Journal of Biotechnology, 20 (1991)291-300
291
© 1991 ElsevierSciencePublishersB.V.All rightsreserved 0168-1656/91/$03.50 ADONIS 0168165691001380 BIOTEC 00673
Production of functional IgM Fab fragments by Saccharomyces cerevisiae J. Edqvist x, S. Keriinen 1, M. Penttil~i and J.K.C. Knowles 1
1, K.B. Str~by
2
1 VTT Biotechnical Laboratory, Espoo, Finland and 2 Department of Microbiology, Universityof Umea, Umea, Sweden
(Received 19 March 1991;accepted20 May 1991)
Summary The aim of this study was to express and secrete functional mouse IgM fragments in yeast. The heavy chain eDNA was truncated at two different sites, yielding genes coding for the complete VII domain. In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3' terminus of the CH1 region. Both shortened genes were coexpressed in Saccharomyces cerevisiae with a eDNA gene encoding a full length mouse Ig light chain. We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments. Antibody; Assembly; IgM; Immunoglobulin; Secretion; Yeast
Introduction Heterologous production of antigen binding proteins is a field in rapid development (see review by Winther and Milstein, 1991). One reason for this increasing interest is the possible commercial applications for antibodies or antigen binding proteins if produced at a low cost. Such applications might include areas where the Correspondence to and present address: J. Edqvist,Dept. of Microbiology,Universityof Ume~,S-901 87
Ume~, Sweden.
292
specific binding of the antibody is useful, as in tumor therapy and diagnostics, in affinity purification and as biocatalysts. Model studies of protein assembly and folding may also utilize heterologously produced antibody proteins and in this case eukaryotic microorganisms might be especially attractive as host cells. The first attempts to produce IgG antibodies in Escherichia coli (Boss et al., 1984) and yeast (Wood et al., 1985) gave low yields of functional antibodies and indicated that the ability of microorganisms to synthesize and secrete functional full size antibodies was limited. Therefore, the interest has turned to production of fragments of antibodies containing only the antigen binding regions (Better et al., 1988; Bird et al., 1988; Horwitz et al., 1988; Huston et al., 1988; Skerra and Pliickthun, 1988). In this article, we demonstrate production and secretion of functional Fab fragments of mouse IgM by the yeast Saccharomyces cereoisiae.
Materials and Methods
Strains and growth conditions E. coli DH5ot (F-, endA1, hsdR17 (rk, mk) , supE44, thi-1, A-, recA1, gyrA96, relA1, A(argF-lacZYA)U169, qb8OdlacZAM15)was used for plasmid propagation. E. coli JM101 and JM109 (Yanisch-Perron et al., 1985) were used in site-directed mutagenesis. S. cereoisiae DBY746 (a, his3A1, leu2-3, 1eu2-112, ura3-52, trp1-289, cyh r) obtained from Dr. D. Botstein, U.S.A., was the host for Fab fragment expression. Bacteria were grown at 37 o C in Luria broth (Lennox, 1955) supplemented with 100 mg 1- ~ ampicillin. Yeast were grown at 30 ° C in YPD or selective SC-media (Sherman et al., 1986). Pre-inocula for expression studies were grown in selective media.
Plasmids Bluescribe M13 + (Stratagene, U.S.A.) was used for site-directed mutagenesis and for subclonings. Yeast expression vector pMA91 (Mellor et al., 1983) was modified to pMA91stop by annealing two synthetic oligonucleotides, 5'-GATCTAGCTAGCTAG-3' and 5'-GATCCTAGCTAGCTA-3' containing stopsignals for translation in all three reading frames and ligating to the BglII-site in pMA91, between the phosphoglyceratekinase (PGK1) promoter and terminator.
DNA techniques Standard recombinant DNA techniques were used (Maniatis et al., 1982). Nucleotide sequences were determined by the dideoxy method (Sanger et al., 1977), modified for plasmid sequencing (Zagursky et al., 1986). The LiCl-method (Ito et al., 1983) was used for transformation of S. cereoisiae.
293
Immunoblottings Standard procedures were used for transfer of proteins from polyacrylamide gels to nitrocellulose (Towbin et al., 1979). Ig chains were detected (Leary et al., 1983) with alkaline phosphatase labeled, heavy chain-specific goat antibodies against mouse IgM (Southern Biotechnology Associates, U.S.A., cat. no. 1020-04).
Enzyme-linked immunosorbent assay (ELISA) ELISA was done essentially as described by Voller et al. (1979). Affinity purified, biotin-labeled and unlabeled heavy chain-specific goat antibodies against mouse IgM (cat. no. 1060-08 and 1060-01) and mouse A light chain (cat. no. 1060-08 and 1060-01) were from Southern Biotechnology Associates (U.S.A.). Streptavidin-biotinylated horseradish peroxidase complex was from Amersham (U.K.) and ortho-phenylene diamine dihydrochloride from Sigma (U.S.A.).
