Bacterial expression of immunoglobulin VH proteins

0161-5890/90 $3.00 + 0.00 Pergamon Press plc

Molecular Immunology, Vol. 27, No. I, pp. 25-35, 1990 Printed in Great Britain.

BACTERIAL

EXPRESSION OF IMMUNOGLOBULIN PROTEINS*

KEIKO UDAKA, MING-MING CHUA, LI-HENG TONG, FRED KARUSH Department

of Microbiology,

V,

and SOL H. GOODGAL

School of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.

(First received I February 1989; accepred in revised .form 14 June 1989)

bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the V, protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-1-sulfonyl (Dns) group. This system has been applied to the expression of the V, genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity

Abstract-A

is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The V, proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield V,L molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x lo6 M -’ and 7 x lo9 Mm’ for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the V, protein itself as well as the modulation of affinity by site-directed mutagenesis.

INTRODUCTION

A fundamental genetic difference between the V, and V, domains of the immunoglobulin molecule is the utilization of the diversity (D) gene segment in the expression of the former together with the incorporation of non-coded nucleotides in the V,D and DJ, junctions. These features increase the potential diversity of the CDR3 of the V, protein by several orders of magnitude relative to that of the V, protein. It may be anticipated therefore that in the germ-line B cell response the broader range of structures of the antigen-binding region of the V, protein would result in a more appropriate complementary structure than could be exhibited by the V, protein. That is, the V, domain would be expected to provide the major portion of the contact amino acid residues engaged in stabilizing interactions with the epitope. This issue has received little experimental attention, partly because of experimental difficulties. It is, furthermore, of particular interest from the point of view of the evolution of immunoglobulin molecules. The interest arises from the commonly held presumption that the ancestral immunoglobulin molecule was a single-

*This work was supported by grant DCB-8417506 (F. K. and S. H. G.) fr&n the National Science Foundation, by Public Health Service Grant AI-09492 CF. K.) from the National Institute of Allergy and Inf&tious Diseases and by a grant from Cytogen Corporation (F. K.). Abbreviations: Dns, 5-dimethylaminonaphthalene-l-sulfonyl; PBS, 0.1 M NaCl + 0.02 M phosphate, pH 7.2; PMSF, phenylmethylsulfonylfluoride.

chain protein with an established recognition function. Indications of the primacy of the V, domain in the germ-line response are provided by early observations of the free energy of binding of the isolated H chain in rabbit IgG antibody specific for the p-azophenyl-p-lactoside (Utsumi and Karush, 1964) and, more recently, by nucleotide sequence studies of hybridomas secreting monoclonal IgM antibodies (Chau et al., 1987). We have undertaken the evaluation of the V, domain in the germ-line response from the point of view of its capacity to exhibit immune recognition. For this purpose we are employing cell lines secreting monoclonal antibodies specific for the 5-dimethylaminonaphthalene-1-sulfonyl (Dns) group. In order to produce sufficient amounts of defined protein we have turned to the use of a bacterial system for the expression of the rearranged V, gene. [See Udaka et al. (1987) for an early report.] In the present report we present a detailed description of the methodology developed for the high-level bacterial expression of the V, protein and its characterization. A number of attempts have been made to express immunoglobulin chains and fragments in Escherichiu coli. In the early efforts the products were produced largely in an insoluble form and yielded only low levels of activity after renaturation (Cabilly et al., 1984; Boss et al., 1984; Kurokawa et al., 1983). In the case of L chain expression it was shown that substantial secretion into the periplasmic space occurred if the cloned cDNA carried the eukaryotic signal peptide (Zemel-Dreasen and Zamir, 1984). More recently a major advance has been described in the feasibility

26

KEIKO LJDAKA et

of the bacterial expression of a functional F, fragment (Skerra and Phickthun, 1988) and a functional Fab fragment (Better et al., 1988). In both cases appropriate bacterial leader sequences were ligated to the coding sequences. These signal sequences provided for the transport of the individual chains into the periplasm where, apparently, the cleavage, folding and assembly required for a functional product occurred. Substantial quantities of these products could be obtained from the periplasmic space and/or the culture supernatant. Most recently it has been demonstrated by two groups (Huston et al., 1988; Bird ef al., 1988) that a single chain F, protein can be expressed in E. coli which exhibits the same reactivity as the corresponding monoclonal antibody. These findings and others describing the expression of eukaryotic single-chain proteins in E. coli (Briggs and Gierasch, 1986; Talmadge et al., 1980) coupled with the results of our investigation demonstrate the utility and feasibility of the high-level expression of immunoglobulin proteins in bacterial systems. MATERlALS

