Gene, 85 (1989) 553-557
`Elsevier
`
`GENE 03344
`
`553
`
`Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab frag(cid:173)
`ments in Escherichia coli
`
`(Carcinoembryonic antigen; inclusion bodies; recombinant antibody; recombinant DNA)
`
`Shmuel Cabilly
`
`Department of Biology, Technion -
`
`Israel Institute of Technology, Technion City, Haifa 32000 (Israel)
`
`Received by A.D. Riggs: May 8 1989
`Revised: July 17 1989
`Accepted: August 3 1989
`
`SUMMARY
`
`Expression in Escherichia coli of recombinant genes coding for the K-chain and the Fd fragment of an
`antibody directed against carcinoembryonic antigen gives rise to Fab dimers. These Fab fragments possess
`antibody activity, as demonstrated by enzyme-linked immunosorbent assay as well as by ligand competition
`assay. Effective production of soluble Fab in Escherichia coli was achieved by a decrease in the growth
`temperature. Following a one step purification by anion exchange chromatography, the bacterially-produced
`Fab retains its activity at 4 o C for at least two months. The relatively simple methodology described in this study
`should be useful for the design and production of antibodies in bacteria.
`
`INTRODUCTION
`
`Genetic engineering provides a means for the
`manipulation of antibody (Ab) structure and specifi(cid:173)
`city. Several forms of recombinant Abs have been
`constructed:
`human/mouse
`chimeric
`Abs
`(Boulianne et al., 1984), enzyme-linked Fab frag(cid:173)
`ments (Neuberger et al., 1984), Fv fragments (Skerra
`and Fluckthun, 1988) and 'single chain antibodies'
`(Bird et al., 1988; Huston et al., 1988). Recombinant
`
`Ab genes have been expressed in Escherichia coli
`(Boss et al., 1984; Cabilly et al., 1984), in yeast
`(Horwitz et al., 1988) and in mammalian lymphoid
`cells (Neuberger et al., 1984). Even though lympho(cid:173)
`cytes are naturally adapted for Ab production, bacte(cid:173)
`ria have potential advantages in terms of recom(cid:173)
`binant gene manipulations, growth, handling and
`economy. However,
`immunoglobulin (Ig) poly(cid:173)
`peptides precipitate in the bacterial cytoplasm as
`inclusion bodies, and solubilization of these bodies
`
`Co"espondence to: Dr. S. Cabilly, Department of Chemical
`Immunology, Weizmann Institute of Science, 76100 Rehovot
`(Israel) Tel. (08)482111; Fax (08)466966.
`
`Abbreviations: Ab, antibody; Ag, antigen; B-Fab, bacterially(cid:173)
`produced Fab; BSA, bovine serum albumin; CEA, carcino(cid:173)
`embryonic antigen; DE-52, diethylaminoethyl-cellulose; EIA,
`enzyme immunoassay; Fab, antigen-binding fragment; Fd, trun-
`
`cated heavy chain; Fd', gene encoding Fd; lg, immunoglobulin;
`k, gene encoding K chain; Fv, variable region fragment; PBS(cid:173)
`NP40, 0.15 M Na0/0.01 M Na ·phosphate pH 7.2/0.05% (v/v)
`Nonidet-P40; PMSF, phenylmethylsulfonyl fluoride; RBS, ribo(cid:173)
`some-binding site; PAGE, polyacrylamide gel electrophoresis;
`SDS, sodium dodecyl sulfate; TS buffer, 10 mM Tris-HO
`pH 8/150 mM Na0/0.05% (v/v) Nonidet-P40; v region, variable
`region.
`
`0378-1119/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
`
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`554
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`has required the use of chaotrophic agents followed
`by renaturation procedures. These procedures give
`very poor yields of Ab activity (Boss et al., 1984;
`Cabilly et al., 1984 ). Recently it was reported that the
`introduction of a bacterial signal sequence into
`Ig-encoding genes promotes the secretion of active
`Fab or Fv fragments (Better et al., 1988; Skerra and
`Fluckthun, 1988).