Results and Discussion
Plasmid constructions In order to study expression and secretion of the Fab fragment of a mouse IgM from hybridoma B1-8 against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP), the heavy chain eDNA was truncated at two different sites and placed under control of the yeast PGK1 promoter and expressed in S. cerevisiae. Plasmids pX and pY (Fig. 1) were both constructed for expression of truncated heavy chains, pY contains DNA coding for the complete CH1 domain and 3 additional amino acids derived from the linker in the expression vector, pX is missing DNA for the last 6 amino acids of the CH1 domain and contains instead 3 amino acids derived from the linker. The difference between pY and pX is shown in Fig. lB. Plasmid pL (Fig. 1) which carries the light chain eDNA, and plasmids pX and pY were transformed into S. cerevisiae. Cells containing the light chain plasmid with either one of the heavy chain plasmids, as well as cells transformed with only one type of plasmid were studied with respect to synthesis of immunoglobulin chains and functional Fab fragments.
Plasmid stability Both of the heavy chain plasmids contain the LEU2 selection marker, while the light chain plasmid carries the HIS3 marker. These two different selection markers were used to ascertain selection of yeast cells containing both heavy chain and light chain plasmids. The expression studies were carried out under non-selective conditions. Plasmid stability in the non-selective medium was determined by plating the cells on non-selective and on selective media, pL, carrying the gene for the light chain, was
294
lost to a much higher extent than pY, containing the truncated heavy chain gene (Table 1). The percentage of plasmid loss was the same for yeast containing pL alone or pL together with pY and almost the same for pL together with pX. Thus, pL is more unstable than pY and the presence of pX or pY has a low or no effect on the stability of pL.
Antigen binding activity secreted by yeast cells The B1-8 antibody has higher affinity for 4-hydroxy-5-iodo-3-nitrophenyl acetyl (NIP), than for the original hapten 4-hydroxy-3-nitrophenyl acetyl (NP) (Imanishi and M~ikel~i, 1974). Therefore the secretion of antigen binding Fab fragment from
I
PGKI P~L~91 prom [ linker
Bs [ AAAAC CAAAAGATC CTAGA ATO
pX / pY
heavy chain
(I0,4 kb)
PGKI pMA91 pro----~ I llnker
Bs A [ , AAAAC CAAAAGATC CTAGAGTCflA AT~ATG light c h i n
pL
cDNA [I •
I
P P
I J
cDNA
(12,o kb)
leu2'
PvuIZ
B_~IT
CH1
~
CH2
GGC AAA AAC AAA OAT CGA TOT AGO TAG 0 K N K D R S S stop Y
GfiC AAA AAC AAA OAT CTO CAT GTO CCC ATT CCA GGA TCT AGO TAG 0 K. N K D L H V P I P 0 S S stop
295 TABLE 1 Stability of expression plasmids Plasmids
(× 10s)
Cells/ml
Leu + (%)
His + (%)
His +, Leu + (%)
pX + pL
1.4
86
27
27
pY + pL
0.2 1.4 2.7
86 79 63
19 14 17
17 12 10
pY
0.2 1.6 2.7
76 81 64
pL
0.2 1.8 2.6
13 11 11
Yeast cells were grown in YPD and then placed on non-selective YPD agar and selective SC agar. Results are obtained from the same cultures, as used for ELISA analyses in Fig. 3.
yeast cotransformed with pY + pL or pX + pL was analyzed by a sandwich ELISA technique, which detects binding to NIP-cap-BSA. Figs. 2 and 3A show that active NIP-binding Fab fragments were secreted from yeast carrying the genes for the longer form of the truncated heavy chain and for the light chain (pY + pL). No activity was detected in culture media of cells carrying pX + pL (Fig. 2). Furthermore, no antigen binding could be detected in culture media of ceils expressing single chains (Fig. 3A). Thus, either single Ig Fig. 1. (A) Constructions of plasmids for expression of IgM Fab fragments in S. cereuisiae. Mouse p. chain eDNA from hybridoma B1-8 was obtained as a 1.8 kb EcoRI insert in plasmid pAB/z-II (Bothwell et al., 1981). The insert was ligated into the EcoRI-site of Bluescribe M13+ (Bs). After removing the polydG tail at the 5'-flank of the coding region by site-directed mutagenesis, the gene was released from Bluescribe by X b a I + BglII (Fragment X) or by X b a I + PvuII (Fragment Y). The XbaI site is located in the cloning cassette of Bluescribe M13 +, the PvuII site is located at the border of the CH1 and the CH2 domains in the heavy chain sequence and the BglII site is 18 bp upstream of the PvuII site (Fig. 1B). The truncated eDNA genes, X and Y, were placed behind the PGK1 promoter on the vector pMA91stop, giving plasmids pX and pY. * Denotes parts of plasmids p X / p L shown in Fig. lB. The Ig ~ light chain eDNA, isolated from the mouse myeloma MOPC-315 was obtained as a 0.8 kb fragment in plasmid pA2-1 (Bothwell et al., 1982). A PstI (P) fragment carrying the light chain eDNA was inserted into the PstI site of Bluescribe M13+. This fragment lacked the 3' part of the coding region and the stop codon, which therefore was inserted as a 42 bp PstI fragment from an AvaI (A) + SspI fragment from p~.2-1. The polydG tail was then removed with site-directed mutagenesis. The gene was released from Bluescribe M13+ with Xbal and HindIII and placed under control of the PUK1 promoter and terminator in pMA91stop. The HIS3 selection marker was inserted at the BstEII (B) site in LEU2 on pMA91stop, to give the opportunity to co-select for light chain plasmids and heavy chain plasmids in the same yeast cells. The light chain expression plasmid is called pL. (B) Nucleotide and amino acid sequences (one letter code) of the truncated mouse ~ genes X and Y. Only the 3' end of the coding regions is shown. For mouse ~ eDNA, only the part of the sequence corresponding to the junction between CH1 and CH2 domain is shown. Domain boundaries are assigned according to Auffray and Rougeon (1980).
