PROTEIN EXPRESSION AND PURIFICATION 7, 447–457 (1996)ARTICLE NO. 0066

A General Bacterial Expression System for FunctionalAnalysis of cDNA-Encoded Proteins

Magnus Larsson,* Eva Brundell,† Louise Nordfors,* Christer Hoog,†Mathias Uhlen,* and Stefan Stahl*,1

*Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden; and†Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden

Received December 18, 1995

existing genes and gene products. The efforts to solveA general system for functional analysis of cDNA- this immense task involve the construction and analy-

encoded proteins is described. The basic concept in- sis of cDNA libraries representing different tissues, cellvolves the expression in Escherichia coli of selected types, developmental stages, etc. The large-scale cDNAportions of cDNAs in an approach toward the under- analysis can be divided into different types of ap-standing of the function of the corresponding proteins. proaches. (i) Full-length cDNA sequencing, which per-A selected cDNA is expressed as part of a fusion pro- haps enables prediction of the function for the encodedtein used for immunization to elicit antibodies, and a gene product if homologies to characterized proteinscorresponding fusion protein, having the cDNA-en- exist. However, only approximately 4000 human genescoded portion in common, for purification of target

of 50,000 to 100,000 have been completely sequencedprotein-specific antibodies. This antiserum could behitherto. (ii) The sequencing of expressed sequence tagsused for functional analysis of the cDNA-encoded pro-(ESTs)2 from cDNA libraries (1,2), and subsequent ho-tein, e.g., by immunohistology. Two general expressionmology matching with databases, is an approach whichvector systems for E. coli have been constructed, bothcould give a hint about the encoded function, if the(i) designed with multiple cloning sites in three differ-gene, or a related one, is previously described. Obvi-ent reading frames, (ii) having their protein produc-ously, the majority of isolated human ESTs representstion controlled by the tightly regulated T7 promoter,

and (iii) enabling affinity purification of the expressed novel genes for which no function can be assigned. Nev-target proteins by fusions to IgG-binding domains ertheless, it is likely that a vast majority of the humanderived from staphylococcal protein A or a serum albu- genes are covered by ESTs present in the different da-min-binding protein derived from streptococcal pro- tabases (3). (iii) Characterization of mRNA expressiontein G, respectively. This novel system has been evalu- profiles, by sequencing of cDNA libraries from differentated by expressing five cDNAs, isolated from pre- cell types (4), gives valuble information only if the genepubertal mouse testis by a differential cDNA library function is known.screening strategy. All five clones could be expressed The genome approaches so far have thus concen-intracellularly in E. coli as fusion proteins with high trated on the accumulation of various sequence data,production levels, ranging from 4 to 500 mg/liter, and

with interpretations relying on analogies to knownaffinity purification yielded essentially full-lengthgenes, proteins, and structures. Since the final under-products. Characterization of affinity-purified anti-standing of a gene function needs to be verified experi-bodies revealed that there exists no cross-reactivitymentally, gene expression of the isolated cDNAs orbetween the two fusion systems and that such antibod-ESTs is required (5). The function of a certain proteinies indeed could be used for immunohistology. The im-could then be investigated directly by full-length cDNAplications for the described system for large-scale

functional analysis of cDNA libraries are discussed. expression or gene knockout experiments. Both theseq 1996 Academic Press, Inc.

2 Abbreviations used: ABP, albumin binding protein derived fromstreptococcal protein G; BSA, bovine serum albumin; EST, expressedAn important aspect of the ongoing genome projects sequence tag; FCA, Freund’s complete adjuvant; FITC, fluorescein

is to determine location, structure, and function of all isothiocyanate; HSA, human serum albumin; ORF, open readingframe; PBS, phosphate-buffered saline; SpA, staphylococcal proteinA; SpG, streptococcal protein G.1 To whom correspondence should be addressed.

4471046-5928/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

LARSSON ET AL.448

approaches are elaborate and time-consuming. A com- MATERIALS AND METHODSplementary and more rational approach to understand

Construction of expression vectors. To construct thegene function when full-length cDNA is not isolatedexpression vectors pT7-TZZ in frames a, b and c, re-would be to produce a portion of a protein and use itspectively, a gene fragment encoding a divalent syn-to raise an antiserum for identifying the protein local-thetic IgG-binding affinity handle, ZZ (20), derivedization in tissues and cells. This latter approach re-from staphylococcal protein A (SpA), was amplifiedquires robust expression systems with high expressionfrom plasmid pEZZT308 (21) using a standard poly-levels and possibilities for rapid and efficient recoverymerase chain reaction (PCR) protocol (22). The oligo-of the produced gene product.nucleotide LAMA7, 5*-CCCTGATCACCATGGGCGC-Bacterial production systems have proven to be pow-AACACGATGAAGCCGTAG-3 *, was used as anerful in terms of high expression levels for the produc-upstream primer, and the oligonucleotides LAMA8,tion of various mammalian proteins (6–8). Affinity pu-5*-GG GG GAT C C G T A G T G A G C G A A G G T A C C-rification of gene products, by expressing a target geneATTCGCGTCTACTTTCGGCGCC-3 *, LAMA9,fused to a gene encoding an affinity tag, consitutes a5*-GGGGGATCCAGTAGTGAGCGAAGGTACCAT-strategy which has been utilized extensively in differ-TCGCGTCTACTTTCGGCGCC-3 *, and LAMA10,ent fields of reseach (9,10). Parallel expression of a gene5*-GGGGGATCCATGTAGTGAGCGAAGGTACCA-in two expression systems, in order to generate twoTTCGCGTCTACTTTCGGCGCC-3 *, respectively, werefusion proteins, having the target protein in common,used as downstream primers, to generate fragmentshas previously been utilized for certain applicationscorresponding to frames a, b, and c. Upstream restric-(11–14). Hey and co-workers (14) have used one fusiontion sites for BclI and NcoI were introduced by LAMA7,protein for immunization and the second for boosting,and sites for KpnI and BamHI by the downstreamto avoid carrier-mediated suppression (15), while oth-primers. In addition, sequences for cleavage of pro-ers have used one fusion protein to elicit an immuneduced fusion proteins by His64Ala–subtilisin (23) wereresponse and the other fusion protein for monitoringintroduced downstream of the ZZ fragment by theof the antibody responses (11–13) and purification ofdownstream primers. The three different ZZ-encodingtarget protein-specific antibodies (11,13).PCR fragments were cleaved with BclI and BamHI andWhen setting up such parallel expression systems,introduced into the BamHI site of plasmid pET21a(/)suitable for functional analysis of cDNA-encoded pro-(Novagen Inc., Madison, WI), resulting in the expres-teins, robustness in the production is of the utmostsion vectors pT7-TZZa, pT7-TZZb, and pT7-TZZc, re-importance. Intracellular production makes it possiblespectively, having their multiple cloning sites in threeto express proteins which cannot be secreted throughdifferent reading frames. Correct insertion was verifiedhydrophobic membranes (16,17). It is also importantby solid-phase DNA sequencing (22).to choose a tightly regulated promoter system, such as

