refolding of a recombinant collagen-targeted tgf-β2 fusion protein expressed inescherichia coli

PROTEIN EXPRESSION AND PURIFICATION 11, 169–178 (1997)ARTICLE NO. PT970784

Refolding of a Recombinant Collagen-Targeted TGF-b2Fusion Protein Expressed in Escherichia coli1

Bo Han,* Frederick L. Hall,† and Marcel E. Nimni*,2

*Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, and Divisionsof Surgical and †Cardiothoracic Research, Children’s Hospital Los Angeles, Los Angeles, California 90027

Received February 3, 1997, and in revised form June 9, 1997

peptides, which function as potent regulators of cellIn this study, a tripartite transforming growth fac- growth, differentiation, and extracellular matrix depo-

tor-b (TGF-b2) fusion protein bearing an N-terminal sition (1,2). There are three major TGF-b isoforms inpurification tag and an auxiliary collagen binding mammalian cells, designated TGF-b1, TGF-b2, anddecapeptide has been constructed and expressed at TGF-b3 (3). The sequence homology among these iso-high levels in Escherichia coli. The resulting recom- forms is greater than 65% and their biological activitybinant protein accumulates in an insoluble and bio- is similar in many in vitro assays (4). However, therelogically inactive inclusion–body complex. The in- are significant differences in potency and physiologicalsoluble protein was solubilized in guanidine hydro- effects, including inhibition of hematopoeitic stem cells,chloride and a Ni-chelating affinity column was regulation of endothelial cells proliferation, and induc-utilized to isolate the 13.5-kDa TGF-b2 fusion pro- tion of mesoderm formation in vertebrate embryos (4–tein, which was then refolded into its native confor-

6), suggesting that particular TGF-b isoforms may bemation under controlled redox conditions. The for-potentially useful for specific therapeutic applications.mation of native homodimers was monitored by non-

Currently, two approaches are used to obtain TGF-reducing sodium dodecyl sulfate–polyacrylamideb1 for biochemical and pharmacological studies. Onegel electrophoresis gradient gels and the bioactivityinvolves extraction from tissues, such as placenta,determined by a quantitative TGF-b assay system us-blood platelets, or bone. However, such biologicaling mink lung epithelial cells transfected with a plas-sources are limited, especially for the minor isoformsminogen activator inhibitor-1 promoter/luciferaselike TGF-b2 and b3 (3). Another approach involves thereporter plasmid. To optimize yields, renaturation

conditions including denaturants, limiting protein expression and purification of recombinant TGF-b1concentrations, redox ratios, dialysis conditions, from mammalian CHO cells (7). Although expressionand refolding kinetics were studied and monitored of recombinant proteins in transformed prokaryotic mi-by bioactivity. These studies demonstrate that re- croorganisms could, theoretically, serve to guaranteecombinant TGF-b2 fusion proteins can be produced an unlimited supply of such recombinant proteins, un-in E. coli and renatured into biologically active ho- fortunately they have not generated native, soluble,modimers. Furthermore, they confirm that the auxil- and biologically active conformations. Instead, overex-iary collagen binding domain effectively targets the

pression of recombinant protein from Escherichia colirecombinant growth factor to type I collagen. Takenoften results in the accumulation of insoluble proteinstogether, these studies advance the technology nec-within bacterial inclusion bodies (8) due in part to theessary to generate large quantities of targeted TGF-

b fusion proteins for specific biomedical applica-tions. q 1997 Academic Press

D-thiogalactopyranoside; PMSF, phenylmethysulfonyl fluoride;VWF, von Willebrand factor; TFA, trifluoroacetic acid; PAI-1, plas-TGF-b3 is a multifunctional growth factor, part of a minogen activator inhibitor-1; HAc, acetic acid; PEG, polyethylene

large and growing family of structurally related poly- glycol; L-Arg, L-arginine. CHO, Chinese hampster ovary; RT-PCR,reverse transcriptase-polymerase chain reaction; BSA, bovine serumalbumin; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Ea-1 This work was supported by NIH AG Grant 02577.

2 To whom correspondence should be addressed. gle’s medium; PBS, phosphate-buffered saline; DTT, dithiothreitol;SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electropho-3 Abbreviations used: TGF-b, transforming growth factor-b; GSH,

reduced glutathione; GSSG, oxidized glutathione; IPTG, isopropyl b- resis; DMF, dimethylformamide; LAP, latency-associated peptide.