296 0.,4
0.3
¢~ 0.2
o.1
pX • pL Plosmids
None
pY°pL
Fig. 2. Secretion of NIP-binding Fab fragments and Ig light chain by yeast. Levels of Ig light chain (ELISA, filled bars) and NIP binding activity (open bars) in the culture media of S. cerecisiae DBY746 harboring none, p X + p L , or p Y + p L expression plasmids. Cells were grown in 200 ml YPD. Supernatants from 20 ml samples from cultures in early stationary growth phase ( 1 - 2 x l0 s cells per ml) were concentrated 27 times by centrifugation in Centriprep-10 concentrators (Amicon, U.S.A.). For detection of the light chain, microtiter plates were coated with unlabeled affinity purified goat anti-mouse A light chain antibodies. 0.2 ml concentrated growth medium was added and diluted stepwise in PBS (0.15 M NaCI, 0.012 M NazHPO 4, 0.003 M NaHzPO 4, pH 7.4) in 2-fold dilutions. Bound light chain was detected by addition of biotin-labeled goat antibodies against mouse A chain, followed by streptavidinbiotinylated horseradish peroxidase complex with substrate solution as in Voller et al. (1979). A492 of 1.0 corresponds to approx. 50 ng of mouse lgM, A. Antigen binding activity was analyzed in the same way, except that the microtiter plates were coated with NIP-cap-BSA.
0.2 0.1
None
03
"B
pY
pL
Anti-light chain binding activity
0':~
pY*pL r - - ' - I
J
J
I
II
j i ~
0.1 rI'tI---1
None
,
~ I I
pY
II
pL
pY ° pL
Plasrnids Fig. 3. Secretion of NIP binding activity (A) and Ig light chain (B), from yeast at different growth states carrying none, pY or pl.,, or p Y + p L expression plasmids. Samples were from cultures in the exponential (filled bars), early stationary (open bars) and stationary growth phase (grey bars), corresponding to 2 x 107, 2 × 10s and 3 x 108 cells per ml, respectively, For experimental details, see legend to Fig. 2.
297 chains did not bind to NIP, or the single chains could not be secreted into the culture medium.
Secretion of light chain Secretion of light chain was analyzed by ELISA (Figs. 2 and 3B). The concentration of light chain in the growth media was estimated by comparing the absorbance of the sample to that of a known amount of commercially available Ig light chain analyzed in the same assay. Cells containing pY + pL, pX + pL or pL alone were all found to secrete light chain at amounts of 5-20 ng ml-~. The low levels are partly explained by the low stability of the light chain plasmid, as shown in Table 1. Highest amounts of secreted light chain were recovered from stationary growth phase cultures (2.5-3 × l0 s cells per ml). When yeast cells coexpressing light and heavy chain were compared, higher amounts of light chain were detected from cultures carrying pX + pL than from pY + pL or pL alone (Fig. 2). Thus, the previously noticed lack of antigen binding activity in the supernatants of pX + pL cultures could neither be explained by lowered production of light chain, nor by blocked secretion of light chain in yeast cells carrying pX + pL.
Expression of truncated Ig chains X and Y The differences that we have observed in NIP binding activity of the two heavy chain constructions may be explained in several ways: (a) Although antibody fragments are formed and secreted, only the Fab containing the longer one of the truncated heavy chains recognizes the hapten. (b) Only the longer heavy chain fragment is able to assemble with the light chain to form a functional Fab fragment. (c) The expression level or half-life of the more truncated heavy chain is significantly lower compared to the longer heavy chain fragment. The detected NIP-binding activity indicated that the longer form of the truncated heavy chain was produced and secreted, but ELISA analyses using heavy chain specific antibodies as capture antibodies gave no signals above the background level (data not shown). Proteins from the yeast cultures were therefore analyzed for heavy chain production with the immunoblot technique. When the blots were treated with heavy chain-specific antibodies against mouse IgM (Fig. 4), a distinct band corresponding to a polypeptide of the expected size of the truncated heavy chain, 25 kDa, was detected in culture supernatants and cell extracts from cells carrying pY + pL. No detectable signal was obtained from pX + pL growth medium samples, although cell extracts were shown both by Northern analyses to contain mRNA from X (data not shown) and to give weak signals corresponding to polypeptides of 23-25 kDa (Fig. 4). Thus, although existing in the cells, the combined pX + pL products are not secreted to the growth medium while this is the case for pY + pL products. This suggests that either the expression of the shorter heavy chain fragment is somewhat impaired, or that its half-life is much shorter than that of the longer
298
A
B C
DE
":
FGHI
J KL +-116 ,,-84 +--58 +--48
+'-36 +--27