`I here report that in E. coli cells growing at 21 oc,
`a single expression plasmid coding for 1e-chains and
`truncated heavy chains (Fd fragments) gives rise to
`high yields of functional Fab fragments.
`
`EXPERIMENTAL AND DISCUSSION
`
`(a) Expression of soluble Fab-fragments in the
`Escherichia coli cytoplasm
`
`In an earlier study (Cabilly et al., 1984), we con(cid:173)
`structed expression vectors for ~e-chains and Fd
`fragments. The signal peptide coding sequence of
`each of the Ig genes was replaced by an ATG start
`codon and, as a result, Ig products accumulated in
`the E. coli cytoplasm. To obtain co-expression of
`both 1e-chain and Fd fragments, E. coli cells were
`co-transformed by two expression plasmids each
`carrying a different selection marker. However, no
`control could be established over the relative amount
`of each plasmid within individual cells. For this
`reason, and due to the possibility that translation of
`the two polypeptides from the same mRN A might
`improve their chance for proper assembly, a single
`plasmid harboring both genes in one transcription
`unit was constructed. Details of the plasmid, marked
`pFabCEA, are presented in Fig. 1.
`Transformants carrying pFabCEA expressed
`both K-chains and Fd fragments (Fig. 2A; note that
`the relative intensities of the two bands might be
`affected by the differential sensitivity of the lg poly(cid:173)
`peptides to detection by rabbit anti-mouse IgG;
`Cabilly et al., 1984 ). The amounts of soluble Ig poly(cid:173)
`peptides in E. coli transformants growing at three
`different temperatures (37, 30 and 21 oq were com(cid:173)
`pared. Relative band density ofimmunoblots (Fig. 3,
`lanes 4-6) show that the amount of soluble lg poly(cid:173)
`peptides is about ten-fold higher in cells growing at
`21 or 30°C as compared to cells grown at 37°C. In
`
`Fig. 1. Structure of the "and Fd expression vector pFabCEA.
`Two previously described expression plasmids (Cabilly et al.,
`1984), pkCEAtrp 207-1* and pyFd'CEAtrp 207-1* were used to
`construct a single transcription unit gene encoding the K-chain
`(k) and the Fd-fragment (Fd'). A Hpal-Sa/I fragment which
`contains the Fd gene was isolated from the yFd'CEA 207-1*
`plasmid and inserted into the K-chain expression vector
`pkCEAtrp 207-1*. The latter vector was linearized with EcoRI,
`followed by filling in 5' overhangs using Klenow polymerase and
`digestion with Sail. That way, the k and Fd' genes were succes(cid:173)
`sively positioned under the control of the trp promoter. Each of
`these genes is preceded by the trp RBS and the distance from
`k-gene termination codon to the RBS of Fd gene is 45 bp. Plas(cid:173)
`mids were prepared and propagated in the E. coli strain HB 101
`(recA 1, endA 1 ).
`
`addition, at the lower temperatures, a significantly
`higher amount of insoluble Igs was also observed
`(Fig. 3).
`Ig polypeptides from the soluble fraction of cul(cid:173)
`tures grown at 21 oc were purified on a DE-52
`column and analysed by non-reducing SDS-PAGE.
`Several bands were detected when the blotted
`membrane was reacted with rabbit anti-mouse lgG
`(Fig. 2B). The broad band at about 47 kDa repre(cid:173)
`sents two nearly overlapping bands (lanes 1, 2).
`These bands correspond in size to Ig dimers. The
`predicted Mr of a ~ejFd dimer is 47 508 and that of
`a K-chain dimer is 46 483. It therefore seems that the
`lower of the two high Mr bands is a ~e-chain dimer,
`whereas the higher one is a ~e/Fd dimer. The Ig
`preparation contains additional lower Mr bands.
`These bands represent either monomers or non(cid:173)
`covalent dimers which dissociate in the presence of
`SDS. In bacteria producing ~e-chain only, a band
`which probably represents ~e-chain dimers was de(cid:173)
`tected (lane 3).