1691046-5928/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

AID PEP 0784 / 6q15$$$281 10-13-97 16:29:13 pepa AP: PEP

HAN, HALL, AND NIMNI170

formation of misfolded polypeptides and/or high molec- anti sense primer for both constructs is ttagatgcatttg-caagact. PCR products were resolved on agarose gels,ular-weight multimers.

Purification and renaturation of insoluble recombi- purified by Geneclean (Bio101), and ligated into therecipient TA cloning vector (Invitrogen). The excisednant proteins requires solubilization, reoxidation, and

proper refolding to generate biologically active struc- inserts were then ligated in frame into a pET28b ex-pression vector (Novagen) which added a histidine pu-tures. Analysis of the crystal structure of TGF-b2 re-

veals an unusual protein conformation which can be rification tag to the amino terminus of each fusion pro-tein. Both pET–TGF-b2–F1 and pET–TGF-b2–F2characterized as an outstretched hand with two fingers

(9). The hydrophobic regions near the finger tips allow constructs were maintained in XL-blue strain of E. coli.The orientation and reading frame of each insert wastwo monomers to come together in an opposite orienta-

tion and form the biologically active homodimer. All confirmed by manual DNA nucleotide sequence analy-sis using the modified dideoxy chain termination meth-nine cysteines of each TGF-b2 monomer form disulfide

bonds: eight are involved in intrachain bonds, while ods (USB).residue 77 forms a single interchain bridge (10). To TGF-b2 expression. For recombinant protein ex-effectively renature TGF-b2 homodimer from E. coli pression, the pET–TGF-b2–F1 and pET–TGF-b2–F2inclusion bodies, covalent disulfide bonds in correct ori- constructs were transformed into the BL21(DE3) strainentation need to be regenerated. The likelihood of form- of E. coli. Primary cultures were grown from singleing correct TGF-b2 homodimer from the process of ran- plated colonies and flasks containing 500-ml culturesdom SS-bond formation is exceedingly low, given that were inoculated with primary cultures at a 1:50 dilu-the number of SS bonds equals nine and only one of tion. Transformed bacteria were cultured in 2YT me-34,459,245 possibilities would therefore represent the dium supplemented with kanamycin (100 mg/ml) untilnative state (11). Recently, our laboratory has success- the A600 reading reached 0.7–1.0, after which proteinfully overcome some of these technical difficulties with expression was induced in the presence of 0.4 mM IPTGthe expression of hTGF-b1 in E. coli (12), but the over- for 5 h at 377C. Bacterial pellets were collected by cen-all yields were very low. trifugation at 5000g for 10 min and stored at 0707C.

In the present study, we engineered two constructs, Isolation of inclusion bodies. The bacterial pelletsTGF-b2–F1 and TGF-b2–F2. The F1 represents TGF- were washed with buffer A (20 mM Tris–HCl, 250 mMb2 active fragment fusion protein. The F2, besides the NaCl, 0.05% NP-40, pH 8.0) and lysed by the additionTGF-b2 active fragment, incorporates a modified colla- of 0.4 mg/ml lysozyme, 5 ng/ml DNase and 0.8 mMgen binding domain derived from von Willebrand’s fac- PMSF in 1/10 of the original volume of cell suspension.tor (12) . We expressed these recombinant proteins at a The lysis was facilitated by sonication and homogeniza-high level in E. coli utilizing the T7 polymerase system, tion with a BrinKmann Polytron operated at 26K forpurified the solubilized proteins from the bacterial in- 30 s on ice, followed by centrifugation at 10,000g forclusion bodies, and refolded them into their native pro- 20 min at 47C to pellet the inclusion bodies.tein conformation as renaturation conditions were de-

Solubilization and purification of TGF-b2 fusion pro-fined, the activity of each construct, as well as the effi-teins from inclusion bodies. Inclusion bodies were sol-ciency of different renaturation methodologies wasubilized in 6 M guanidine–HCl, 0.1 M phosphate buffer,evaluated using a quantitative bioassay. Finally, wepH 8.0, at room temperature in 1/10th the original vol-confirmed that the TGF-b2–F2 construct binds effi-ume of cells and allowed to stand at room temperatureciently to type I collagen.for 1.5 h with occasional vortexing. The solubilized fu-sion proteins were separated from insoluble debris by