Fig. 4. Western analysis of expression of truncated Ig heavy chains in yeast. Culture media from 200 ml of yeast cells grown in YPD was treated with RNaseA (20 /~g), precipitated with cold TCA (final concentration 10%, 1 h at 0 °C), washed with cold acetone and dissolved in sample buffer (0.18 M Tris-HCI, pH 6.8, 14% glycerol, 14%/3-mercaptoethanol, 2% SDS, 0.15 mg ml-I bromophenol blue). Cell extracts were prepared from 10 ml yeast cultures at different growth stages. Cells, washed once in water, were dissolved in 400/~1 2% SDS, 1/2 volume of glass beads were added and the cells were vortexed and boiled twice for 90 s. Samples were run overnight (60 V) in a 7.5-15% gradient SDS/polyacrylamide gel (Laemmli, 1970). Proteins were transferred from polyacrylamide gels to nitrocellulose (Towbin et al., 1979). Blots were blocked by incubation in TBST (10 mM Tris-HCI, pH 6.8, 0.15 M NaCI, 0.05% Tween 20)+1% BSA for 2 h at ~ ° C . Ig chains were detected with heavy chain-specific alkaline phosphatase labeled goat antibodies (1:200 diluted in TBST) against mouse IgM. Samples are from culture supernatants (lanes A-C) and cell extracts (lanes D-K). Cells from control yeast strain lacking expression plasmids (lanes A, J and K), from yeast carrying pY+pL (lanes B, G, H and I) and from yeast carrying pX + pL (lanes C, D, E and F). Samples were taken from yeast cultures in exponential growth phase (lanes F, I and K), early stationary growth phase (lanes E, H and J) and stationary growth phase (lanes A, B, C, D and G). Control Sigma M-3273 mouse IgM (lane L). heavy chain fragment. If so, it may be due to i m p r o p e r folding of the p r o t e i n or inability to form a f u n c t i o n a l complex with the light chain. Based o n s e q u e n c e comparisons, the hinge region, which separates the F a b from the Fc part, is not very p r o n o u n c e d in IgM in contrast to IgG. T h e r e f o r e correct folding of the m o l e c u l e might be sensitive to t r u n c a t i o n close to the F a b sequence. F u r t h e r conclusions o n how t r u n c a t i o n affects expression a n d f u n c t i o n of IgM F a b f r a g m e n t s has to await studies o n the expression of several t r u n c a t e d forms of the heavy chain in yeast. However, the results p r e s e n t e d here are to o u r
299
knowledge the first ones to show that biologically active and functional IgM Fab fragments can be produced in microorganisms.
Acknowledgements Plasmids pAB/z-II and pA2-1 were generous gifts from Dr. D. Baltimore. We thank Drs. A.J. Kingsman and S.M. Kingsman for giving us plasmid pMA91 and we are grateful to Prof. O. M~ikel~i for providing us with NIP-cap-BSA. This work was financially supported by Stiftelsen Svensk Etanolutveckling and by Nordic Yeast Research Program.
References Auffray, C. and Rougeon, F. (1980) Nucleotide sequence of a cloned eDNA corresponding to secreted p. chain of mouse immunoglobulin. Gene 12, 77-86. Better, M., Chang, C.P., Robinson, R.R. and Horwitz, A.H. (1988) Escherichia coli secretion of an active chimeric antibody fragment. Science 240, 1041-1043. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. (1988) Single-chain antigen binding proteins. Science 242, 423-426. Boss, M.A., Kenten, J.H., Wood, C.R. and Emtage, J.S. (1984) Assembly of functional antibodies from immunoglobulin heavy and light chains synthesized in E. coil Nucl. Acids Res. 12, 3791-3806. Bothwell, A.L.M., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewski, IC and Baltimore, D. (1981) Heavy chain variable region contribution to the NP b family of antibodies: somatic mutation evident in a 72a variable region. Cell 24, 625-637. Bothwell, A.L.M., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewski, K. and Baltimore, D. (1982) Somatic variants of murine immunoglobulin A light chains. Nature 298, 380-382. Horwitz, A.H., Chang, C.P., Better, M., Hellstrom, K.E. and Robinson, R.R. (1988) Secretion of functional antibody and Fab fragment from yeast cells. Proc. Natl. Aead. Sci. U.S.A. 85, 8678-8682. Huston, J.S., Levinson, D., Mudgett-Hunter, M.,Tai, M.-S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85, 5879-5883. Imanishi, T. and M~ikelii,O. (1974) Inheritance of antibody specificity. I. Anti-(4-hydroxy-3-nitrophenyl) acetyl of the mouse primary response. J. Exp. Med. 140, 1498-1511. Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Leary, J.J., Brigati, D.J. and Ward, D.C. (1983) Rapid and sensitive colorimetrie method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bioblots. Proe. Natl. Acad. Sci. U.S.A. 80, 4045-4049. Lennox, E.S. (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1, 190-206. Maniatis, T., Fritseh, E.F. and Sambrook, J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Mellor, J., Dobson, M.J., Roberts, N.A., Tuite, M.F., Emtage, J.S., White, S., I.owe, P.A., Patel, T.,, Kingsman, A.J. and Kingsman, S.M. (1983) Efficient synthesis of enzymatieally active calf ehymosin in Saccharomyces cerevisiae. Gene 24, 1-14.