Hybridoma

AND METHODS

cell lines

Two hybridomas were used to provide a source of mRNA. They produce anti-Dns antibodies, one of which, DBl-314.3, is an IgM with a relatively low intrinsic binding constant for N-t-Dns-lysine (KA = 2.6 x lo6 Mm’). The other, DB3-226.2, is an IgGl with high affinity (KA = 7.7 x lo9 Mm’). The production of these hybridomas and the determination of the binding constants have been previously described (Fan and Karush, 1984). Both antibodies contain a lambda 1 light chain and the nucleotide sequences of the variable regions of DBl-314.3 have been established (Chua et al., 1987). The nucleotide sequence of the V, domain of DB3-226.2 has also been reported (Burns et al., 1990).

al.

Isolation qf mRNA The preparation of mRNA described (Chua et al., 1987). Synthesis

has been previously

of ds-cDNA

The first strand cDNA of the V, regions was synthesized by the primer extension method as described by Reilly et al. (1984). For DBl-314.3 the oligonucleotide primer Q-1 7 (5’ dGAAGACATTTGGGAAGG 3’) is complementary to bases 11 to 27 of the CHl region of the p chain. For DB3-226.2 the primer Q-15 (5’ dCCGGTCACCTATCTG 3’) is complementary to bases 22 to 36 of the CHl region of the y chain. Ten to thirty micrograms of poly-A’-mRNA were annealed with phosphorylated primer end-labeled with [1;-“P]ATP. Reverse transcription was effected by the addition of 4 U/pg mRNA of AMV reverse transcriptase (Life Sciences, St Petersburg, FL). Second-strand cDNA was synthesized by the method of Gubler and Hoffman (1983) with the use of RNase H and E. coli DNA polymerase I and blunt ending was effected with T4 DNA polymerase. Full-length cDNA was detected as the main band, approximately 500 bp, on the preparative 6% PAGE gel. The band was cut out and the cDNA electroeluted with the ISCO electroconcentrator (ISCO, Lincoln, NE). The isolated product was used for cloning. Cloning of cDNA

E. coli strains HMS174 (F-, hsdR, recA, rif’), ED8739 (F-, metB, hsdS, supE, supF) and BL21 (DE3) (F-, hsdS, gal, imm”, Anin5) were kindly provided by Dr William F. Studier (Brookhaven National Laboratory, Upton, NY). Doctor Studier also provided the bacteriophage lambda derivative CE6 (imm lambdac 1857, ind 1, Sam7) and the plasmid expression vectors pAR3038, 3039 and 3040.

The cloning of the Vn-containing cDNA was carried out by blunt end ligation into the SmaI site of pUC18 and transformation of E. coli JM107 (Yanisch-Perron et al., 1985) by the method of Hanahan (1983). Recombinant clones were identified as b-galactosidase-negative colonies by reaction with the fluorogenic substrate 4-methyl-umbelliferylb-D-galactoside (Sigma, St Louis, MO) as described by Youngman (1985). Clones containing full-length cDNA of the V, region including a leader sequence and a 5’ untranslated region were selected by the size of the insert and the restriction pattern (PstI, ScaI) predicted from the nucleotide sequence previously determined (Chua et al., 1987). Confirmation of the cloned V, gene was obtained by nucleotide sequencing using the method of Maxam and Gilbert (1980).

Oligonucleotides,

Transfer qf V, genes into expression

E. coli strains,

bacteriophage,

enzymes

lambda and plasmids

and chemicals

Oligonucleotides were provided by the DNA Synthesis Service (Department of Chemistry, University of Pennsylvania). Restriction enzymes and other enzymes were purchased from New England Biolabs (Beverly, MA), BRL (Gaithersburg, MD), Pharmacia Inc. (Piscataway, NJ) or Boehringer Mannheim (Indianapolis, IN). Radioactive chemicals were purchased from Amersham Corp. (Arlington Heights, IL).

oectors

A series of plasmid vectors pAR3038, 3039 and 3040 developed by Rosenberg et a/. (1987) were utilized as expression vehicles. These vectors carry a T7 expression cassette consisting of a T7 promoter and an initiation signal in the 5’ half, termination signals for transcription and translation in the 3’ half and a unique BamHI site in between for insertion of exogenous genes. These vectors are adjusted for different reading frames.