EXPERIMENTAL PROCEDURES centrifugation at 10,000g. Purification of solubilizedTGF-b2 fusion proteins was accomplished by Ni-NTATGF-b2 constructs. Cytoplasmic RNA extractedchromatography (Qiagen): The protein solution wasfrom human MG-63 osteosarcoma cells (13) was re-initially purified as a batch followed by loading onto averse transcribed into cDNA by using a specific TGF-Ni-NTA column and the Ni-NTA beads allowed to settleb2 antinsense primer. Polymerase chain reactiondown for 30 min. The column was washed with 2 vol(PCR) was used for amplification of TGF-b2 cDNAsof 8 M urea in buffer A, pH 8.0, and followed by 2 vol(Perkin–Elmer): For TGF-b2–F1, the sense primer isof 8 M urea in buffer A, pH 6.8. TGF-b2s were elutedggtgctagcgctttggatgcggcctattg. For TGF-b2–F2, the fu-with 8 M urea in buffer A, pH 4.2. Protein concentrationsion protein incorporating a high-affinity collagen bind-was monitored by the method of Bradford (Bio-Rad)ing domain, we utilized a 50-nucleotide primer, catatutilizing BSA as a standard.tggcgcgaaccgagcttcatggctctgagtggtgctagcgctttgg, de-

signed to include two parts: 20 nucleotides from the TGF-b2 refolding. After Nickel chelate affinity pu-rification of the His-tagged protein, samples were di-TGF-b2 sequence linked in frame with 30 nucleotides

encoding a modified collagen binding peptide. (14). The luted to appropriate concentrations (10–100 mg/ml) in


TGF-b2, COLLAGEN BINDING, E. coli EXPRESSION, AND REFOLDING 171

FIG. 1. Design and construction of genetically engineered TGF-b2 fusion proteins. (A) Tripartite TGF-b2 fusion proteins (1–3) weregenerated by RT-PCR, cloned into TA cloning vectors, and then ligated into a pET28b expression vector (Novagen) utilizing EcoRI andNdeI sites, which incorporated an N-terminal 6xHis tag into position 1. (B) The TGF-b2–F2 protein contained a high-affinity collagenbinding domain modified from von Willebrand factor. This ECM binding domain was flanked by linker regions containing glycine to minimizesteric hindrance. A potential thrombin site included in the vector design.

8 M urea, pH 8.0. A redox buffer (buffer A with 0.2 acetonitrile with 0.05% TFA. Samples in acetonitrilewere then either frozen directly or lyophilized and re-mM GSSG/2.0 mM GSH was formulated (see Results),

chilled on ice, and slowly added to the purified TGF- constituted into 1% BSA/40 mM HCl prior to freezing.b2 fusion proteins to dilute the urea to 1.3 M (6X dilu- TGF-b2 bioassay. Mink lung epithelial cells trans-tion) and initiate the oxidative refolding. After vigorous fected with a plasminogen activator inhibitor-1 (PAI-mixing, the recombinant TGF-b2 fusion proteins were 1) promoter–luciferase reporter construct (15) wereallowed to anneal into a thermodynamically stable a generous gift of Dr. D. B. Rifkin (New York Univer-structure at 47C for various time intervals under speci- sity Medical Center). PAI-1–Luc transformed minkfied conditions. After prolonged oxidative refolding, lung cells were cultured as monolayers in 10% FBS/samples were dialyzed either against buffer B (500 mM DMEM containing 200 mg/ml geneticin. TrypsinizedNaCl, 20 mM Tris–HCl, 10% glycerol, pH 8.0) or 10% cells were aliquoted into 48-well plates at a density

of 9 1 104 cells/well and allowed to attach for 5.0 hat 377C. The cell cultures were washed twice withDMEM and TGF-b was added in a 0.5% FBS/DMEMtest medium. After 17 h, the medium was removed,the cells were rinsed twice with ice cold PBS, and 50ml of cell lysis buffer (Promega) was added and al-lowed to stand at room temperature for 10–15 min.Ten microliters of each resulting cell extract was gen-tly mixed with 50 ml of the luciferase assay reagent(Promega luciferase testing kit) at room temperaturein 0.5 ml Eppendorf tubes. Luciferase activity, re-ported as relative light units (cpm/ cell), was quanti-fied by liquid scintillation counting.