300
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. Sherman, F., Fink, G.R. and Hicks, J.B. (1986) Methods in Yeast Genetics. A Laboratory Manual. Cold Spring Harbor, NY. Skerra, A. and Pliickthun, A. (1988) Assembly of a immunoglobulin Pv fragment in Escherichia coli. Science 240, 1038-1041. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. Voller, A., Bidwell, D.E. and Bartlett, A. (1979) The enzyme linked immunosorbent assay (ELISA), Vol. 1, Dynatech Europe, U.K. Winter, G. and Milstein, C. (1991) Man-made antibodies. Nature 349, 293-299. Wood, C.R., Boss, M.A., Kenten, J.H., Calvert, J.E., Roberts, N.A. and Emtage, J.S. (1985) The synthesis and in vivo assembly of functional antibodies in yeast. Nature 314, 446-449. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33, 103-119. Zagursky, R.J., Berman, M.L, Baumeister, K. and Lomax, N. (1986) Rapid and easy sequencing of large linear double stranded DNA and supercoiled plasma DNA. Gene Anal. Teeh. 2, 89-94.
291
© 1991 ElsevierSciencePublishersB.V.All rightsreserved 0168-1656/91/$03.50 ADONIS 0168165691001380 BIOTEC 00673
Production of functional IgM Fab fragments by Saccharomyces cerevisiae J. Edqvist x, S. Keriinen 1, M. Penttil~i and J.K.C. Knowles 1
1, K.B. Str~by
2
1 VTT Biotechnical Laboratory, Espoo, Finland and 2 Department of Microbiology, Universityof Umea, Umea, Sweden
(Received 19 March 1991;accepted20 May 1991)
Summary The aim of this study was to express and secrete functional mouse IgM fragments in yeast. The heavy chain eDNA was truncated at two different sites, yielding genes coding for the complete VII domain. In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3' terminus of the CH1 region. Both shortened genes were coexpressed in Saccharomyces cerevisiae with a eDNA gene encoding a full length mouse Ig light chain. We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments. Antibody; Assembly; IgM; Immunoglobulin; Secretion; Yeast
Introduction Heterologous production of antigen binding proteins is a field in rapid development (see review by Winther and Milstein, 1991). One reason for this increasing interest is the possible commercial applications for antibodies or antigen binding proteins if produced at a low cost. Such applications might include areas where the Correspondence to and present address: J. Edqvist,Dept. of Microbiology,Universityof Ume~,S-901 87
Ume~, Sweden.
292
specific binding of the antibody is useful, as in tumor therapy and diagnostics, in affinity purification and as biocatalysts. Model studies of protein assembly and folding may also utilize heterologously produced antibody proteins and in this case eukaryotic microorganisms might be especially attractive as host cells. The first attempts to produce IgG antibodies in Escherichia coli (Boss et al., 1984) and yeast (Wood et al., 1985) gave low yields of functional antibodies and indicated that the ability of microorganisms to synthesize and secrete functional full size antibodies was limited. Therefore, the interest has turned to production of fragments of antibodies containing only the antigen binding regions (Better et al., 1988; Bird et al., 1988; Horwitz et al., 1988; Huston et al., 1988; Skerra and Pliickthun, 1988). In this article, we demonstrate production and secretion of functional Fab fragments of mouse IgM by the yeast Saccharomyces cereoisiae.
Materials and Methods
Strains and growth conditions E. coli DH5ot (F-, endA1, hsdR17 (rk, mk) , supE44, thi-1, A-, recA1, gyrA96, relA1, A(argF-lacZYA)U169, qb8OdlacZAM15)was used for plasmid propagation. E. coli JM101 and JM109 (Yanisch-Perron et al., 1985) were used in site-directed mutagenesis. S. cereoisiae DBY746 (a, his3A1, leu2-3, 1eu2-112, ura3-52, trp1-289, cyh r) obtained from Dr. D. Botstein, U.S.A., was the host for Fab fragment expression. Bacteria were grown at 37 o C in Luria broth (Lennox, 1955) supplemented with 100 mg 1- ~ ampicillin. Yeast were grown at 30 ° C in YPD or selective SC-media (Sherman et al., 1986). Pre-inocula for expression studies were grown in selective media.
Plasmids Bluescribe M13 + (Stratagene, U.S.A.) was used for site-directed mutagenesis and for subclonings. Yeast expression vector pMA91 (Mellor et al., 1983) was modified to pMA91stop by annealing two synthetic oligonucleotides, 5'-GATCTAGCTAGCTAG-3' and 5'-GATCCTAGCTAGCTA-3' containing stopsignals for translation in all three reading frames and ligating to the BglII-site in pMA91, between the phosphoglyceratekinase (PGK1) promoter and terminator.
DNA techniques Standard recombinant DNA techniques were used (Maniatis et al., 1982). Nucleotide sequences were determined by the dideoxy method (Sanger et al., 1977), modified for plasmid sequencing (Zagursky et al., 1986). The LiCl-method (Ito et al., 1983) was used for transformation of S. cereoisiae.
293
Immunoblottings Standard procedures were used for transfer of proteins from polyacrylamide gels to nitrocellulose (Towbin et al., 1979). Ig chains were detected (Leary et al., 1983) with alkaline phosphatase labeled, heavy chain-specific goat antibodies against mouse IgM (Southern Biotechnology Associates, U.S.A., cat. no. 1020-04).