`
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`Page 2
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`

`
`3
`
`kDa
`
`-48 •
`
`-26
`
`1
`
`2
`
`t
`
`8
`
`A
`
`1 2 3 4
`
`5 6
`
`•
`
`555
`
`kDa
`
`-26
`-24
`
`Fig. 2. Immunoblot of B-Fab under reducing and non-reducing
`conditions. Panel A: analysis of Igs from B-Fab-producing cells
`under reducing conditions. Panel B: non reducing gel. Lane 1, Ig(cid:173)
`polypeptides from Fab-producing bacteria; lane 2, a preparation
`(the same as in lane 1) that has been further purified by affinity
`chromatography on anti-mouse IgG-agarose; lane 3, a prepara(cid:173)
`tion from K-chain-producing bacteria. Transformants of the
`E. coli strain W31l0, AE2 tonB-trpE, were grown at 21 oc in M9
`medium supplemented with 4% L broth to a cell density of
`A 550 = 1.0. The harvested cell pellet from a 1 liter culture was
`suspended in 5 ml of disruption buffer containing 20 mM Tris(cid:173)
`HQ, pH 8, 20 mM EDT A, 1 mM PMSF and 1 mM leupeptin,
`disrupted by a French Pressure Cell, and centrifuged at
`40000 x g for 30 min. The supernatant was dialysed against
`20 mM Tris · HCl, pH 8, 5 mM NaCl, and purified by two con(cid:173)
`secutive runs on a DE-52 column (5 ml, Whatman) pre(cid:173)
`equilibrated and eluted with the same buffer. The first 10 ml of
`unbound material was collected, concentrated to 1.5 ml and dia(cid:173)
`lysed against TS buffer. Samples representing 1 ml bacteria,
`A 550 = 1.0 were separated on O.l%SDS-12% PAGE, and the
`proteins transferred to nitrocellulose membranes (Schleicher &
`Schuell, BA 83). The membranes were blocked with 1% BSA in
`TS buffer, incubated with affinity purified rabbit anti mouse IgG
`( 1 : 1000) for 1 h at 3 7 o C, washed three times for I 0 min with TS
`buffer, incubated with 1:1000 dilution of goat anti-rabbit IgG
`alkaline phosphatase conjugate for 1 h, and washed again with
`TS buffer. The bands were visualized by the method of Leary
`et al. (1983).
`
`(b) Ag binding by bacterially-produced Fab
`
`DE-52-purified lgs from E. coli extracts were
`analysed for Ag (CEA) binding by EIA. As shown
`in Fig. 4, B-Fab binds to CEA in a saturable manner.
`In contrast to B-Fab, no Ag binding was detected
`when either K-chains or Fd-fragments, were analysed
`under the same conditions (Fig. 4 ). The specificity of
`B-Fab is also demonstrated by its ability to compete
`for the binding of hybridoma-produced 1251-anti(cid:173)
`CEA to the antigen (Fig. 5). This demonstrates that
`
`Fig. 3. Expression of lg polypeptide in E. coli as a function of the
`growth temperature. Bacteria ( 10 ml) were grown to a cell density
`of A 550 = 1.5. The cell pellet was suspended in 1 ml disruption
`buffer (see Fig. 2) containing 1 mg lysosyme and incubated for
`30 min. at 4°C. Following freezing and thawing four times,
`cellular DNA was fragmented by shearing through a 27-gage
`syringe needle and the lysate centrifuged for 15 min in an
`Eppendorf centrifuge at 4 o C. One tenth of either the particulate
`on the soluble fractions was applied to 0.1% SDS-12% PAGE.
`Immunoblots were developed by alkaline phosphatase Ab con(cid:173)
`jugate as described in Fig. 2. The particulate fractions (lanes 1-3)
`and the soluble fractions (lanes 4-6) were derived from cultures
`grown at 37°C(Ianes 1,4), 30°C(lanes 2,5) and 2JOC (lanes 3,6).