27

Bacterial expression of immunog~obulin Vu proteins

Cloned V, fragments were excised from pUC18 by digestion with EcoRI and BamHI and then inserted into pAR3038 for DBl-314.3 and into PAR3039 for DB3-226.2 following the addition of an EcoRIBamHI adaptor (Boehringer-Mannheim, Indianapolis, IN) as shown in Fig. 1. These constructs are designated pAR3 14 and pAR226, respectively. E. coli HMS174 was selected as the host cell for the expression vectors. Expressive of Vx genes in E. coli The expression of the Vu gene was based on the induction in the HMS174 host cell of T7 RNA polymerase by infection with the lambda phage derivative CE6 which carries the T7 RNA polymerase gene (Rosenberg et al., 1987). The CE6 phage was prepared by injection of the suppressor host ED8739. Since the CE6 phage carries the Sam7 mutation the

1 liai BarnHI

5’uT

+I

EcoRl- BamHl adaptor

EC&I

Fig. 1. Transfer of the cDNAs of Vu regions from DBl314.3 and DB3-226.2 cloned in pUCl8 into the T7 expression vectors pAR3038 and pAR3039. The Vu regions were excised from the vector pUCl8 and inserted into the BamHI site of the T7 expression cassette of pAR3038 and PAR3039 after the addition of the EcoRI-BamHI adaptor. The plasmid containing the Vu of DBl-314.3 is termed pAR314 and the one with the V,, of DB3-226.2 is labeled pAR226. P, sequence containing the T7 promoter and initiation signals for translation; S’UT, L, S-untranslated region and leader sequence of the IgH chain gene; C, 1, C, I peptide originating from the ohgonucleotide primer; T, termination signals for transcription and translation,

host cell was lysed, allowing the precipitation of the released phage particles with polyethylene glycol (Maniatis et at., 1982). The system used for the expression of the V, gene consisted of the combination of CE6 (Sum7) and HMS174 (non-suppressor), chosen to avoid lysis of the host cell after infection with the phage. The quantity of the V, gene product relative to the total bacterial protein was estimated by scanning the SDS-PAGE gel of the whole cell lysate stained with Coomassie brilliant blue with the Soft Laser Scanning Densitometer (Biomed Instrument, Inc., marketed by LKB Instruments, Inc., Gaithersburg, MD). N-terminus trimming

of V, genes

The strategy for trimming the nucleotide sequence 5’ to the authentic Vu sequence was to eliminate the stretch of nucIeotides between the NheI site immediately following the ATG codon derived from the expression vector and a restriction site in the leader sequence close to the N-terminus of the Vu gene. The procedure is illustrated in Fig. 2 and was carried out after transfer of the expression cassette to pUCl8. In the case of DBl-314.3 a Sal1 restriction site was created at nucleotide -8 by changing C at -6 to G by site-directed mutagenesis with the mutagenic oligonucleotide 5’ dCTGGGAGTCGACACCTG 3’ following the method of plasmid heteroduplex formation (Inouye and Inouye, 1987). In the case of DB3-226.2 the AccI site at nucleotide -7 of the original sequence was used for trimming. After elimination of the intervening sequence each staggered end was blunt-ended for ligation by filling in with T4 DNA polymerase (Figs 2 and 3b). This construct was labeled pUE226. Three variants of DBI-314.3 were constructed, with five codons [pUE314(5)], four codons [pUE314(4)] or two codons [pUE314(2)] preceding the first codon (CAG) of the V, gene (Fig. 3). For pUE314(5) the NheI end was filled in and the Sal1 end partially filled in followed by trimming with SI nuclease. For pUE314(4) the Sal1 overhang was trimmed with SI nuclease followed by blunt end ligation to the filled-in NheI end. The construction of pUE314(2) started with pUE314(4) in which the NheI site was eliminated and a NarI site created by changing T at -3 to G by site-directed mu~genesis using a uracil-containing Ml3 recombinant as a template (Kunkel et al., 1987) (See Fig. 3a). The oligonucleotide used was 5’ dGACCTGGGCGCCATATG 3’. Nucleotides -3 (G) and -4 (C) were eliminated by digestion with NarI and SI nuclease. Confirmation of the sequences described above was provided either by analysis with restriction enzymes or sequencing by the dideoxy method (Sanger et al., 1977) with the use of the Sequenase kit (Tabor and Richardson, 1987) (U.S. Biochemicals, Cleveland, OH).