Enzyme-linked immunosorbent assay (ELISA) ELISA was done essentially as described by Voller et al. (1979). Affinity purified, biotin-labeled and unlabeled heavy chain-specific goat antibodies against mouse IgM (cat. no. 1060-08 and 1060-01) and mouse A light chain (cat. no. 1060-08 and 1060-01) were from Southern Biotechnology Associates (U.S.A.). Streptavidin-biotinylated horseradish peroxidase complex was from Amersham (U.K.) and ortho-phenylene diamine dihydrochloride from Sigma (U.S.A.).
Results and Discussion
Plasmid constructions In order to study expression and secretion of the Fab fragment of a mouse IgM from hybridoma B1-8 against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP), the heavy chain eDNA was truncated at two different sites and placed under control of the yeast PGK1 promoter and expressed in S. cerevisiae. Plasmids pX and pY (Fig. 1) were both constructed for expression of truncated heavy chains, pY contains DNA coding for the complete CH1 domain and 3 additional amino acids derived from the linker in the expression vector, pX is missing DNA for the last 6 amino acids of the CH1 domain and contains instead 3 amino acids derived from the linker. The difference between pY and pX is shown in Fig. lB. Plasmid pL (Fig. 1) which carries the light chain eDNA, and plasmids pX and pY were transformed into S. cerevisiae. Cells containing the light chain plasmid with either one of the heavy chain plasmids, as well as cells transformed with only one type of plasmid were studied with respect to synthesis of immunoglobulin chains and functional Fab fragments.
Plasmid stability Both of the heavy chain plasmids contain the LEU2 selection marker, while the light chain plasmid carries the HIS3 marker. These two different selection markers were used to ascertain selection of yeast cells containing both heavy chain and light chain plasmids. The expression studies were carried out under non-selective conditions. Plasmid stability in the non-selective medium was determined by plating the cells on non-selective and on selective media, pL, carrying the gene for the light chain, was
294
lost to a much higher extent than pY, containing the truncated heavy chain gene (Table 1). The percentage of plasmid loss was the same for yeast containing pL alone or pL together with pY and almost the same for pL together with pX. Thus, pL is more unstable than pY and the presence of pX or pY has a low or no effect on the stability of pL.
Antigen binding activity secreted by yeast cells The B1-8 antibody has higher affinity for 4-hydroxy-5-iodo-3-nitrophenyl acetyl (NIP), than for the original hapten 4-hydroxy-3-nitrophenyl acetyl (NP) (Imanishi and M~ikel~i, 1974). Therefore the secretion of antigen binding Fab fragment from
I
PGKI P~L~91 prom [ linker
Bs [ AAAAC CAAAAGATC CTAGA ATO
pX / pY
heavy chain
(I0,4 kb)
PGKI pMA91 pro----~ I llnker
Bs A [ , AAAAC CAAAAGATC CTAGAGTCflA AT~ATG light c h i n
pL
cDNA [I •
I
P P
I J
cDNA
(12,o kb)
leu2'
PvuIZ
B_~IT
CH1
~
CH2
GGC AAA AAC AAA OAT CGA TOT AGO TAG 0 K N K D R S S stop Y
GfiC AAA AAC AAA OAT CTO CAT GTO CCC ATT CCA GGA TCT AGO TAG 0 K. N K D L H V P I P 0 S S stop
295 TABLE 1 Stability of expression plasmids Plasmids
(× 10s)
Cells/ml
Leu + (%)
His + (%)
His +, Leu + (%)
pX + pL
1.4
86
27
27
pY + pL
0.2 1.4 2.7
86 79 63
19 14 17
17 12 10
pY
0.2 1.6 2.7
76 81 64
pL
0.2 1.8 2.6
13 11 11
Yeast cells were grown in YPD and then placed on non-selective YPD agar and selective SC agar. Results are obtained from the same cultures, as used for ELISA analyses in Fig. 3.
yeast cotransformed with pY + pL or pX + pL was analyzed by a sandwich ELISA technique, which detects binding to NIP-cap-BSA. Figs. 2 and 3A show that active NIP-binding Fab fragments were secreted from yeast carrying the genes for the longer form of the truncated heavy chain and for the light chain (pY + pL). No activity was detected in culture media of cells carrying pX + pL (Fig. 2). Furthermore, no antigen binding could be detected in culture media of ceils expressing single chains (Fig. 3A). Thus, either single Ig Fig. 1. (A) Constructions of plasmids for expression of IgM Fab fragments in S. cereuisiae. Mouse p. chain eDNA from hybridoma B1-8 was obtained as a 1.8 kb EcoRI insert in plasmid pAB/z-II (Bothwell et al., 1981). The insert was ligated into the EcoRI-site of Bluescribe M13+ (Bs). After removing the polydG tail at the 5'-flank of the coding region by site-directed mutagenesis, the gene was released from Bluescribe by X b a I + BglII (Fragment X) or by X b a I + PvuII (Fragment Y). The XbaI site is located in the cloning cassette of Bluescribe M13 +, the PvuII site is located at the border of the CH1 and the CH2 domains in the heavy chain sequence and the BglII site is 18 bp upstream of the PvuII site (Fig. 1B). The truncated eDNA genes, X and Y, were placed behind the PGK1 promoter on the vector pMA91stop, giving plasmids pX and pY. * Denotes parts of plasmids p X / p L shown in Fig. lB. The Ig ~ light chain eDNA, isolated from the mouse myeloma MOPC-315 was obtained as a 0.8 kb fragment in plasmid pA2-1 (Bothwell et al., 1982). A PstI (P) fragment carrying the light chain eDNA was inserted into the PstI site of Bluescribe M13+. This fragment lacked the 3' part of the coding region and the stop codon, which therefore was inserted as a 42 bp PstI fragment from an AvaI (A) + SspI fragment from p~.2-1. The polydG tail was then removed with site-directed mutagenesis. The gene was released from Bluescribe M13+ with Xbal and HindIII and placed under control of the PUK1 promoter and terminator in pMA91stop. The HIS3 selection marker was inserted at the BstEII (B) site in LEU2 on pMA91stop, to give the opportunity to co-select for light chain plasmids and heavy chain plasmids in the same yeast cells. The light chain expression plasmid is called pL. (B) Nucleotide and amino acid sequences (one letter code) of the truncated mouse ~ genes X and Y. Only the 3' end of the coding regions is shown. For mouse ~ eDNA, only the part of the sequence corresponding to the junction between CH1 and CH2 domain is shown. Domain boundaries are assigned according to Auffray and Rougeon (1980).