TGF-b2–F2 collagen binding assay (In vitro 3H-la-FIG. 2. Expression and purification of recombinant fusion proteins. beled recombinant TGF-b2–F1 and TGF-b2–F2) Bac-Protein expression is visualized on reducing SDS–PAGE gel; lane

terial inclusion bodies with TGF-b2–F1 and –F2 con-1, molecular weight markers; lanes 2 and 5, bacterial proteins ofstructs were isolated by centrifugation and labeled bytransformed TGF-b2–F1 and TGF-b2–F2, respectively, before in-

duction; lanes 3 and 6, after 5 h induction by 0.4 M IPTG for 5 h. a method developed in our laboratories (16). Briefly,The arrow indicates the position of the induced TGF-b2–F1 and inclusion bodies were dehydrated in DMF in a smallTGF-b2–F2 monomers; lanes 4 and 7, after purification by Ni-NTA screw-cap glass test tube and purged with nitrogen. Toaffinity chromatography, resulting in pure TGF-b2–F1 and TGF-

this suspension was added 0.5 ml of fresh preparedb2–F2 as monomer bands. There is a slight upshift of TGF-b2–F2due to the presence of auxiliary collagen binding domain. 3H-NaBH4 (100 mCi, Amersham) / DMF solution. The



FIG. 3. Dimer formation and biological activity under various redox conditions (GSH/GSSG ratio). (A) The TGF-b2–F2 fusion proteinspurified from E. coli inclusion bodies were folded under specified GSH/GSSG ratios for 24 h at 47C and the results were display on areducing SDS–PAGE gel. Lower bands (Ç15 kDa) represent monomers while upper bands (Ç30 kDa) represent dimers. Lane 1, 10 mM

GSH and 1 mM GSSG. Lane 2, 2 mM GSH and 1 mM GSSG. Lane 3, 2.0 mM GSH and 0.2 mM GSSG. Maximal levels of monomer to dimerconversion occurred when the GSH/GSSG ratio was 2 mM/0.2 mM which was confirmed by the assessment of resulting biological activities(B) for both TGF-b2–F1 and TGF-b2–F2. After refolding under different redox conditions for 48 h, samples were dialyzed against 10%acetonitrile and 0.5% TFA, reconstituted in 0.1% BSA/40 mM acetic acid (HAc), and bioassayed fro TGF-b activities. Activity reported asluciferase activity units (cpm/cell), the biological activity was determined to be optimal a GSH/GSSG ration of 2.0 mM/0.2 mM, which wouldcorrelate the efficiency of dimer formation.

suspension was immediately vortexed and then al- RESULTSlowed to stand at room temperature for 1 h. Pellets Construction, Expression, and Purification of TGF-b2were washed with DMF and then PBS. Labeled recom- Fusion Proteinsbinant proteins were purified and renatured as de-scribed. The bioactivity was also checked. In this study, we used RT-PCR and recombinant

DNA technologies to engineer a chimeric TGF-b2 fu-In 24-well plates, 200 ml of 3.0 mg/ml type I collagen(pH 7.0) derived from pepsin treated bovine tendon (17) sion protein (TGF-b2–F2) which includes a high-affin-

ity collagen binding domain (Fig. 1). The strategicallywere incubated at 377C for 30 min for fiber formation.Twenty microliters of serially diluted 3H-TGF-b1–F1 modified collagen binding decapeptide was derived

from a von Willebrand’s factor domain (VWF) involvedand 3H-TGF-b1–F2 was added on the top of the colla-gen gel and allowed to stand for 1 h at 377C. Gels were in the recognition of exposed vascular collagen se-

quences (12,14). Thus, the TGF-b2–F2 construct,extensively washed with 2.0 ml PBS three times, eachfor 30 min. One half milliliter of 100U/ml collagenase which incorporates the collagen binding decapeptide

WREPSFMALS, was designed specially for targetingwas then added and incubated at 377C for 1 h. Twentymicroliters of digested solution was counted (Beckman the growth factor to collagen. The cysteine of the origi-

nal sequence was replaced conservatively by a methio-2000). The activity of collagenase released TGF-b2–F1and TGF-b2–F2 was assayed. In a separate assay a nine, in order that this auxiliary domain should not

interfere with the elaborate disulfide bond formation0–5 M salt gradient was also applied to the wells tostudy the release profile. required for TGF-b2 renaturation and dimerization.



yields and recovery of the recombinant proteins. Ex-tensive purification of the recombinant proteinsTGF-b2–F1 and TGF-b2–F2 (which include an N-terminal (His)6 leader sequence) was afforded bymetal chelate (Ni-NTA) chromatography performedunder denaturing conditions in the presence of 6 M

guanidine.Since renaturation of biological activity was more

successful in 8 M urea (see Discussion), the bound re-combinant proteins were washed on the column with8 M urea, pH 8.0, followed by a more stringent washwith 8 M urea, pH 6.8, followed finally by elution of thepurified proteins in 8 M urea at pH 4.2 (see Fig. 2,lanes 4 and 7). Approximately 3 to 5 mg of purifiedrecombinant TGF-b2 (3.75 mg TGF-b2–F1 or 5.25 mgTGF-b2–F2) was routinely recovered from a 50-ml bac-terial culture following Ni-NTA purification.