`
`1 .50 . - - - - - - - - - - - - - - - . ,
`
`10
`
`E c:
`0 v
`<(
`
`1.00
`
`0.50
`
`0.00
`
`10
`100
`Dilutions of 8-Fab
`Fig. 4. A dose response curve ofB-Fab binding to CEA. Analysis
`was done by EIA. (e), B-Fab; T, purified extracts from bacteria
`producing either K-chain or Fd fragments (see Fig. 2). The
`amount oflg chains in the different 'non-diluted' DE-52 purified
`extracts was brought to about 3 Jlg/well.
`CEA (25 ng in 50 Ill of 0.1 M carbonate-bicarbonate buffer,
`pH 9.3) was dried at 37°C in wells of a polyvinylchloride micro(cid:173)
`titer plate and nonspecific sites were blocked with 2% BSA.
`Serial dilutions of lg purified from E. coli extracts were added
`and incubated for 90 min at 37°C, then washed extensively with
`phosphate-buffered saline containing 0.05% Nonidet-P40
`(PBS-NP40). Goat anti-mouse Fab-alkaline phosphatase con(cid:173)
`jugate was used to determine specific ligand binding. Parallel
`series of diluted lg preparations were incubated in the absence
`of antigen for background subtraction.
`
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`Page 3
`
`

`
`556
`
`the bacterially-produced Fab has the same antigen
`binding site as that of the monoclonal antibody from
`which it is was derived.
`On the basis of these results, the estimated amount
`of functional B-Fab obtained after DE-52 purifica(cid:173)
`tion from 1 ml of bacteria at A 550 = 1.0, is about
`100 ng. This amount is equivalent to 5-10% of the
`total protein in the DE-52 eluate or about 1.5 JJ.gfmg
`protein.
`The activity of the DE-52-purified B-Fab remained
`stable upon storage at 4 o C for more than two
`months.
`
`(c) Conclusions
`
`(1) This report shows that functional Fab frag(cid:173)
`ments can be obtained from extracts of E. coli trans(cid:173)
`formants.
`(2) The amount oflg polypeptides and its soluble
`fraction is much higher in E. coli cells growing at
`21 oc or 30°C rather than at 37°C.
`(3) The soluble Fab fragments isolated from
`E. coli appear as covalent dimers (Fig. 2B). It seems
`that in the highly reducing environment of the E. coli
`cytoplasm (Politt and Zalkin, 1983), the Ig poly(cid:173)
`peptides exist as non-covalently linked dimers, and
`
`;e
`
`0 -"0 c:
`
`:I
`0
`.0
`.0
`<X
`,.....,
`>-4
`It)
`
`~ ........
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`1000
`
`10
`100
`8-Fob (ng)
`Fig. 5. Inhibition curve of 1251-labeled anti-CEA binding to CEA
`with B-Fab as inhibitor. Increasing amounts of DE-52 purified
`B-Fab were added together with 50 ng of 1251-anti-CEA (Johnson
`and Thorpe, 1982) to CEA precoated wells. Following incubation
`for 90 min, the wells were washed with PBS-NP40, cut out and
`taken for radioactive measurement in a gamma counter. The
`total amount of lg polypeptides in the DE-52 purified extracts
`(indicated as B-Fab on the abscissa) was estimated by scanning
`immunoblots which included standards of hybridoma-produced
`anti-CEA and were visualized by goat anti-mouse Fab-alkaline
`phosphatase conjugate.
`
`that the covalent dimers are formed by air oxidation
`following cell rupture.
`( 4) The Ig dimers seen in E. coli are not limited to
`pairs of K/Fd. Dimers of K-chains were seen in
`extracts of K-chain producing bacteria (Fig. 2B,
`lane 3) and Fd dimers were formed in Fd producing
`cells (unpublished data). However, the much higher
`association constant of K/Fd dimers compared to
`that of the homodimers (Darrington, 197 8 ), implies
`predominance of K/Fd dimers in cells producing
`equal amounts of both chains.