296 0.,4
0.3
¢~ 0.2
o.1
pX • pL Plosmids
None
pY°pL
Fig. 2. Secretion of NIP-binding Fab fragments and Ig light chain by yeast. Levels of Ig light chain (ELISA, filled bars) and NIP binding activity (open bars) in the culture media of S. cerecisiae DBY746 harboring none, p X + p L , or p Y + p L expression plasmids. Cells were grown in 200 ml YPD. Supernatants from 20 ml samples from cultures in early stationary growth phase ( 1 - 2 x l0 s cells per ml) were concentrated 27 times by centrifugation in Centriprep-10 concentrators (Amicon, U.S.A.). For detection of the light chain, microtiter plates were coated with unlabeled affinity purified goat anti-mouse A light chain antibodies. 0.2 ml concentrated growth medium was added and diluted stepwise in PBS (0.15 M NaCI, 0.012 M NazHPO 4, 0.003 M NaHzPO 4, pH 7.4) in 2-fold dilutions. Bound light chain was detected by addition of biotin-labeled goat antibodies against mouse A chain, followed by streptavidinbiotinylated horseradish peroxidase complex with substrate solution as in Voller et al. (1979). A492 of 1.0 corresponds to approx. 50 ng of mouse lgM, A. Antigen binding activity was analyzed in the same way, except that the microtiter plates were coated with NIP-cap-BSA.
0.2 0.1
None
03
"B
pY
pL
Anti-light chain binding activity
0':~
pY*pL r - - ' - I
J
J
I
II
j i ~
0.1 rI'tI---1
None
,
~ I I
pY
II
pL
pY ° pL
Plasrnids Fig. 3. Secretion of NIP binding activity (A) and Ig light chain (B), from yeast at different growth states carrying none, pY or pl.,, or p Y + p L expression plasmids. Samples were from cultures in the exponential (filled bars), early stationary (open bars) and stationary growth phase (grey bars), corresponding to 2 x 107, 2 × 10s and 3 x 108 cells per ml, respectively, For experimental details, see legend to Fig. 2.
297 chains did not bind to NIP, or the single chains could not be secreted into the culture medium.
Secretion of light chain Secretion of light chain was analyzed by ELISA (Figs. 2 and 3B). The concentration of light chain in the growth media was estimated by comparing the absorbance of the sample to that of a known amount of commercially available Ig light chain analyzed in the same assay. Cells containing pY + pL, pX + pL or pL alone were all found to secrete light chain at amounts of 5-20 ng ml-~. The low levels are partly explained by the low stability of the light chain plasmid, as shown in Table 1. Highest amounts of secreted light chain were recovered from stationary growth phase cultures (2.5-3 × l0 s cells per ml). When yeast cells coexpressing light and heavy chain were compared, higher amounts of light chain were detected from cultures carrying pX + pL than from pY + pL or pL alone (Fig. 2). Thus, the previously noticed lack of antigen binding activity in the supernatants of pX + pL cultures could neither be explained by lowered production of light chain, nor by blocked secretion of light chain in yeast cells carrying pX + pL.