Renaturation of hrTGF-b2–F1 and rhTGF-b2–F2

The glutathione redox system. Proteins expressedin E. coli often accumulate in inclusion bodies as aggre-gates of insoluble forms, which do not posses biologicalactivity, within the reducing environment of the bacte-rial cytoplasm (18). Since control of redox conditions isa key factor in successfully refolding and recombinantTGF-b2 fusion proteins, we tested various concentra-tions of reduced and oxidized glutathione (GSH, GSSG)FIG. 4. Bioactivity of renature TGF-b2–F1 and TGF-b2–F2 as-as ‘‘oxido-shuffling’’ reagents to increase the rate andsayed under standard dialysis conditions. Commercial TGF-b1 (R&Dyield of correct protein disulfide bond formation. Assystem) was used to generate a standard curve for the MLC Bioassay.

Renatured recombinant TGF-b2–F1 and TGF-b2–F2 were diluted shown in Fig. 3, the concentration and ratio of reduced/1:5 and then serially diluted in assay medium. Under identical condi- oxidized glutathione exherts a major influence on thetions to yield relative biological activity: TGF-b2–F1 is 134.8 ng/ml, resulting biological activity. Nonreducing SDS–PAGEand TGF-b2–F2 is 155 ng/ml.

gels revealed that a redox system composed of 2.0 mM/0.2 mM of GSH/GSSG yielded maximal formation ofTGF-b2 dimers under these conditions (Fig. 3A). Sub-Flanking linkers were designed to include glycine resi-sequent bioassays confirmed that a 2 mM/0.2 mM GSH/dues to minimize steric hindrances. The resulting ge-GSSG ratio resulted in the highest recovery of biologi-netically engineered TGF-b2 fusion proteins contain acal activity (Fig. 3B). By contrast, the utilities of DTTcontiguous series of functional domains: a purificationand b-mercaptoethanol as reducing agents to controltag, a thrombin protease site, and a collagen bindingthe rate of the redox reactions were found to be limiteddomain followed by the mature fragment of TGF-b2.(data not shown).Recombinant proteins can be expressed at high

Protein concentration. Associated with the kineticslevels in bacteria transformed with plasmids bearingand thermodynamics of monomer renaturation, inthe T7 polymerase system, BL21(DE3) strain. In thevitro protein refolding, misfolded intermediates, andpresence of 0.4 mM IPTG, the levels of recombinantmultimeric aggregates compete with the renaturationTGF-b2 expression approached 40% of cellular pro-of TGF-b2. In this study, the limiting concentration fortein, as determined by SDS–PAGE and proteinoptimal recovery of native TGF-b2–F1 was found to bestaining (Fig. 2). The reducing SDS–PAGE gel12 mg/ml. For TGF-b2–F2, the limiting concentrationshows a representative induction of the 12.5-kDaturned out to be somewhat lower (10 mg/ml), possiblyTGF-b2 monomer for TGF-b2–F1 (Fig. 2, lane 3) anddue to an increase in hydrophobic interactions associ-13.5 kDa for the TGF-b2–F2 construct bearing theated with the auxiliary collagen binding decapeptide.auxiliary VWF-derived decapeptide (Fig. 2, lane 6).

The bulk of the recombinant proteins were found in Dialysis conditions. For recovery of active recombi-nant TGF-b2–F2 which includes the auxiliary collagenthe insoluble inclusion bodies harvested after cell

lysis by centrifugation. For solubilization 6 M guani- binding decapeptide and the (His)6 purification tag, pH8.0 was found to be optimal for recovering native con-dine was determined to be optimal in terms of overall



FIG. 5. Effects of dialysis and lyophilization on bioactivity of renatured TGF-b2 fusion proteins. Refolded TGF-b2–F1 (A) and TGF-b2–F2(B) were dialyzed against specified concentrations of acetonitrile acidified with 0.5% TFA or with 0.1% BSA/40 mM HAc. After lyophilization,recombinant proteins were reconstituted in 1% BSA/4 mM HCl prior to bioassay. TGF activity is expressed as luciferase activity units (cpm/cell).