`(5) Since in the B-Fab expression plasmid the
`K-chain coding sequence is located upstream from
`the Fd fragments, an excess of K-chains may be
`expected. This would explain the apparent produc(cid:173)
`tion of K-chain dimers in Fab-producing cells
`(Fig. 2B).
`( 6) This method opens a route for
`in situ
`screening of E. coli colonies producing Fab frag(cid:173)
`ments with particular specificities.
`
`ACKNOWLEDGEMENT
`
`I thank Dr. D. Cassel, D. Sahar, N. Halachmi
`and R. Amit for valuable discussions. This research
`was supported by a grant from the Israel Cancer
`Research Fund and a grant from the Ministry of
`Commerce and Industry.
`
`Better, M., Chang, C.P., Robinson, R.R. and Horwitz, A.H.:
`E. coli secretion of an active chimeric antibody fragment.
`Science 240 (1988) 1041-1043.
`Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S.,
`Kaufman, B.M., Lee, S., Lee, T., Hope, S.H., Riordan, G.S.
`and Whitlow, M.: Single chain antigen-binding proteins.
`Science 242 (1988) 423-426.
`Boss, M.A., Kenten, J.H., Wood, C.R. and Emtage, J.S.:
`Assembly of functional antibodies from immunoglobulin
`heavy and light chains synthesized in E. coli. Nucleic Acids
`Res. 12 (1984) 3791-3806.
`Boulianne, G.L., Hozumi, N. and Shulman, M.J.: Production of
`functional chimaeric mouse/human antibody. Nature 312
`( 1984) 643-646.
`Cabilly, S., Riggs, A.D., Pande, H., Shively, J.E., Holmes, W.E.,
`Rey, M., Perry, L.J., Wetzel, R. and Heyneker, H.L.: Genera(cid:173)
`tion of antibody activity from immunoglobulin polypeptide
`
`•
`
`REFERENCES
`
`BEQ 1032
`Page 4
`
`

`
`chains produced in E. coli. Proc. Nat!. Acad. Sci. USA 81
`(1984) 3273-3277.
`Dorrington, K.J.: The structural basis for the functional ver(cid:173)
`satility of immunoglobulin G. Can. J. Biochem. 56 (1978)
`1087-1101.
`Horwitz, A.H., Chang, C.P., Better, M., Hellstrom, K.E. and
`Robinson, R.R.: Secretion of functional antibody and Fab
`fragment from yeast cells. Proc. Nat!. Acad. Sci. USA 85
`( 1988) 8678-8682.
`Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.,
`Novotny, J., Margolies, M.N., Ridge, R.I., Bruccolery, R.E.,
`Haber, E., Crea, R. and Oppermann, H.: Protein engineering
`of antibody binding sites: Recovery of specific activity in an
`anti-digoxin single chain Fv analogue produced in E. coli.
`Proc. Nat!. Acad. Sci. USA 85 (1988) 5879-5883.
`Johnson, A. and Thorpe, R.: Immunochemistry in Practice.
`Blackwell Scientific Publications, London, 1982.
`
`557
`
`Kane, J.F. and Hartley, D.L.: Formation of recombinant protein
`inclusion bodies in E. coli. Trends Biotechnol. 6 (1988)
`95-101.
`Leary, J.J., Brigati, D.J. and Ward, D.C.: Rapid and sensitive
`colorimetric method for visualising biotin-labeled DNA
`probes hybridized to DNA or RNA immobilized on nitro(cid:173)
`cellulose: bio-blots. Proc. Nat!. Acad. Sci. USA 80 (1983)
`4045-4049.
`Neuberger, M.S., Williams, G.T. and Fox, R.O.: Recombinant
`antibodies possessing novel effector functions. Nature 312
`( 1984) 604-608.
`Pollitt, S. and Zalkin, H.: Role of primary structure and disulfide
`bond formation in P-lactamase secretion. J. Bacteriol. 153
`(1983) 27-32.
`Skerra, A. and Fluckthun, A.: Assembly of a functional immuno(cid:173)
`globulin F Jragment in E. coli. Science 240 ( 1988) 1038-1040.
`
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