The pT7-ABP expression vectors, also in threethe phage T7 promoter (18), which makes it possibleframes, were constructed from the pT7-TZZ vector ofto produce proteins that normally would be deleteriousthe corresponding frame. A DNA fragment encodingto the host cell (19). When selecting an affinity systemthe albumin-binding protein (ABP) (residues 146–choosing an affinity tag with a high affinity for a spe-266), from the serum albumin binding region fromcific ligand is desired. This affinity tag should allowstreptococcal protein G (SpG) (21,24), was obtained byaffinity purification in buffers containing chaotropicPCR amplification from plasmid pVABP308 (25), usingagents such as guanidine hydrochloride or urea, usedLA MA17, 5* -CC GAA TTCG CTA GCT TAGC TG-for solubilization of proteins precipitated into inclusionAAGCTAAAGTCTTAG-3 *, as an upstream primer andbodies. Furthermore, the affinity tags should be resis-LAMA18, 5*-CCGGTACCAGGTAATGCAGCTAAAAT-tant to proteolysis.TTCATC-3 *, as a downstream primer, respectively. AnHere, we present two general expression vector sys-upstream restriction site for NheI and a downstreamtems, constructed in three different reading frames andsite for KpnI were introduced by LAMA17 anddesigned for intracellular production in EscherichiaLAMA18, respectively. The PCR fragment was cleavedcoli. The expression systems are evaluated for the pro-with restriction enzymes NheI and KpnI and intro-duction of five cDNA-encoded proteins from a cDNAduced into the appropiate pT7-TZZ vector (frame a, b,library prepared from prepubertal mouse testis. Pro-or c, respectively) previously digested with the sametein production is characterized in terms of productionrestriction enzymes. Correct insertion was verified bylevels, solubility of the target proteins, and productsolid-phase DNA sequencing (22). The resulting vectorsquality after affinity purification. Purified fusion pro-were denoted pT7-ABPa, pT7-ABPb, and pT7-ABPc,teins have been used to elicit antibodies and polyclonalrespectively.sera, purified using the corresponding fusion protein,

have been used for functional analysis by immunohis- Generation and selection of cDNA clones. cDNA wassynthesized from poly(A/) RNA isolated from prepuber-tology.

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

FUNCTIONAL cDNA ANALYSIS 449

tal and adult mouse testis and cloned into the Lambda frame, respectively. E. coli BL21(DE3)pLysS cells (Nova-gen Inc., Madison, WI) transformed with the vectors en-uni-ZAP vector (Stratagene, La Jolla, CA), as pre-

viously described (2,26). Lambda uni-ZAP cDNA clones coding the different fusion proteins were grown overnightat 377C in shake-flasks containing 50 ml 30 g/liter Trypticcorresponding to mRNAs overexpressed in testis were

selected by a differential screening method and ana- soy broth (Difco, Detroit, MI), supplemented with 5 g/liter yeast extract (Difco), 100 mg/ml ampicillin, and 34lyzed by Northern blot hybridization (2,26,27). Selected