formation and biological activity. At pH 6.5 precipita- which conditions no appreciate loss of activity seemsto occur.tion occurred, and at pH 11.0 no precipitation was seen,

but TGF-b activity was lost. We determined that 10% Refolding kinetics. As a rule, folding is slow whenglycerol was beneficial as a stabilizing agent, while it involves intermolecular interactions and/or isomer-other additives like PEG, sucrose, and L-arginine were ization steps (19). We used 0.05 M iodoacetamide tonot as effective (data not shown). Buffer A, at pH 8.0, block disulfide bond exchange at different time pointssupplemented with 250 mM NaCl and 10% glycerol af- (20). Nonreducing SDS–PAGE gels (Fig. 6A) showedforded optimal recoveries upon gradual protracted dial- that the TGF-b2–F2 dimer started to form within 0.5ysis (Fig. 4). h of refolding at 47C. With increasing time the Ç30-

Alternatively, we endeavored to assess the utility of kDa bands begins to shift upward to form a distinctiveadding organic solvents to the dialysis buffer, i.e., ace- doublet. Initial dimers seem to form within the firsttonitrile and 0.5% trifluoroacetic acid. Indeed, it was 0.5 h of diluting the denaturant, but isomerization intofound that acetonitrile did help maintain the native a stable conformation occurs over a period of hours,conformation of the TGF-b2 dimer and prevent aggre- judging from parallel bioassays (see Fig. 6B). The bio-gation. Furthermore, after dialysis, acetonitrile could logical activity increased with folding time over thebe easily removed by lyophilization. Therefore, differ- first 24 h, reached maximal levels atÇ48 h, and exhib-ent acetonitrile concentrations and conditions were ited little change over the next 24 h.evaluated in a series of comparative studies (Fig. 5).Importantly, TGF-b2 fusion proteins in the resulting Collagen Binding Activity of TGF-b2–F2lyophilized powders could be reconstituted in a 4 mM

HCl solution supplemented with 0.1% BSA, but the To investigate whether TGF-b2–F2 is physiochemi-cal and biochemically different from TGF-b2–F1, a se-recovery of bioactivity after lyophilization was found to

be about five times less than that after standard dial- ries of collagen binding/release assays were carried out.Binding TGF-b2–F2 to collagen is dose-dependentysis. Samples are routinely stored at 0207C, under



from a sequence of von Willebrand’s Factor, expressedthis recombinant fusion protein in E. coli, and rena-tured its biological activity in vitro under experimen-tally optimized conditions. Although expression of re-combinant proteins by bacterial systems promises anunlimited source of useful proteins and growth factors,the production of disulfide-containing proteins by bac-terial systems is often complicated by low yields andthe formation of insoluble inclusion bodies (19,21). Inthis study, two TGF-b2 constructs were expressed inE. coli and transformed with an expression plasmidwhich utilizes the T7 polymerase system to efficientlyexpress recombinant proteins. The pET28-b–TGF-b2vector constructs utilized in the study exhibits two no-table advantages. First, the T7 polymerase system af-fords high levels of protein expression under the controlof the IPTG inducible lac UV5 promoter. Second, thepurification of TGF-b2 fusion proteins to homogeneityprior to renaturation was facilitated by the inclusionof an in-frame (His)6 purification tag in the expressionvector.

The renaturation of protein aggregates in bacterialinclusion bodies into biologically active conformationsinvolves a number of interdependent steps: solubiliza-tion, oxidative refolding, and withdrawal of denatur-ants, each of which represents a challenge of strategyFIG. 6. Renaturation of TGF-b2 fusion proteins over time. Bioactiv-and optimization (22,23). In the case of TGF-b iso-ity is presented as a feature of time in oxidative refolding conditionsforms, the processes are complicated by the number ofprior to dialysis and bioassay. Aliquots (10-ml) of TGF-b2–F1 and

TGF-b2–F2 were removed at specific intervals, dialyzed against 10% cysteine residues involved in disulfide bond formation,acetonitrile/0.05% TFA, lyophilized, and reconstituted into 1.0 ml 1% which stabilize an intricate ‘‘knot’’ structure character-BSA/4 mM HCl prior to bioassay. As shown in nonreducing SDS– istic of this family of growth factors (9). In the presentPAGE gels (inset), initial dimer formation occurs within 0.5 h (lane

study, we determined that a strong ionic denaturant,2); however, increase refolding time (lanes 3 and 4) are associated6 M guanidine, was more effective than 8 M urea inwith the appearance of dimers which exhibit lower electrophoresis

mobility (upper arrow). solubilizing the inclusion bodies. Guanidine also ap-peared to be preferable to urea since the latter maycontain isocyanate, leading to carbamylation of free