28

KEIKO UDAKA et al. -153

I

+1

-57

-93

5’UT

I

Leader

end of mRNA

EcoRV

3'

.L

DW. 314.3

I

AccI 1

Fig. 2. T7 expression cassettes containing the cDNAs of the V, regions in pAR226 and pAR314. The entire expression cassette can be excised by digestion with BglII and EcoRV and transferred to other vectors. The sequences derived from the vectors are indicated by a straight line or boxes for the T7 promoter and Shine-Dalgarno sequence (SD). The cDNA sequences are shown as bars. The 153 base pair sequence 3’ of the initiation codon ATG and 5’ of the N-terminus of V,, most of which was removed by N-terminus trimming, is illustrated. The restriction sites involved in N-terminus trimming are enlarged for detail. The nucleotide targeted for site-directed mutagenesis to create a SaII site in DBI-314.3 is indicated by a black circle. Translation termination codons for different reading frames in the region 3’ of the V, insert are boxed. The AccI site used for trimming the 5’ sequence of DB3-226.2 is indicated.

C-terminus

trimming

of’ V, genes

In the 3’ half of the expression cassette there are multiple termination codons for translation adjusted to different reading frames. In the case of pUE3 14(5), (4) and (2), derived from pAR3038, the proximal termination signal is functional, leading to an additional five amino acids beyond the Cp peptide specified by the corresponding polynucleotide primer. On the other hand pUE226, derived from pAR3039, uses a distal termination signal and gives rise to 22 additional amino acid residues beyond the Cy peptide specified by the corresponding primer. The use of BamHI and Sl nuclease led to the removal of eight codons from the Cy sequence and added 15 codons from the expression cassette. This construct was designated pUF226 and the expressed V, product contained 144 residues.

Preparation

qf mono&ma1

antibody to Cy peptide

In order to identify the expressed V,, product associated with DB3-226.2 by Western blotting, monoclonal antibodies were prepared against a peptide corresponding to the N-terminal 12-mer of the CHI region of the ~1 heavy chain (Ala-Lys-Thr-Thr-Pro-Pro-Ser-ValTyrPro-Leu Ala), designated the Cy peptide. Two derivatives of this peptide were synthesized by the solid phase method (Marglin and Merrifield, 1970; Stewart and Young, 1984), one with Cys at the N-terminus (Cys-Cy peptide) and the other with N-(2,4-dinitrophenyl)-cysteic-Cys at the N-terminus (DNP-cysteicCys-Cy peptide). For immunization the Cys-C; peptide was conjugated to tetanus toxoid (kindly provided by Wyeth Laboratories, Marietta, PA) with the use of the bifunctional reagent SPDP, ,“I’-

29

Bacterial expression of immunoglobulin V, proteins

a

was

reactive

with

the Vu protein

by the ELBA

assay.

t+ “H

Preparation of antiserum to the Cp peptide

pUE 314(L)

A

m

*f

S

S

A+?

Tc?

r

“H Q

?AG

Polyclonal rabbit antiserum to a peptide corresponding to the N-terminal sequence (13-mer) of the Q 1 domain (Q peptide) was prepared as previously described (Ghose and Karush, 1985).

V ?~TC

Western blotting

NheI

“H I+ I( QCTC? ‘dAG $TC

pUE 311(2) nATG G 7

NQrI vr c&c

Q

b

jZ?J

r&i

v

%AG +&c

pUE 226 InTc(

;?$~f&T+

T:ii

r

“H

+b”,

%r,

Fig. 3. Nucleotide sequences of V, genes from DBl-314.3 and DB3-226.2 at the translation initiation site after Nterminus trimming of pAR314 and pAR226. N-terminus trimming was started from the expression plasmids pAR3 14 and pAR226 that contain V, genes from DBl-314.3 and DB3-226.2, respectively. Nucleotides preceding the N-terminus of V, genes were removed by restriction digestion as described in Materials and Methods. Construction of the expression vectors with trimmed V, was completed by ligation of the NheI end, following the initiation codon ATG, with the SaII end for pAR314 or the AccI end for pAR226. (a) Three variations of pAR314 were constructed as described in Materials and Methods. pUE314(5) and (4) were the result of trimming different numbers of nucleotides from the staggered restriction ends. pUE314(2) was derived from pUE314(4). After removal of the NheI site, a NarI site was created by sitedirected mutagenesis at the point marked by a black circle. Removal of the NarI site resulted in the completed construct. (b) The plasmid from pAR226 was modified as described in Materials and Methods and designated pUE226. The corresponding amino acids are shown by single letters above the codons. The initiation codon ATG is boxed. The N-terminus of the V, gene is indicated by arrows.

succinimidyl-3-(2_pyridinedithio)-propionate (Pierce Chemical Co., Rockford, IL) as described (Karush and Tang, 1988). The second derivative, DNPcysteic-Cys-Cy, was reacted with iodoacetate to block the SH groups and purified by HPLC. Amino acid anlaysis yielded the theoretical composition. It was used as the adsorbed antigen in the ELISA assay for anti-Q peptide antibodies. The immunization and fusion procedures were similar to those described previously (Mandal and Karush, 1981). Among three fusions approximately 80 hybridomas showed reactivity with

the Cy peptide

of which

only

one (Cy 2-236)