Lambda uni-ZAP clones were converted into pBlue- mg/ml chloramphenicol. The overnight cultures were di-luted 1:20 and used to inoculate 500 ml of the same me-script (SK0) cDNA clones.dia. The cultures were grown in shake-flasks at 377C.Sequence analysis. The isolated clones containingExpression of the recombinant fusion proteins were in-the cDNA inserts were sequenced from the 5* endduced at mid-log phase (A600 nmÉ 1) by addition of isopro-using semiautomated solid-phase DNA sequencingpyl-b-D-thiogalactoside (IPTG) (Pharmacia Biotech) to a(28), to establish open reading frames (ORFs). Prim-final concentration of 1 mM. Cells were harvested 3.5 hers RIT30, 5*-biotin-AAAGGGGGATGTGCTGCAAG-after induction, by centrifugation at approximately 5000gGCG-3 *, and RIT27, 5*-GCTTCCGGCTCGTATGTT-for 10 min. The pelleted cells were stored at0207C. TheGTGTG-3 *, complementary to regions downstreamcells were thawed and resuspended in 30 ml TST (50 mMand upstream of the multilinker region of pBluescriptTris–HCl, pH 7.5, 0.2 M NaCl, 0.05% Tween 20), followed(SK0) (Stratagene), were used for the PCR amplifica-by sonication (25). After centrifugation at 20,000g for 10tion performed directly on bacterial colonies. A two-min, the supernatants containing soluble gene productsstep PCR program with denaturation at 967C for 15were filtered (1.2mm, Millipore Corp., Bedford, MA), priors and annealing and extension at 727C for 2 min wasto the affinity purification procedure. Insoluble materialsperformed on a Perkin–Elmer Gene Amp Systemwere pelleted by centrifugation at approximately 20,000g9600. The biotinylated, double-stranded PCR prod-for 10 min. The pelleted material containing precipitateducts were immobilized on paramagnetic beads withproteins were solubilized by adding guanidinium hydro-covalently coupled streptavidin, Dynabeads M280–chloride and b-mercaptoethanol to final concentrationsStreptavidin (Dynal AS, Oslo, Norway). A neodym-of 6 M and 10 mM, respectively, followed by incubationium–iron–boron permanent magnet (Dynal AS) wasat room temperature for 2 h on a magnetic stirrer. Afterused to sediment the beads. The eluted nonbiotinyl-centrifugation at 20,000g for 10 min, the supernatantsated strand was used as template for a fluoresceinwere diluted (in TST containing 0.1 M guanidinium hy-isothiocyanate (FITC)-labeled sequencing primer, 5*-drochloride) to a final concentration of 0.5 M guanidiniumFITC-TTCACACAGGAAACAGCTATGACC-3 *. Thehydrochloride. After centrifugation at 10,000g for 10 min,sequencing reactions were performed on a roboticfiltered (1.2 mm) supernatants were applied directly toworkstation (Biomek 1000, Beckman Instruments,the affinity chromatography columns. The affinity puri-Fullerton, CA) and an Automated Laser Fluorescentfication of the ZZ and ABP fusion proteins, on IgG–Seph-DNA Sequencer (Pharmacia Biotech, Uppsala, Swe-arose (Pharmacia Biotech) or HSA–Sepharose (21), re-den) was used for detection of the Sanger fragments.spectively, was performed essentially as described earlierSolid-phase sequencing (22,28) was also employed for(11,30). The columns were washed with 100 ml TST andverification of the expression vectors, using the bio-50 ml 5 mM NH4Ac, pH 5.5, before elution of bound mate-tinylated primer LAMA14, 5*-biotin-ATGCTAGTTA-rial with 0.2 M HAc, pH 3.3 (ZZ fusions), or 0.5 M Hac,TTGCTCAGCGGTGG-3 *, and LAMA27, 5*-ATCCC-pH 2.8 (ABP fusions), respectively. Protein content wasGCGAAATTAATACGACTCAC-3 *, for a PCR ampli-estimated by absorbance measurements (A280 nm) and rel-fication generating a biotinylated product coveringevant fractions were lyophilized.the region of interest. The oligonucleotide LAMA16,

5*-FITC-TGTTAGCAGCCGGATCTC-3 *, was used as Immunization of rabbits. New Zealand White rab-bits were immunized intramuscularly with 500 mg ofsequencing primer. Alternatively, LAMA11, 5*-TTA-

ATACGACTCACTATAGG-3 *, was used as a sequenc- the ZZ-fusion proteins in phosphate-buffered saline(PBS) and Freund’s complete adjuvant (FCA). Boostering primer on the eluted nonbiotinylated strand in

the opposite direction, and then fluorescein-15-dATP injections were given 4, 8, and 12 weeks after the initialinjection with the same amount of fusion protein butwas used as an internal label (29).with Freund’s incomplete adjuvant instead of FCA. TheSubcloning, expression, and affinity purification ofrabbits were bled 10 days after the second and thirdfusion proteins. The cDNA fragments were cleaved outbooster injections. Antisera from the last bleeding werefrom the corresponding pBluescript (SK0) vector withused in this study.EcoRI and XhoI, except in the case of cDNA clone II, for

which EcoRI and an internal HindIII site were used. The Affinity purification of polyclonal sera. Total rabbitsera were diluted 1:30 in TST and applied on a proteincDNA fragments were then inserted into the pT7-TZZ

and pT7-ABP expression vectors in the appropriate AG–Sepharose column (Pharmacia Biotech). After

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

LARSSON ET AL.450

washing with 100 ml TST and 50 ml 5 mM NH4Ac, pH5.5, the total IgG was eluted with 0.5 M HAc, pH 2.8.The HAc was exchanged for PBS using a PD-10 column(Pharmacia Biotech). Purified ABP fusion proteins (ap-proximately 3 mg), containing the different cDNA-en-coded proteins, were bound to HSA–Sepharose (21)and covalently crosslinked to the matrix using glutardi-aldehyde (0.07% final concentration) (11). The reactionwas stopped after 5 min by addition of 1% glycine solu-tion. The gel was washed in consecutive steps withPBS, 0.5 M Hac, pH 2.8, and 0.2 M glycine, pH 2.8. Afraction of approximately 6 mg of total IgG (originatingfrom an immunization with a certain ZZ-fusion protein)was diluted in PBS to a final volume of 35 ml andloaded on a column with 1 ml of HSA–Sepharose hav-ing the corresponding ABP-fusion protein immobilized.After extensive washing with PBS, retained antibodieswere eluted with 0.2 M glycine, pH 2.8, and immedi-ately neutralized in 2 M Tris. Buffer exchange to PBSwith 0.5% bovine serum albumine (BSA) was per-formed using a PD-10 column (Pharmacia Biotech).

Immunological analysis. Affinity-purified ABP-Iand ABP-II fusion proteins were separated by SDS–PAGE under reducing conditions. The separated pro-

FIG. 1. A flow chart representation of the basic concept for func-teins were transfered to Immobilon-P membranestional analysis of cDNA-encoded proteins. See text for details.

