, Ohio State university, Columbus, OH. It secretes reduced amounts of exoproteases (M. Sarvas, 1995) ** The "intraceilular" B. subtilis expression vector used was pKTH288 (4.45 kbp). It was derived from pKTH23 9 (Palva et al., 1 98 1) by insertion of a 14-nucleotide linker at its EcoEU site. *** The secretion B. subtfis expression vector used was pKTH132 (4.55 kbp). It was derived from PUB 1 10 by insertion of a 500 bp insert on its BamHI site containing the promoter, ribosome binding site and the signal sequence of the amy gene of B. amyloliqefaciem (Palva et al., 1982; Sarvas, 1995.)
Table 2. Equilibrium dialysis assay: Binding of radiolabeled
ferricrocin to FhuA and BacFhuD-His,
Sample counted [55Fe-femcrocin] (CPM)
control dialysis bag
BacFhuD-His, (4.5 pg)
dialysis buffer 1 74
dialysis buffer
control dialysis bag 150
Figure 1. Schematic representation of the cell wall of Gram-positive and Gram-negative
bacteria. (Adapted and modified &om Pugsley, 1993)
Milieu
Protein layer
Cell Wall
Milieu
I i
Protein layer
Lipopolysacharide
Outer membrane
Cell Wall & periplam
Cytoplmnic Cytoplasmic membrane memb m e
Cytoplasm Cytoplasm
Gram-positive bacterium Gram-negative bacterium
Figure 2. Diagrammatic representation of a periplasmic binding protein. The strucme
represented is from the Haemophilus influenzae ferric iron-binding protein (hFBP).
HFBP binds a single ~ e " in a cleft formed between two globular domains rich in a-
helices and P-strands. The structure of this protein has been recently solved (Bruns et d.,
1997). P-sheets are represented in yellow while a-helices are in blue. The base of the
cleft is constituted by two P-strands (in yellow), as it is typical with other periplasrnic
binding proteins. This illustration was produced by Dr. Chris Bruns (Bruns el a/., 1997)
[This Figure was kind!y made available on the World Wide Web (internet) by Dr. Bruns].
Figure 3. Diagrammatic representation of the structural organization of a typical ATP
transporter. Two transmem brane domains (typically) span the membrane six times. The
short motif ("EAA" loop), situated on a cytoplasmic loop, which is conserved between
many transporters is represented by a * (see text). The ATP-binding domains are located
on the cytoplasmic side of the membrane and may interact with the "EAA" loop region
(see text). It should be noted that in this illustration the ABC transported is represented
as having four separate domains. However, sometimes these can be hsed together (see
text) [Adapted and modified from Higgins, 19921.
EXTRACELLULAR
CYTOPLASM
Figure 4. Model of transport mec hanisrn across the cytoplasmic membrane involving the
CM complex and a periptasmic binding protein. This model is the three state model
proposed by Davidson et al. (1992). The transport model depicted in this figure is
described in the text. (Adapted and modified from Davidson er al., 1992)
State I - State I1 - State I11
ATP hydrolysis k'
State I
ATP hydrolysis u"
Figure 5. Strategy for overexpression and purification of BacFhuD-His,
CLONING OF
* (Hid6
PCR AMPLIFY f
HIII
TRANSFORM ALPHA-AMYLASE PFCFO~ PROMOTER
B. subtilis
8M UREA INCLUSION
BODIES
PARTIAL SIGNAL SEQUENCE
AGAROSE
Figure 6. Inclusion bodies from B. swbtilis IH6 140 strains overexpressing BacFhuD and
BacFhuD-His,. Protein samples were loaded on a 10 % SDS-PAGE and stained with
Coomassie Brilliant Blue (CBB). Lane M, molecular weight markers; lanes 1- 2,
BacFhuD-His, from B. subtilis inclusion bodies ( 5 and 15 pg, respectively); lanes 3 - 4,
have the same as lanes 1 - 2, except that the proteins were treated with P-ME prior to
loading on the gel; lanes 5 - 6, BacFhuD-His, From B. strbtilis inclusion bodies ( 2 and 5
pg, respectively); lanes 7 - 8, same as lanes 5 - 6, except that proteins were treated with
P-ME prior to loading on the gel.
Figure 7. Purification of BacFhuD-His, from urea-solubilized inclusion bodies by ~ i * ' -
NTA chromatography employing a Biologic FPLC (Biorad). Samples were loaded onto a
10 % SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M, protein molecular
weight markers; lane I, BacFhuD-His, from B. subtilis inclusion bodies (3 pg); lane 2,
urea-solubilized inclusion bodies tiom B. subtilis expressing BacFhuD-His, (5 pg)
[starting material for FPLC-mediated Ni2'-NTA chrornatopphy]: lane 3. material that
did not bind to the Ni2'-NTA matrix (flow-through), lane 4, Ni2+-NTA purified BacFhuD-
His, from inclusion bodies (5 pg) [protein was not treated with P-ME prior to loading on
the gel]; lanes 5 - 6. N~'*-NTA purified BacFhuD-His, from inclusion bodies (5 pg and
15 pg, respectively) [protein was treated with P-ME prior to loading on the gel].
Figure 8. Identification of BacFhuD-His, by immunoblotting. After separation on a 10
% SDS-PAGE (protein setup identical to that of the gel in the previous figure), proteins
were transferred to nitrocellulose. probed with Pab 993, and detected with a secondary
polyclonal anti-rabbit Ab conjugated to alkaline phosphatase. Lane M, pre-stained
protein molecular weight markers; lane I, BacFhuD-His, from B. subrilis inclusion bodies
(600 ng); lane 2, urea solubilized inclusion bodies from B. sztbfilis expressing BacFhuD-
His, (1 pg) [starting material for FPLC-mediated Ni2'-NTA chromatography]; lane 3,
material that did not bind to the Ni2'-NTA matrix (flow-through), lane 4, Ni2'-NTA
purified BacFhuD-His, from inclusion bodies (1 pg) [protein was not treated with P-ME
prior to loading on the gel]; lanes 5 - 6, Ni"-NTA purified BacFhuD-His, from inclusion
bodies (1 pg and 3 pg, respectively) [protein was treated with P-ME prior to loading on
the gel].