Samples were analyzed by SDS gel electrophoresis in 12.5% polyacrylamide slab gel using the discontinuous buffer system described by Laemmli (1970) with the addition of 120 mM NaCl in the separating gel for better resolution of proteins of low mol. wt as described by Coleman et al. (1983). After transfer to nitrocellulose paper and blocking (Szewczyk and Kozloff, 1985) the hybridoma tissue culture supernatant containing Cy2-236 antibody or rabbit antiCp peptide serum was added. After removal of unreacted antibody peroxidase-labeled goat antimouse IgG or anti-rabbit IgG was added and the location of the Vu product visualized with the color reagent 4-chloro-l-naphthol (Sigma). Isolation of the VH product

The following procedure was developed to separate the V, product from bacterial proteins. The bulk of the bacterial proteins was extracted according to a modification of the method of Kurokawa et al. (1983). Cells were collected from 80 ml of culture and resuspended in 6ml of 25mM Tris-HCI, pH 8.0, containing 1 mM PMSF, 10% sucrose, 0.01 M EDTA, 0.2M NaCl, 0.1% NP-40, 0.2% Triton X100 and 0.5 mg/ml lysozyme. After incubation on ice for 30 min and at 37°C for 3 min, the suspension was sonicated on ice intermittently for 2 min and centrifuged at 12,000g for 20 min. Almost all of the V, protein remained in the pellet. The pellet was suspended in 1 ml of 25 mM Tri-HCl, pH 8.0, with 10% sucrose. The mixture was treated with 5pg of DNase, 10 pg of RNase and MgCl, (10 mM). After incubation on ice for 30 min 0.5 A4 EDTA (0.15 ml) was added and incubation continued for a further 10 min. Following centrifugation in the microfuge for 10 min the pellet was washed with saline and then incubated at 37°C for 2 hr in 1 ml of a solution containing 8 M urea, 20mM Tri-HCl, pH 8.0, 10 mM EDTA and 1 mM PMSF. After centrifugation in the microfuge for 10min the supernatant contained most of the Vu product. Isolation of the Vn product was achieved by gel filtration through a Sephacryl S-200 column in 8 M urea, 20 mM Tris-HCI, pH 8.0, and 10mM EDTA. Preparation of L chain

Anti-Dns hybridoma antibodies prepared from ascites as previously described (Fan and Karush, 1984) were adsorbed on a Dns-Lys-Affi-Gel 10 column (Fan and Karush, 1984) and extensively

KEIKO UDAKA et

30

washed with PBS to remove non-antibody protein. Due to the high affinity of DB3-226.2 (IgGl) for the Dns-lysyl group and the difficulty, therefore, of its elution, the adsorbed antibody was reduced in situ with 10 mM DTT and alkylated with 25 mM iodoacetate. Elution was effected either with 8 M urea or 1 N propionic acid. The dissociated antibody was fractionated on Sephadex-200 column (1.5 x 90 cm) in I A4 propionic acid, 4.5 M urea and 50 mM NaCl. The theoretical amount of L chain was obtained. For preparation of the L chain of DBl-314.3 the antibody was purified on a Dns-Lys-Al&Gel 10 column using 0.5 M naphthylacetate at pH 7.0 for elution. Following removal of the naphthylacetate by dialysis the antibody solution was reduced and alkylated as described above and fractionated as noted. The yield of L chain was approximately 70% of theoretical. Recombination

of V,, protein and L chain

The association of the V, protein and L chain was carried out by the method of Hamel et al. (1987). Equal molar amounts of homologous Vu and L chain were added together in 1 M propionic acid, 4.5 M

al.

urea and 50 mM NaCI. Association occurred during dialysis of the reaction mixture, first against water and then followed by 20 mM sodium acetate buffer, pH 5.5. Finally, IO-fold concentrated PBS, sufficient to yield 1 x PBS was added gradually over a 4-hr period giving rise usually to a turbid solution. The resulting supernatant after centrifugation was the V,L preparation used for affinity measurements. These preparations contained excess L chains. Afinity measurements V, L recombinants

for binding of Dns-lysine

by

The binding constants for complex formation between V,L and Dns-lysine were determined by the method of resonance energy transfer described in detail elsewhere (Karush and Tang, 1988). The measurements were made with the above V, L preparations without isolation of purified V,L protein since L chain itself showed no measurable binding under the experimental conditions employed. It was necessary, therefore, to determine the actual concentration of the binding species in the V, L preparation. A maximum value was ascertained by linear extrapolation of the initial slope of the binding curve to its