(Millipore Corp., Bedford, MA), and blocked with 3%BSA in PBS with 0.05% Tween 20 (PBST) for 1 h atroom temperature. The membranes were probed with

phot microscope, and photographed with Kodak TMAXpurified rabbit IgG diluted 1:100, for 1 h at room tem- 400 film.perature. Membranes were washed in PBST. The mem-branes were developed by adding swine anti-rabbit IgG

RESULTSconjugated with horseradish peroxidase (P399, Dako-The Basic Conceptpatts, Copenhagen, Denmark) diluted 1:10,000, fol-

lowed by incubation for 40 min at room temperature. The presented system designed for functional analy-After washing in PBST, analysis was performed using sis of cDNA-encoded proteins is outlined in Fig. 1. Se-the ECL Western blotting detection system (Amersham lected clones are sequenced, at least 300–400 nucleo-Corp., Buckinghamshire, UK). tides from the 5* ends to establish ORFs, and matched

Immunofluorescence microscopy of mouse testicular with databases to investigate possible homologies withsections were performed as follows. BALB/c mice were previously reported proteins. Selected cDNA clones areperfused using 1% paraformaldehyde (31). The testis thereafter sucloned in parallel into vectors of the twowere removed and fixed in ice-cold paraformaldehyde expression systems having the appropriate readingfor 6 h, after which the testis were immersed overnight frame (Fig. 1). Upon intracellular expression in E. coli,in 0.3 M sucrose. The testis were froozen on dry ice, two different fusion proteins are produced. One vectorsectioned (7-mm slices) at 0257C, and analyzed using system expresses the target protein fused to an IgG-indirect immunofluorescence microscopy. Germ cells at binding affinity tag (ZZ) (20) derived from SpA, and thedifferent stages of development were isolated as de- second vector system yields the target protein fused toscribed before (27) and centrifugated onto a glass slide an HSA-binding affinity tag (ABP) (21,24,30) derivedusing a cytospin centrifuge. The cells were fixed in from SpG. The use of both these tags has been exten-freeze-cold methanol:acetone (50:50) for 5 min. The af- sively documented (9,15,32,33), and the rationale forfinity-purified anti-ZZ-I IgG were diluted 1:20. The sec- selecting them as affinity tags for this system is de-ondary antibodies were FITC-conjugated swine anti- scribed below. The expressed fusion proteins are affin-rabbit IgG (diluted 1:50) (Boehringer Mannheim, ity purified directly either after cell disruption, if theMannheim, Germany). The slides were mounted in a fusion product is predominantly soluble, or after solubi-78% glycerol mounting medium, containing 1 mg/ml lization and renaturation of inclusion bodies, in such

cases when the fusion product aggregates in inclusionparaphenylene diamine, examined in a Nikon Labo-

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

FUNCTIONAL cDNA ANALYSIS 451

FIG. 2. Expression vectors. Schematic representation of the pT7-TZZ (A) and pT7-ABP (B) expression vectors, respectively, constructedwith a multiple cloning site (MCS) in three reading frames, a, b, and c (C). The expression vectors are designed for intracellular productionof target proteins fused to an affinity fusion partner; the IgG-binding ZZ (A) derived from staphylococcal protein A (SpA) or the serumalbumin-binding ABP (B) derived from streptococcal protein G (SpG). The expression is in both vector systems under control of the T7promoter (PT7)/lac operator (lacO) sequence located upstream of the sequence encoding the affinity tags, followed by a MCS suitable forinsertion of genes encoding desired target proteins. Also encoded by the vector is the lac repressor (LacI) for tight repression prior to theinduction with IPTG, and b-lactamase (Bla) for selection by ampicillin. The vectors also contain the phage f1 intergenic region (ori f1),enabling the preparation of single-stranded DNA for DNA sequencing. The multiple cloning site exists in three reading frames (C). Encodedamino acids are denoted in a one-letter code above the nucleotide sequences. The overlined sequence represents the cleavage site forHis64Ala–subtilisin (H64A) (23), which can be used to cleave the produced fusion protein to release the target protein in such cases wherethis is desired.

body form. The inherent properties of the target gene sites constructed in three reading frames introduceddownstream of the affinity tags to allow direct insertionproduct will thus obviously decide which strategy to

employ. The ZZ-P-fusion proteins are, after purification of cDNAs using alternative restriction enzymes com-mon for vectors frequently used for cDNA library con-by affinity chromatography on IgG–Sepharose, used

for immunization (Fig. 1). The corresponding ABP-P- structions. To allow enzymatic cleavage of producedfusion proteins for applications where removal of thefusion protein can be immobilized, during the purifica-

tion procedure to the HSA–Sepharose column, and affinity tag would be desired, both sets of vectors con-tain a cleavage site for H64A–subtilisin (23,33,34).used for the isolation and purification of antibodies re-

active with the cDNA-encoded portion, P (Fig. 1). Such The ZZ and ABP affinity tags are selected since theyshare several advantageous features: (i) they are bothaffinity-purified antibodies would be suitable for differ-relatively small in size, both being 14 kDa, which en-ent analyses, including Western blots and immunohis-sures a high product to tag ratio; (ii) the highly specifictology studies aimed to elucidate the localization andinteractions with their ligands, IgG (ZZ-fusions) orfunction of the cDNA-encoded protein.HSA (ABP-fusions), respectively, enable efficient puri-fication by affinity chromatography (33); (iii) both tagsThe Expression Vectorsalso allow efficient recovery of gene products of low

The two different sets of novel expression vectors are solubility since affinity chromatography can be per-outlined in Fig. 2. The transcription is under the con- formed after guanidine hydrochloride/urea-mediatedtrol of the tightly regulated phage T7 promoter system solubilization and renaturation of precipitated prod-(18). Both vector systems are designed for intracellular ucts (17,25) and (iv) ZZ and ABP fusions can be de-expression of recombinant proteins as fusions to one tected by blotting techniques after SDS–PAGE analy-of two different affinity tags: (i) the IgG-binding ZZ sis by taking advantage of their IgG(Fc) and HSA bind-domains derived from SpA (20) or (ii) the ABP from ing properties, respectively. ZZ fusions are efficiently

stained using a commercially available complex of rab-SpG (21,24,30). The vector systems have multicloning

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

LARSSON ET AL.452

TABLE 1

Characteristics for the Five Selected cDNA Clones

Accession Determined sequence (nt)/No. cDNA clone No.a full-length mRNAb (nt) Found homologiesc

I MMTSG15T3 X61814 680/1200 NoII MMTSG67X X61876 1200/2200 Nuclear pore targeting complex component of Mus musculus