(Fig. 7A). TGF-b2–F2 can be released from the colla- amino groups within the polypeptide. However, in eval-gen gel after collagenase digestion with biological activ- uating the recoveries from subsequent refolding steps,ity (Fig. 7B). TGF-b2–F1, which lacks the collagen we found that the TGF-b2 activity recovered from gua-binding domain, binds to collagen with less affinity nidine–HCl solutions was much lower than from urea.through nonspecific interactions. Exposed to a salt gra- Others also have reported that guanidine–HCl maydient, TGF-b2–F2 will not elute until the salt concen- interfere with the refolding process (22). For this rea-tration reaches 4 M (data not shown). This was also son, we utilized guanidine–HCl initially as a denatur-the case with TGF-b1–F2 fusion protein (14). These ant to solubilize the inclusion bodies and optimize re-experiments demonstrate that the collagen binding do- covery of the recombinant proteins and then replacedmain enables TGF-b2–F2 to be sequestered by the col- the 6 M guanidine–HCl with 8 M urea in the course oflagen matrix and that it can be released by collagenase affinity purification. Oxidative refolding was initiateddigestion to display its biological activity. by dilution to 1.3 M urea, pH 8.0, in an optimized urea–

redox buffer.Overall Yields of Refolding Proteins Since removal of the denaturant by simple dialysisTable 1 shows the step by step yields and specific caused abundant precipitation we performed an initial

activity. sixfold dilution of the denaturant (from 8 M urea to 1.3M urea) with an optimized redox buffer system, which

DISCUSSION maintained both the urea concentration and redox con-ditions for a protracted time interval (48 h). The solubi-In this study, we engineered a novel TGF-b2 fusion

protein containing a collagen binding domain adapted lized proteins rearranged into native conformations



FIG. 7. Binding curve of renatured TGF-b2–F2 for collagen matrices. (A) 3H-labeled fusion proteins bind to collagen in a dose-dependentmanner. (B) To release bound TGF-b2 fusion proteins from the collagen gels, 0.5 ml of collagenase (100 U/ml) was used to digest the collagenand release soluble collagen fragments. The solubilized TGF-b2–F1 and TGF-b2–F2 proteins were assayed and the activity units wereexpressed as luciferase counts (cpm/cell).

and the biological activity increased dramatically after native as well as misfolded TGF-b2, we endeavoredto optimize these conditions. Small molecules such asthis prolonged period without encountering overt pro-glycerol, sucrose, PEG (200–2000), BSA, L-Arg are of-tein precipitation.ten added in dialysis buffers to prevent precipitationIn the final step, dialysis was used to remove the(19,24,25). In this study, we found 10% glycerol to bedenaturant and the redox reagents, followed by centrif-advantageous. Also, salt concentrations and pH areugation to collect misfolded aggregates. Since proteinvery important considerations during dialysis, as pro-precipitation at this step represents a major loss ofteins stability is often dependent on the ionization stateof side residues (26). In these studies, we determined

TABLE 1 that dialysis into 0.5 M NaCl, pH 8.0, 10% glycerolproved optimal for recovery of biological active TGF-bYields and Purity at Different Stepsfusion proteins. Conceivably, the lack of native propep-of Purification and Renaturationtide sequence (27) and/or the presence of auxiliarydomains may hinder the process of renaturation. Nev-Fraction TGF-b2–F1 TGF-b2–F2ertheless, by manipulating redox conditions, protein

Inclusion bodies 8.92 mg 13.4 mg concentrations, pH, ionic strength, small molecule ad-After Ni-NTA column 3.75 mg 42.0%a 5.25 mg 39.2%a

ditives, and dialysis conditions, we have improved theStarting concentration

yields from Ç0.5% in our earlier attempts with TGF-for refolding 12 mg/ml 10 mg/mlb1–F2 to an average of between 25 and 30% TGF-b2–After dialysis (soluble) 2.8 mg/ml 23%a 92.6 mg/ml 26%a