Fig. 4. SDS-PAGE (left) and Western blot (right) analyses Panel (a), total cellular protein synthesized with pAR3039 (lane 1, left and lane 1, right). Cellular protein of HMS174, after infection by CE6, transformed with the plasmid pUE226 (lane 2, left and lane 2, right). Total cellular protein was obtained by suspending the cell pellet in 0.1 M Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% sucrose and heating at 100°C for 5 min. After centrifugation the supernatant was analyzed. The tissue culture supernatant from an anti-Q peptide hybridoma was used to detect the Vu product in the Western blot analysis. Pane1 (b) lanes 1 (left and right) used the product generated by pUE226. The sonicated cell pellet was extracted with 8 M urea, 20 mM Tris-HCI, pH 8.0, 10 mM EDTA, 1 mM PMSF. The sample was loaded on the gel without boiling in SDS and mercaptoethanol. Lanes 2 (left and right) used the second peak from the chromatographic fractionation in 8 M urea of the 8 M urea extract of HMS 174 transformed with pUE226. Lanes 3 (left and right) used the recombined product prepared from the V, of pUE226 and the 226.2 L chain.

Bacterial expression of immunoglobulin V, proteins intersection with the final linear portion of the binding curve representing the signal of unbound ligand. The final value was obtained by reiterative caiculation to yield a heterogeneity index that differed from unity by not more than 0.1% (Karush and Tang, 1988). RESULTS

Full-length cDNA (approximately 500 base pairs) was obtained with both the Cp and Cy primers from the mRNA of DBl-314.3 and DB3-226.2, respectively. In addition to the rearranged Vu segment itself, the cloned sequences contained a leader sequence of 57 base pairs and a 5 untranslated region of 36 base pairs for 314.3 and 37 bp for 226.2. It was also found that the first three base pairs from the 5’ end of the Q-17 primer and one base pair from the Cy -15 primer had been deleted by exonuclease activity during the cloning procedure. Expression of I’, genes in E. coli The expression of the Vu genes in E. coli was successively carried out using the system developed by Rosenberg et al. (1987). In this system the T7 RNA polymerase required for Vu expression is provided through infection with the lambda phage derivative CE6 which carries the polymetase gene. The level of expression is maximum and reproducible

a

31

if the multiplicity of infection is maintained between 5-1O:l (CE6: E. cob) and vigorous aeration is provided. For the expressed product derived from DB3-226.2 (pUF226) the fully trimmed Vu insert was used for expression. This product is seen as a new band in SDS-PAGE of mol. wt of about 15,500 (Fig. 4) and constitutes as much as 10% of total bacterial protein. The product was further characterized by Western blotting using a monoclonal antiCy peptide antibody. It is evident (Fig. 4) that the 226 product is clearly identified as a protein reactive with the monoclonal antibody. The product expressed by pAR314, reflecting the largest translatable sequence, was detected on SDS-PAGE as a new band of mol. wt of approx. 19,000 (Fig. 5) and constituted about 3% .of the total bacterial protein. The 314 product can also be identified (Fig. 5) using the polyclonal antiserum, although this serum gives rise to bands due to the presence of anti-E. coli antibodies, as is observed also with non-immune rabbit serum. An amino acid analysis was also carried out with a purified 226 product prior to the C-terminal trimming (pUE226). It therefore contained 22 amino acid residues beyond the C peptide corresponding to the original primer employed. Thus, in addition to the 120 residues for the Vn itself there were five additional ones at the ~-te~inus and 34 additional ones at the C-terminus. The calculated amino acid composition for this product was in satisfactory agreement with the experimental result.

b

Fig. 5. SDS-PAGE and Western blot analysis of lysates of E. coli cells with different plasmid constructs of the V, gene from DBl-314.3. After induction for the expression of the V, gene the lysates were prepared by boiling in the presence of 1% SDS and 2-mercapt~thanol. Each lane was Ioaded with the extract of 4 x 10’ cells in a 12.5% polyacryIamide gel. The gel was stained with Coomassie brilliant blue (a) or subjected to Western blot analysis (b) with rabbit poiyclonal anti+ peptide antiserum prepared as described in Materials and Methods. The bands corresponding to the Vu protein are indicated by arrows. Lane 1 (mol. wt standards), IgM heavy chain (H), mol. wt 70,000; bovine serum albumin (BSA), mol. wt 68,000; Fd fragment of IgM, mol. wt 25,000; L chain, mol. wt 23,000; egg white lysozyme (Lys Z), mol. wt 14,500. Lane 2, HM174 carrying pAR3038 without a V, insert. Lane 3, pAR314. Lane 4, pUE314(5). Lane 5, pUE314(4). Lane 6, pUE314(2).