III MMTSG118X X61832 1000/2400 NoIV MMTEST638 Z31216 500/1600 Tektin A1 and B1 from Strongylocentrotus purpuratusV MMTEST641 Z31219 470/1300 Six human ESTs

a Accession numbers in the EMBL database.b Estimated by Northern blot.c Homologies found on nucleotide and/or amino acid level.

bit anti-horseradish-peroxidase-IgG and horseradish clone IV and V the exact number of expressed aminoacids is not known since the DNA sequencing from theperoxidase. ABP fusions are stained using biotinylated

HSA and conjugated streptavidin–alkaline phospha- 5* end did not reach the translation termination signals(stop codons). The protein production characteristicstase. Furthermore, both tags have through extensive

documented use proven to be highly stable to proteoly- in terms of expression levels for the fusion proteins,solubility of the encoded gene fusion products, fractionsis (9,15,17,25,32,33) and they contain no cysteines

which could cause formation of unwanted disulfide- selected for further experiments, and protein stabilitybridged complexes. In addition, both ZZ and ABP share of selected fractions are presented in Table 2. The pro-stuctural similarities. Both tags comprise two struc- tein expression levels were generally high but variable,tural domains, each of which has a globular fold, con- ranging from 4 to 500 mg per liter culture. Neverthe-sisting of a triple a-helical bundle (35,36). Both N- and less, shake-flask cultivations were in all cases enoughC-termini of the two tags can be used for fusion without to ensure material for immunization of rabbits. Theinterfering with the folding. The structural integrity of solubility of the different fusion proteins was also vari-the tags is probably part of the reason for their stability able. Clones I and II gave almost exclusively precipi-to proteolysis. tated material, clone III only soluble material, while

clones IV and V displayed intermediate properties.Characterization of Selected cDNA ClonesTo evaluate the potential of the presented system, a

To evaluate the efficacy of the novel expression vector more detailed characterization of the protein expres-systems for functional cDNA analysis, five different sion from clones I and II, as ZZ and ABP fusions, re-cDNA clones (Table 1) were selected from cDNA librar- spectively, was performed (Fig. 3). Both these clonesies prepared from prepubertal or adult mouse testis by behaved similarly. Tight regulation of the expressiona differential cDNA library screening strategy (2,26, was indeed achieved since no gene products could be27). The selected clones are for simplicity designated detected by SDS–PAGE analysis of whole-cell lysatesI–V in this study but full names and EMBL database prior to induction (Figs. 3A and 3B, lanes 1 and 4,accession numbers are given in Table 1. The nucleotide respectively), while strong bands appeared at expectedsequences of the selected clones were determined by a sizes in the induced cultivations. The recombinant genePCR-based, semiautomated, solid-phase DNA sequenc-

fusion products constituted approximately 30–40% ofing strategy direct from bacterial colonies (28). Thistotal cell protein (Figs. 3A and 3B, lanes 2 and 5, re-sequencing strategy allowed simultaneous sequencingspectively) as determined from the Coomassie-stainedfrom two different directions. Established ORFs weregels. After single-step recovery by affinity chromatog-matched for homologies with sequences in the publicraphy on IgG–Sepharose (ZZ fusions) or HSA–Sepha-databases. Three of five clones showed significant ho-rose (ABP fusions), respectively, from gunidine hydro-mologies to previously reported sequences, while no ho-chloride-solubilized and subsequently renatured mate-mologies were found for two of the sequences (Table 1).rial (17,25), only full-length gene products could beAll five cDNA clones were nevertheless subcloned intovisualized by the SDS–PAGE analysis. Following thethe appropriate frame of the two vector systems, re-basic concept (Fig. 1), the affinity-purified fusion pro-spectively (Table 2).teins, ZZ-I and ZZ-II, were used to immunize rabbits in

Evaluation of the System by Expression of Selected order to elicit antisera reacting with the cDNA-encodedcDNA Clones proteins corresponding to clones I and II, respectively.

Antiserum resulting from the ZZ-I immunizationThe reading frames and number of expressed aminoacids for the cDNA clones are indicated in Table 2. For could be affinity purified on immobilized ABP-I (Fig.

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

FUNCTIONAL cDNA ANALYSIS 453

TABLE 2

Experimental Results from the Expression of Selected cDNA Clones

Expression Number of aa Expression level Solubility Fraction Protein stability ofcDNA clone frame expressed (mg/liter) (%) selected selected fractiona

I c 150 500 õ10 Insoluble ///II c 97 500 õ10 Insoluble ///

III c 204 4 100 Soluble //IV b ú166 20 ú50b Insoluble /V a ú156 120 ú50b Insoluble ///

a ///, only full-length protein detected on SDS–PAGE. //, full-length protein predominant on SDS–PAGE. /, full-length proteindetected on SDS–PAGE.

b Uncertain values since soluble proteins were severely degraded.

1) to yield antibodies specific for the cDNA-encoded for functional cDNA analysis, antisera elicited from ZZ-I immunization, with subsequent purification on immo-protein, thus avoiding background reactivity to the car-

rier protein. Antibodies reactive to clone II were pre- bilized ABP-I, were utilized in immunohistology analy-ses. The expression of the protein corresponding topared similarly. To investigate if the system induced

cross-reactive antibodies, anti-ZZ-I antiserum was cDNA clone I (Tables 1 and 2) in the testis of adultmice was investigated by using immunofluorescenceused to stain ABP-I (Fig. 3C, lane 1) and ABP-II (Fig.