Figure 9. Detection of secreted BacFhuD (secBacFhuD) by irnmunoblotting. Overnight
growth media (2xLw) was transferred to nitrocellulose (employing a milliblot
apparatus, see text), probed with Pab 993, and detected with a secondary polyclonal anti-
rabbit Ab conjugated to alkaline phosphatase; to the control wells labelled N (negative
control), 100 p1 sterile 2xLW were added to 8 M 100 pl urea; to the contol wells
labelled P (positive control). 100 pls of 8 M urea were mixed with 100 pl IB protein
(BacFhud in Tris-EDTA); Wells la through Id contain growth media from a candidate
thought to synthesize and secrete the recombinant FhuD (see text).
Figure 10. Analysis of renatured BacFhud-His,. Samples were loaded on a 8 % SDS-
PAGE and stained with Coomassie Brilliant Blue. Lane M, molecular weight markers;
lane 1, BacFhuD-His, f?om B. subtilis inclusion bodies (4 pg); lanes 2 - 5, BacFhuD-His,
after removal of urea by stepwise dialysis (750 ng, 3 pg, 1.5 pg and 6 pg, respectively).
Lanes 2 - 3 and lanes 4 - 5 represent proteins from two dialysis experiments. Proteins
were not treated with B-ME prior to loading to the gel.
Figure 1 1. FPLC-mediated linear gradient GnHCl removal experiment profile (Biologic,
Biorad). The (left) Y-axis represents a 280 am OD reading and it allows for the protein to
be tracked for the duration of the protocol (flow-through, wash, linear gradient, and elution
stages) The X-axis represents the duration of the experiment. Fractions were collected at
different stages of the experiment and are indicated by the fraction numben.
Figure 12. Analysis of renatured BacFhuD-His, obtained by FPLC-mediated linear
gradient removal of GnHCI. Protein samples were loaded onto a 10 % SDS-PAGE and
stained with Coornassie Brilliant Blue. Lane M, protein molecular weight markers; lane
1 GnHC1-solubilized inclusion bodies from B. subtilis expressing BacFhuD-His, (starting
material for FPLC-mediated N~''NTA chromatography); lane 2, material that did not bind
to NI'WTA column (flow-through); lanes 3 (Fraction 23) - 4 (fraction 24), soluble
BacFhuD-His, that was eluted from the Ni2WTA column by application of 250 mM
imidazole after the linear gradient removal of GnHCl(2.25 pg and 3.0 pg, respectively)
Figure 13. FPLC-mediated fast (one-step) batch-method GnHCl removal experiment
profile (Biologic, Biorad). The (left) Y-axis represents a 280 nm OD reading and it allows
for the protein to be tracked for the duration of the protocol (flow-through, wash steps, and
elution stages) The X-axis represents the duration of the experiment. Fractions were
collected at different stages of the experiment and are indicated by the fraction numbers.
Figure 14. Analysis of BacFhuD-His, removed by FPLC-mediated, fast (one-step)
removal of GnHCl. Protein samples were loaded onto a 10 % SDS-PAGE and stained
with Coomassie Brilliant Blue. Lane M, protein molecular weight marken; lane 2
GnHCI-solubilized inclusion bodies fiom B. subtilis expressing BacFhuD-His, (starting
material for FPLC-mediated N i 2 + N T ~ chromatography); lane 3, material that did not bind
to M'VTA matrix after "batch" binding (flow-through); lanes 1 (fraction 13) and 4
(fraction 14) contain soluble BacFhuD-His, that was eluted fiom the N~'?JTA column by
application of 400 rnM imidazole after one-step removal of the GnHCl(3.7 pg and 2.25
pg, respectively)
Figure 15. Analysis of BacFhuD-His, renatured in a large scaie,"batch"-binding, FPLC-
mediated one-step removal of GnHC1. Protein samples were loaded onto a 10 % SDS-
PAGE and stained with Coomassie Brilliant Blue. Lane M, protein molecular weight
markers; lanes 1 and 3 contain soluble BacFhuD-His, (fiom two consecutive fractions)
that was eluted fiom the NP'NTA column by application of 400 mM irnidazole after one-
step removal of the GnHCl(9 pg and 1.8 pg. respectively): lanes 2 and 4 contain the
soluble BacFhuD-His, from the same two consecutive fractions that were loaded on the
gel after dialyzing out the 400 mM imidazole and 5 mM P-ME (9 pg and 1.8 pg,
respectively).
Figure 16. Analysis of digestion of BacFhuD-His, using proteinase K. Protein samples
were loaded onto a 10 % SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1
contains soluble BacFhuD-His, (5 pg) which was incubated for 10 min at 37 C (in the
absence of proteinase K) prior to loading; lane 2 contains proteinase K (500 ng); lanes 3,
5 and 7 were loaded with soluble BacFhuD-His, (5 pg) which had been pre-incubated
with proteinase K (3 7 C) for 2, 5 and 1 0 min., respectively.; lanes 4 ,6 and 8 were
loaded with soluble BacFhuD-His, (5 pg) which had been pre-incubated with proteinase
K (37 O C) for 2 , s and 10 min. in the presence of femcrocin (30 pM).