32

KEIKO UVAKA

et al.

PuriJication of’ the V,{ product The Vn product isolated by the procedure described above exhibits minimal solubility in nondenaturing solvents. Its substantial recovery was achieved only by solubilization in 8 M urea. In this solvent effective separation of the V, protein from bacterial protein was accomplished by gel filtration through Sephacryl S-200. In Fig. 6 the second peak represents largely the V, protein in a non-covalent dimeric form. Its identification was provided by SDS-PAGE (Fig. 4) and by Western blotting. Similar behavior was observed for both the 314 and 226 products. For the 226 product the content of protein in the V, peak was I mg per 100 ml of cell culture. For the 314(5) product it was 0.3 mg per 100 ml of culture. Recombination

of’ VII and L proteins

The formation of the V,L species was inferred from the increased solubility of the V, protein as a result of the presence of the L chain in the recombination procedure. More significant is the acquisition of binding activity (see below) not exhibited by the individual components. It is estimated from the recovery of soluble protein that only between 10% and 20% of the initial V, protein was retained in the form of the V, L molecule. SDS-PAGE analysis and Western blotting confirm the presence of both components in the recombinant preparation (Fig. 4). Binding qf Da-l~~sine

by V,!L

Fluorescence titrations were carried out as described with one VnL preparation derived from DB3-226.2 and one from DBI-314.3. The V, products of constructs pUF226 and pUE314(5) were used for these V,, preparations. The titration curves are shown in Fig. 7a and 7b, respectively. Analysis of the binding data was done as previously described (Fan and Karush, 1984) and involved

Fig. 6. Gel filtration of 8 M urea extract of Vn-containing sonicated pellet on Sephacryl S-200 (1.5cm x 90 cm) in 8 M urea, 20 mM Tris-HCl. pH 8, 10 mM EDTA. The arrows indicate the elution position of marker proteins: a, rabbit IgG; b, alkylated equine L chain; c, lysozyme. The second peak contained only V, protein by SDSPAGE analysis.

V, L (226) 50-

I 2 ti

30-

IO-

0 70

5

1b)

I

IO

15

I

20

Minutes

502 8 I b 2 30-

IO-

o!o

5

IO

15

Minutes

Fig. 7. Fluorescence titration curves with Dns-lysine. The ligand (200~1) was added continuously to 2.0 ml of the antibody solution at 25’C over a period of 20 min. (a) The V,L was prepared with the V, derived from the pUF226 plasmid. The concentration of ligand used for the titration was 5.0 x 1Om6M. The calculated antibody site concentration was 8.8 x lo-” M. (b) The VnL was prepared with the V, urevared from the pUE3 14(S) plasmid. The concenwas tration L 0; the ligand . used for. the titration 1.00 x 10e4 M. The calculated antibody site concentration was 4.55 x 10-‘M.

reiterative calculation. In this procedure homogeneity of the V,L population was assumed and the site concentration was used as a variable parameter. The results of the calculations indicated that the active species constituted between 10% and 30% of the total protein. In the case of V,,L (226) the resulting intrinsic association constant (KA) was 7x 109M-‘comparedtoavalueof7x 109M-‘for the parent antibody DB3-226.2 (Fan and Karush, 1984). This agreement is rather fortuitous since the uncertainty in site concentration probably generates an uncertainty in KA of a factor of two. This uncertainty is probably involved in the difference between a KA of 7 x IOhM-’ for VnL(314) compared to 2.6 x IO’M ’ for DB3-314.3. For both cases it can reasonably be inferred that the bacterial V, product in the form of the VnL protein is equivalent in its specific reactivity to that exhibited by the V, domain in the parent IgG or IgM molecule.