3C, lane 2) in immunoblotting. In addition, anti-ZZ-II microscopy. Paraformaldehyde-fixed cryosections oftestis were stained with the affinity-purified anti-ZZ-antiserum was used to stain ABP-I (Fig. 3D, lane 1)

and ABP-II (Fig. 3D, lane 2). It was clearly demon- I antibody. An intense anti-ZZ-I antibody signal wasdetected within the nuclei of cells located just insidestrated that no cross-reactivity was induced (Figs. 3C

and 3D), since the antisera reacted specifically with the basal lamina of the seminiferous tubules (Fig. 5A,arrow). The location of these cells within the tubulestheir corresponding ABP fusion protein, resulting in

bands of expected sizes. The ABP-II fusion protein strongly suggests that they represent spermatogonialcell types (37). To be able to monitor the intracellularseemed to exist also in a dimeric form since one larger

sized band also appeared. The affinity-purified ZZ fu- distribution of the protein (cDNA clone I) in intact germcells, testicular cells were isolated from adult mousesions of clones III–V are shown in Fig. 4. As indicated

in Table 2, ZZ-III was recovered from the soluble frac- testis using centrifugal elutriation and Percoll gradientcentrifugation (27). In Fig. 5B, a spermatogonial celltion while ZZ-IV and ZZ-V, respectively, were recovered

from insoluble material. The faint band at the top of and a spermatocyte have been stained with the anti-ZZ-I antibody. An intense nuclear signal is seen in thelane 1 (Fig. 4) corresponds probably to E. coli DnaK, a

molecular chaperone which has been demonstrated to spermatogonial cell, whereas no signal is apparent inthe spermatocyte, a result that is in accordance withcopurify with certain soluble misfolded recombinant

gene products. The quality of the recovered ZZ fusion the experimental result seen in Fig. 5A. The intra-nuclear distribution of the protein corresponding toproteins are obviously variable, but this is not crucial

in this context, since recovered degradation products cDNA clone I in the spermatogonial cell is restrictedto a small number of nuclear bodies. These results dem-also originate from the same cDNA-encoded gene prod-

uct. The affinity-purified material could in all cases be onstrate that antibodies generated by the presentedsystem could be suitable for functional cDNA analysis.used for immunizations to raise antibodies that reacted

strongly with the corresponding ABP fusion (data notshown).

DISCUSSIONTaken together, the results suggest that the descibedsystem for parallel production of cDNA-encoded pro- The described new system is aimed for functionaltein fragments with subsequent generation and purifi- analysis of cDNA-encoded proteins, by the generationcation of antibodies reactive to the corresponding pro- of affinity-purified antibodies which are highly specifictein might be a suitable strategy to provide tools for for a certain cDNA-encoded protein. The basic conceptfunctional analysis of cDNA-encoded proteins. involves the expression of a selected cDNA sequence in

two parallel expression systems, both utilizing the T7Immunohistology as an Approach toward Functional promoter system for tight control of the protein produc-

Analysis tion. The cDNA-encoded protein is thus produced asone part common for two different fusion proteins. OneIn order to evaluate if antisera generated by the

strategy outlined in Fig. 1 could prove to be efficacious of the fusion proteins contains an affinity tag ZZ, de-

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

LARSSON ET AL.454

rived from SpA, which allows efficient recovery by af-finity chromatography on IgG–Sepharose, while thesecond produced fusion protein utilizes the ABP tagderived from SpG for affinity recovery on HSA–Sepha-rose. The three-dimensional structure of the minimalalbumin-binding motif (46 amino acids) of SpG (24) wasrecently resolved by nuclear magnetic resonance (36).In this study, we have utilized a new albumin-bindingtag comprising two such minimal binding motifs joinedby the native linker.

According to the basic concept, the produced and af-finity-purified ZZ-fusion proteins can be used for immu-nizations, while the corresponding ABP-fusion proteinsare utilized, in an immobilized form, for affinity purifi-cation of antibodies reactive with selected cDNA-en-coded protein. The parallel expression systems do notseem to induce antibodies that cross-react (Figs. 3Cand 3D), and affinity-purified antibodies have indeedproved to be suitable for immunohistology to analyzeexpression patterns in cells and tissues (Fig. 5). Analternative strategy for the generation of antibodiessuitable for immunohistology could be to utilize therapidly evolving phage display technology to select re-combinant antibody fragments from large combinato-rial libraries (38). Purified fusion proteins, such as theZZ- or ABP-fusion proteins, respectively, could thus beused for the panning to select binders to cDNA-encodedproteins. This strategy would circumvent the time-con-suming immunization procedures.

The robustness of the expression systems is mostprobably crucial for the efficacy of the whole concept.We have chosen intracellular expression in contrast toother systems also employing parallel expression forsimilar purposes (11,12). This should ensure that ahigher fraction of investigated cDNA-encoded proteinsare successfully produced. Furthermore, we have stud-

FIG. 3. Characterization by SDS–PAGE analysis (12%, re- ied the ratio between soluble and precipitated geneduced conditions) of the protein expression of cDNA clones I (A)and II (B) as ZZ- and ABP-fusion proteins, respectively, followedby immunoblotting (C and D) to evaluate potential cross-reactiv-ity of antibodies raised toward one of the ZZ-fusion proteins. (A)Lane 1, whole-cell lysate prior to induction from an E. coli cultiva-tion harboring the construct encoding ZZ-I. Lane 2, the samecultivation after 3.5 h of induced protein production. Lane 3, ZZ-I after a single-step affinity purification on IgG–Sepharose. Lane4, whole-cell lysate prior to induction from an ABP-I cultivation.Lane 5, the same cultivation after induced protein production.Lane 6, ABP-I after a single-step affinity purification on HSA–Sepharose. Lane M, marker proteins with molecular masses inkilodaltons indicated to the left. (B) The same content lane bylane as in (A) but for cDNA clone II, thus expressed as ZZ-II(lanes 1–3) and ABP-II (lanes 3–6). Coomassie brilliant blue wasused for staining. (C and D) Immunoblotting analysis of reactiv- FIG. 4. Characterization by SDS–PAGE analysis (12%, reduced con-ity with ABP-I and ABP-II, respectively. Rabbit antisera raised ditions) of expressed and IgG affinity-purified ZZ-fusion proteins. Laneagainst ZZ-I or ZZ-II, and affinity purified on ABP-I or ABP-II, 1, ZZ-III fusion protein recovered from the soluble cytoplasmic fraction.respectively, were utilized for the immunoblotting. (C and D) Lane 2, ZZ-IV, and lane 3, ZZ-V fusion proteins, respectively, recoveredLane 1, affinity-purified ABP-I, and lane 2, affinity-purified ABP- by affinity chromatography, from solubilized and renatured intracellu-II, immunoblotted with anti-ZZ-I antibodies (C) or anti-ZZ-II an- lar precipitates. Arrowheads indicate full-length products. Lane M,tibodies (D). Note that the double band in lane 2 (D) indicates a marker proteins with molecular masses in kilodaltons indicated to thenonreduced dimer of ABP-II. left. Coomassie brilliant blue was used for staining.