Specific activity (after F2, before lyophilization.lyophilizing) 134.8 ng/ml 4.8%a 155 ng/ml 5.9%a TGF-b is a multifunctional protein that regulates

many physiological events, including cell proliferationa Yield calculated from the previous step. Overall average yield: and, extracellular metabolism, soft and hard connec-for 50 ml bacterial culture broth, TGF-b2–F1 is 3.75 mg 1 23%tive tissue repair, and inflammatory processes (1–3).1 4.8% Å 41.4 mg and TGF-b2–F2 is 5.25 mg 1 26% 1 5.9% Å 80.5

mg. Excess of TGF-b may be partly responsible for fostering



activities in comparison with transforming growth factor-beta 1aberrant wound healing and the tissue damage causedand -beta 2. Mol. Endo. 3, 1977–1986.by scaring and the pathological consequences has been

5. Qian, S. W., Burmester, J. K., Merwin, J. R., Madri, J. A., Sporn,termed the ‘‘dark side’’ of tissue repair (28). Since TGF-M. B., and Roberts, A. B. (1992) Identification of a structural

b is a pleotropic agent which can stimulate, inhibit, and domain that distinguish the action of the type 1 and 2 isoformsmodulate cellular events in a time- and concentration- of transforming growth factor beta on endothelial cells. Proc.

Natl. Acad. Sci. USA 89, 6290–6294.dependent manner, it is important to control its deliv-ery and bioavailability to advance its use as a potential 6. Merwin, J. R., Roberts, A., Kondaiah, P., Tucker, A., and Madri,

J. (1991) Vascular cell responses to TGFb-3 mimic of TGF-b 1therapeutic agent. Normally, TGF-b is secreted by cellsin vitro. Growth Factors 5, 149–158.as a biologically inactive latent precursor that becomes

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b, a latency-associated peptide, and the latent TGF- ovary cells, isolation, and characterization. Protein. Express.b binding protein (29–31). Many extracellular matrix Purif. 4, 130–140.components bind to TGF-bs in a reversible manner and 8. Williams, D. C., Van-Frank, R. M., Muth, W. L., and Burnett,

J. P. (1982) Cytoplasmic inclusion bodies in Escherichia coli pro-may serve as a reservoir for the growth factors (32). Inducing biosynthetic human insulin proteins. Science 215, 687–our study, we have verified that the active fragment of689.TGF-b2–F1 does bind nonspecifically to type I colla-

9. Sun, D., Piez, K. A., Ogawa, Y., and Daviers, D. R. (1992) Crsytalgen. On the other hand TGF-b2–F2, which contains astructure of transforming growth factor-b2: an unusual fold for

specific collagen binding domain, has a much greater the superfamily. Science 257, 369–373.affinity for collagen matrices when compared to TGF- 10. Schlunegger, M. P., and Grutter, M. G. (1992) An unusual fea-b2–F1. The high-affinity targeting of TGF-b2–F2 to ture revealed by the crystal structure at 2.2A resolution of hu-

man transforming growth factor-beta 2. Nature 358, 430–434.specific sites may be useful clinically, since the darkside, or inappropriate TGF-b activity, is implicated in 11. Anfisen, C. B., and Scheraga, H. A. (1975) Experimental and the-

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(14), these studies extend the methodologies needed to cell or tissue samples. J. Bio. Chem. 257, 8569–8572.generate large quantity of biologically active TGF-b 14. Tuan, T., Cheung, D. T., Wu, L., Yee, A., Gabriel, S., Han, B.,

Nimni, E. M., and Hall, F. (1996) Engineering, expression andisoforms from prokaryotic microorganisms. They alsorenaturation of targeted TGF-b fusion protein. Connect. Tiss.provide support to the concept that growth factors mayRes. 34, 1–9.be targeted to specific tissues, wounds, grafts, and pros-

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16. Cheung, D. T., Benya, P. D., Gorn, A., and Nimni, M. E. (1981)ACKNOWLEDGMENTSAn efficient method for in vitro labeling proteins to high specificredioactivity using 3H-NaBH4 in dimethylformamide. Anal. Bio-We greatly acknowledge the contributions of Lingtao Wu and Lingchem. 116, 69–74.Liu for preparations of the TGF-b2 constructs and Tai-Lan Tuan

and Judy Zhu for their help in developing the bioassay. This work 17. Nimni, M. (1980) The molecular organization of collagen and itsis in partial fulfillment of the requirements for a Doctor in Philosophy role in determining the biological properties of the connectiveDegree in Biochemistry and Molecular Biology (B.H.). tissue. Biorheology 17, 51–82.

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