Bacterial expression of immunoglobulin V, proteins DISCUSSION

This study has demonstrated the feasibility of the high-level expression of the V, protein in E. coli with acquisition of the appropriate tertiary structure. This structure is acquired by a substantial fraction of the V, product following its extraction with 8 M urea and recombination with L chain. In coordination with the homologous L chain, this structure provides a functionally equivalent binding region to that contained in the parental monoclonal antibody. This equivalence is particularly striking and significant for the DB3-226.2 system because of the high intrinsic association constant involved. It suggests that expression in E. coli need not degrade high-affinity systems whether expression provides a V, protein, an Fv product or the Fab fragment. That expression in E. coli does not modify affinity in a low-affinity system (approx. 1 x 10’ n/r-‘) has been demonstrated for the anti-phosphorylcholine F, product (Skerra and Pliickthun, 1988). In this connection our findings support those of others (Inbar et al., 1972; Skerra and Pliickthun, 1988) to the effect that the CHl domain is not required for the structural integrity of the V, region. There are a number of parameters that appear to affect the level of expression of exogenous genes in prokaryotes, including the rate and extent of transcription. the stability of mRNA, the efficiency of translation and the stability of the gene product. Theoretically the T7 promoter system that was used to express V, genes in E. coli should have yielded high-level expression. However, it was found that sequences 3’ of the T7 promoter and 5’ of the amino terminus greatly affected the expression of the V, gene. The V, genes that were initially tested for the expression in pAR314 and pAR226 contained approximately 150 base pairs 5’ to the N-terminus of the bonajde Vn protein. The level of expression of the V, of pAR314 was estimated as 3% of the total bacterial protein whereas that of pAR226 was 1% or less. After removal of most of the 150 base pairs, a significant alteration was observed in the level of V, expressions. The plasmid pUE314(4) produced a truncated V, with four amino acids 5’ to the start codon of the V, but with a yield of only l-2%, whereas the truncated V,, from pUE226 that contained five amino acids at the 5’ terminus increased the yield of V, to at least 10% with and without C-terminal trimming. Inasmuch as pUE226 with five amino acids increased the yield of V, an attempt was made to modify the amino acid sequence at the 5’ end of the V, 314. The Vhs with two amino acids pUE314(2) and five amino acids pUE314(5) at the 5’ end were expressed and found to yield less than 1% V, product for 314(2) and 10% for 314(5). These data suggest that the sequence 5’ to the first amino acid of the V, protein strongly influences its level of expression. A sequence analysis of the pAR226 that contained 150 nucleotides 5’ of the first amino acid of

33

V, (226) revealed an eight base pair palindrome that preceded the leader region of the Vn gene and could be responsible for a low efficiency of translation of the V, protein. The palindrome was not present in the 150 base pairs 5’ to V, of pAR314. There is a report (Sung et al., 1986) that efficient translation requires the presence of an “open” AUG and appropriate secondary structure around the ShineDelgarno sequence (the ribosomal binding site). The V, gene of pUE314(4) contains the sequence 5’CTCC3’ that could interact with the ShineDelgarno sequence and interfere with translation. However removal of the 5’CTCC3’ sequence in the construct pUE314(2) actually lowered the level of Vn expression to 1% or less of total bacterial protein. Furthermore, pUE314(5) with a 10% level of expression contains the sequence CTCC. The presence of five extra codons on the N-terminus of the Vn314(5) is associated with high-level expression, but it was not clear whether the high-level expression was due to the composition of the five amino acids at the N-terminus of the gene product or to some function related to nucleotide spacing. The most serious limitation of our system at the present time is the low solubility of the V, product. It is anticipated that the difficulty will be overcome either by a genetic approach or by chemical modification. The first alternative would involve site-directed mutagenesis such that one or more non-essential hydrophobic residues would be converted to charged residues, e.g. arginine. For example, an attractive choice for this purpose is the invariant tryptophan residue in the Jn segment since this residue normally makes extensive contact with hydrophobic residues of the V, region (Amzel and Poljak, 1979). In the absence of the V, region it probably contributes substantially to the insolubility of the V, product. With respect to chemical modification polyalanylation with N-carboxyl-D,L-alanine anhydride has been used for protein solubilization without affecting binding activity (Karush and Sela, 1967). The availability of a soluble V, protein would be useful for several purposes. It would make it possible to evaluate the affinity potential of the single-chain protein. Such an analysis would have a bearing on the issue of the existence of an ancestral single-chain recognition unit whose duplication and divergence underlie the evolution of contemporary immunoglobulin molecules. In addition, a single-chain recognition molecule could be readily subjected to site-directed mutagenesis based on molecular modeling with a view to modulating affinity (Roberts et al., 1987). Finally, a single-chain recognition molecule, engineered for high affinity and chemical attachment sites at its C-terminus, should be particularly advantageous for in viao targeting. It might, for example, avoid much of the non-specific localization, particularly in the liver, commonly observed with current antibody-based delivery systems for diagnostic and therapeutic use (Rodwell et al., 1986).

34

KEIKO UDAKA et al.

Acknowledgemenfs-We are greatly indebted to Sally Karush for the meticulous preparation of the manuscript and to Marilyn Mitchell for assistance in nucleotide sequencing.

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