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

FUNCTIONAL cDNA ANALYSIS 455

FIG. 5. Immunohistology analysis of the expression of the protein corresponding to cDNA clone I in the seminiferous tubules of mousetestis and in isolated germ cells. The affinity-purified anti-ZZ-I antibodies were used to stain a mouse testis section (A) or purified germcells (B). No signal was seen when a preimmune serum was used (data not shown). The relative locations of the cells labeled in (A) weredetermined by comparing their Hoechst 33258 labeling (data not shown), whereas the identities of the cells in (B) were determined by theirHoechst 33258 staining as well as their May and Grunwald’s–Giemsa staining pattern (data not shown).

products after expression in the two expression sys- selves (33). This dramatic variability in solubility be-tween different gene products necessitates recovery oftems. Although we have expressed portions of only a

few cDNAs, the solubility of the produced fusion pro- both soluble and precipitated material, in order to in-vestigate which fraction (if not both) to collect for fur-teins ranges from completely soluble to a majority of

the gene product precipitated (Table 2). This is obvi- ther studies. Alternatively, a more straightforwardstrategy could be evaluated, solubilizing the whole cellsously due to inherent properties of the cDNA-encoded

proteins, since both tags are highly soluble by them- with guanidinum hydrochloride and thereafter recov-

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

LARSSON ET AL.456

12. Martineau, P., Charbit, A., Leclerc, C., Werts, C., O’Callaghan,ering the total amount of produced fusion proteins asD., and Hofnung, M. (1991) A genetic system to elicit and monitorif they would have been precipitated. True large-scaleantipeptide antibodies without peptide synthesis. Bio/Technol-

functional analysis of cDNA libraries could thus be en- ogy 9, 170–172.visioned, via recombinant protein production and sub- 13. Zueco, J., and Boyd, A. (1992) Protein A fusion vectors for usesequent generation of antibodies specific for each se- in combination with pEX vectors in the production and affinity

purification of specific antibodies. Gene 121, 181–182.lected cDNA-encoded protein. Automation of some of14. Hey, A. W., Johnsen, J. I., Johansen, B., and Traavik, T. (1994)the required unit operations might prove to be benifi-

A two fusion partner system for raising antibodies against smallcial. The different ongoing genome projects have gener-immunogens expressed in bacteria. J. Immunol. Methods 173,ated enormous amounts of partially sequenced cDNAs,149–156.

and there exists a significant need for various alterna-15. Sjolander, A., Stahl, S., and Perlmann, P. (1993) Bacterial ex-tive systems to take care of this information to eluci- pression systems based on protein A and protein G designed

date the function of the cDNA-encoded proteins. We for the production of immunogens, application to Plasmodiumfalciparum malaria antigens. ImmunoMethods 2, 79–92.believe that the general concept for functional cDNA

16. Sjolander, A., Stahl, S., Lovgren, K., Hansson, M., Cavelier, L.,analysis described here might prove to be suitable inWalles, A., Helmby, H., Wahlin, B., Morein, B., Uhlen, M., Ber-this context.zins, K., Perlmann, P., and Wahlgren, M. (1993) Plasmodiumfalciparum: The immune response in rabbits to the clustered

ACKNOWLEDGMENTS asparagine-rich protein (CARP) after immunization in Freund’sadjuvant or immunostimulating complexes (ISCOMs). Exp. Par-This work was financed by support from the Swedish Natural Re-asitol. 76, 134–145.search Council, the Swedish Medical Research Council, and the Pro-

17. Murby, M., Nguyen, T. N., Binz, H., Uhlen, M., and Stahl, S.tein Engineering Program funded by the Swedish National Board(1994) Production and recovery of recombinant proteins of lowfor Industrial and Technical Development (NUTEK). We thank Dr.solubility, in ‘‘Separations for Biotechnology 3’’ (Pyle, D. L., Ed.),Per-Ake Nygren for valuable advice and fruitful discussions.pp. 336–344, Bookcraft Ltd., Bath, UK.

18. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff,REFERENCES J. W. (1990) Use of T7 RNA polymerase to direct expression of

cloned genes, in ‘‘Methods in Enzymology’’ (Goeddel, D. V., Ed.),1. Adams, M. D., Kelley, J. M., Gocayne, J. D., Dubnick, M., Poly-Vol. 185, pp. 60–89, Academic Press, San Diego.meropoulos, M. H., Xiao, H., Merril, C. R., Wu, A., Olde, B.,

19. Sano, T., and Cantor, C. R. (1991) A streptavidin–protein AMoreno, R. F., Kerlavage, A. R., McCombie, W. R., and Venter,chimera that allows one-step production of a variety of specificJ. C. (1991) Complementary DNA sequencing: Expressed se-antibody conjugates. Bio/Technology 9, 1378–1381.quence tags and human genome project. Science 252, 1651–

1656. 20. Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A.,Holmgren, E., Henrichson, C., Jones, T. A., and Uhlen, M. (1987)2. Hoog, C. (1991) Isolation of a large number of novel mammalian

genes by a differential cDNA library screening strategy. Nucleic A synthetic IgG-binding domain based on staphylococcal proteinA. Protein Eng. 1, 107–113.Acids Res. 19, 6123–6127.

3. Uhlen, M. (1995) Whose genome is it anyway? Trends Biotechnol. 21. Nygren, P.-A., Eliasson, M., Palmcrantz, E., Abrahmsen, L., and13, 160–162. Uhlen, M. (1988) Analysis and use of the serum albumin binding

domains of streptococcal protein G. J. Mol. Recognit. 1, 69–74.4. Matsubara, K., and Okubo, K. (1993) cDNA analyses in the hu-man genome project. Gene 135, 265–274. 22. Hultman, T., Stahl, S., Hornes, E., and Uhlen, M. (1989) Direct

solid phase sequencing of genomic and plasmid DNA using mag-5. Gross, G., and Hauser, H. (1995) Heterologous expression as anetic beads as solid support. Nucleic Acids Res. 17, 4937–4946.tool for gene identification and analysis. J. Biotechnol. 41, 91–

110. 23. Carter, P., Nilsson, B., Burnier, J. P., Burdick, D., and Wells,J. A. (1989) Engineering subtilisin BPN* for site-specific proteol-6. Josephson, S., and Bishop, R. (1988) Secretion of peptides fromysis. Proteins Struct. Funct. Anal. 6, 240–248.E. coli: A production system for the pharmaceutical industry.

Trends Biotechnol. 6, 218–224. 24. Nilsson, J., Nilsson, P., Williams, Y., Pettersson, L., Uhlen, M.,and Nygren, P.-A. (1994) Competitive elution of protein A fusion7. Hockney, R. C. (1994) Recent developments in heterologous pro-proteins allows specific recovery under mild conditions. Eur. J.tein production in Escherichia coli. Trends Biotechnol. 12, 456–Biochem. 224, 103–108.463.

25. Murby, M., Samuelsson, E., Nguyen, T. N., Mignard, L., Binz,8. Rudolph, R. (1995) Successful protein folding on an industrialH., Uhlen, M., and Stahl, S. (1995) Hydrophobicity engineeringscale, in ‘‘Principles and Practice of Protein Folding’’ (Cleland,to increase solubility and stability of a recombinant protein fromJ. L., and Craik, C. S., Eds.), pp. 283–298, Wiley, New York.respiratory syncytial virus. Eur. J. Biochem. 230, 38–44.9. Uhlen, M., Forsberg, G., Moks, T., Hartmanis, M., and Nilsson,

26. Yuan, L., Liu, J.-G., and Hoog, C. (1995) Rapid cDNA sequencingB. (1992) Fusion proteins in biotechnology. Curr. Opin. Biotech-in combination with RNA expression studies in mice identifiesnol. 3, 363–369.a large number of male germ cell-specific sequence tags. Biol.10. Ford, C. F., Souminen, I., and Glatz, C. E. (1991) Fusion tailsReprod. 52, 131–138.for the recovery and purification of recombinant proteins. Protein

27. Starborg, M., Brundell, E., and Hoog, C. (1992) Analysis of theExpression Purif. 2, 95–107.expression of a large number of novel genes isolated from mouse11. Stahl, S., Sjolander, A., Nygren, P.-A., Berzins, K., Perlmann,prepubertal testis. Mol. Reprod. Dev. 33, 243–251.P., and Uhlen, M. (1989) A dual expression system for the gener-

ation, analysis and purification of antibodies to a repeated se- 28. Hultman, T., Bergh, S., Moks, T., and Uhlen, M. (1991) Bidirec-tional solid-phase sequencing of in vitro-amplified plasmid DNA.quence of the Plasmodium falciparum antigen Pf155/RESA. J.

Immunol. Methods 124, 43–52. BioTechniques 10, 84–93.

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP

FUNCTIONAL cDNA ANALYSIS 457

29. Ansorge, W., Voss, H., Wiemann, S., Schwager, C., Sproat, B., 34. Forsberg, G., Baastrup, B., Rondahl, H., Holmgren, E., Pohl, G.,Hartmanis, M., and Lake, M. (1992) An evaluation of differentZimmermann, J., Stegemann, J., Erfle, H., Hewitt, N., and Rupp,

T. (1992) High-throughput automated DNA sequencing facility enzymatic cleavage methods for recombinant fusion proteins,applied on des(1-3)insulin-like growth factor I. J. Protein Chem.with fluorescent labels at the European Molecular Biology Labo-

ratory. Electrophoresis 13, 616–619. 11, 201–211.35. Tashiro, M., and Montelione, G. T. (1995) Structures of bacterial30. Nilsson, J., Larsson, M., Stahl, S., Nygren, P.-A., and Uhlen, M.

(1996) Multiple affinity domains for the detection, purification immunoglobulin-binding domains and their complexes with im-munoglobulins. Curr. Opin. Struct. Biol. 5, 471–481.and immobilization of recombinant proteins. J. Mol. Recognit.,

in press. 36. Kraulis, P. J., Jonasson, P., Nygren, P.-A., Uhlen, M., Jendeberg,L., Nilsson, B., and Kordel, J. (1996) The serum albumin binding31. Harlow, E., and Lane, D. (1988) ‘‘Antibodies: A Laboratory Man-

ual,’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, domain of streptococcal protein G is a three-helical bundle: Aheteronuclear NMR study. FEBS Lett. 378, 190–194.NY.

32. Stahl, S., Nygren, P.-A., Sjolander, A., and Uhlen, M. (1993) 37. Russel, L. D., Ettlin, R. A., Hakim, A. P. S., and Clegg, E. D.(1990) ‘‘Histological and Histopathological Evaluation of the Tes-Engineered bacterial receptors in immunology. Curr. Opin. Im-

munol. 5, 272–275. tis,’’ Cache River Press, Clearwater, FL.38. Clackson, T., and Wells, J. A. (1994) In vitro selection from pro-33. Nygren, P.-A., Stahl, S., and Uhlen, M. (1994) Engineering pro-

teins to facilitate bioprocessing. Trends Biotechnol. 12, 184–188. tein and peptide libraries. Trends Biotechnol. 12, 173–184.

/ 6q06$$0589 05-07-96 10:03:16 pepa AP-PEP