Expression of truncated Ig chains X and Y The differences that we have observed in NIP binding activity of the two heavy chain constructions may be explained in several ways: (a) Although antibody fragments are formed and secreted, only the Fab containing the longer one of the truncated heavy chains recognizes the hapten. (b) Only the longer heavy chain fragment is able to assemble with the light chain to form a functional Fab fragment. (c) The expression level or half-life of the more truncated heavy chain is significantly lower compared to the longer heavy chain fragment. The detected NIP-binding activity indicated that the longer form of the truncated heavy chain was produced and secreted, but ELISA analyses using heavy chain specific antibodies as capture antibodies gave no signals above the background level (data not shown). Proteins from the yeast cultures were therefore analyzed for heavy chain production with the immunoblot technique. When the blots were treated with heavy chain-specific antibodies against mouse IgM (Fig. 4), a distinct band corresponding to a polypeptide of the expected size of the truncated heavy chain, 25 kDa, was detected in culture supernatants and cell extracts from cells carrying pY + pL. No detectable signal was obtained from pX + pL growth medium samples, although cell extracts were shown both by Northern analyses to contain mRNA from X (data not shown) and to give weak signals corresponding to polypeptides of 23-25 kDa (Fig. 4). Thus, although existing in the cells, the combined pX + pL products are not secreted to the growth medium while this is the case for pY + pL products. This suggests that either the expression of the shorter heavy chain fragment is somewhat impaired, or that its half-life is much shorter than that of the longer
298
A
B C
DE
":
FGHI
J KL +-116 ,,-84 +--58 +--48
+'-36 +--27
Fig. 4. Western analysis of expression of truncated Ig heavy chains in yeast. Culture media from 200 ml of yeast cells grown in YPD was treated with RNaseA (20 /~g), precipitated with cold TCA (final concentration 10%, 1 h at 0 °C), washed with cold acetone and dissolved in sample buffer (0.18 M Tris-HCI, pH 6.8, 14% glycerol, 14%/3-mercaptoethanol, 2% SDS, 0.15 mg ml-I bromophenol blue). Cell extracts were prepared from 10 ml yeast cultures at different growth stages. Cells, washed once in water, were dissolved in 400/~1 2% SDS, 1/2 volume of glass beads were added and the cells were vortexed and boiled twice for 90 s. Samples were run overnight (60 V) in a 7.5-15% gradient SDS/polyacrylamide gel (Laemmli, 1970). Proteins were transferred from polyacrylamide gels to nitrocellulose (Towbin et al., 1979). Blots were blocked by incubation in TBST (10 mM Tris-HCI, pH 6.8, 0.15 M NaCI, 0.05% Tween 20)+1% BSA for 2 h at ~ ° C . Ig chains were detected with heavy chain-specific alkaline phosphatase labeled goat antibodies (1:200 diluted in TBST) against mouse IgM. Samples are from culture supernatants (lanes A-C) and cell extracts (lanes D-K). Cells from control yeast strain lacking expression plasmids (lanes A, J and K), from yeast carrying pY+pL (lanes B, G, H and I) and from yeast carrying pX + pL (lanes C, D, E and F). Samples were taken from yeast cultures in exponential growth phase (lanes F, I and K), early stationary growth phase (lanes E, H and J) and stationary growth phase (lanes A, B, C, D and G). Control Sigma M-3273 mouse IgM (lane L). heavy chain fragment. If so, it may be due to i m p r o p e r folding of the p r o t e i n or inability to form a f u n c t i o n a l complex with the light chain. Based o n s e q u e n c e comparisons, the hinge region, which separates the F a b from the Fc part, is not very p r o n o u n c e d in IgM in contrast to IgG. T h e r e f o r e correct folding of the m o l e c u l e might be sensitive to t r u n c a t i o n close to the F a b sequence. F u r t h e r conclusions o n how t r u n c a t i o n affects expression a n d f u n c t i o n of IgM F a b f r a g m e n t s has to await studies o n the expression of several t r u n c a t e d forms of the heavy chain in yeast. However, the results p r e s e n t e d here are to o u r
299
knowledge the first ones to show that biologically active and functional IgM Fab fragments can be produced in microorganisms.
Acknowledgements Plasmids pAB/z-II and pA2-1 were generous gifts from Dr. D. Baltimore. We thank Drs. A.J. Kingsman and S.M. Kingsman for giving us plasmid pMA91 and we are grateful to Prof. O. M~ikel~i for providing us with NIP-cap-BSA. This work was financially supported by Stiftelsen Svensk Etanolutveckling and by Nordic Yeast Research Program.
References Auffray, C. and Rougeon, F. (1980) Nucleotide sequence of a cloned eDNA corresponding to secreted p. chain of mouse immunoglobulin. Gene 12, 77-86. Better, M., Chang, C.P., Robinson, R.R. and Horwitz, A.H. (1988) Escherichia coli secretion of an active chimeric antibody fragment. Science 240, 1041-1043. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. (1988) Single-chain antigen binding proteins. Science 242, 423-426. Boss, M.A., Kenten, J.H., Wood, C.R. and Emtage, J.S. (1984) Assembly of functional antibodies from immunoglobulin heavy and light chains synthesized in E. coil Nucl. Acids Res. 12, 3791-3806. Bothwell, A.L.M., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewski, IC and Baltimore, D. (1981) Heavy chain variable region contribution to the NP b family of antibodies: somatic mutation evident in a 72a variable region. Cell 24, 625-637. Bothwell, A.L.M., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewski, K. and Baltimore, D. (1982) Somatic variants of murine immunoglobulin A light chains. Nature 298, 380-382. Horwitz, A.H., Chang, C.P., Better, M., Hellstrom, K.E. and Robinson, R.R. (1988) Secretion of functional antibody and Fab fragment from yeast cells. Proc. Natl. Aead. Sci. U.S.A. 85, 8678-8682. Huston, J.S., Levinson, D., Mudgett-Hunter, M.,Tai, M.-S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85, 5879-5883. Imanishi, T. and M~ikelii,O. (1974) Inheritance of antibody specificity. I. Anti-(4-hydroxy-3-nitrophenyl) acetyl of the mouse primary response. J. Exp. Med. 140, 1498-1511. Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Leary, J.J., Brigati, D.J. and Ward, D.C. (1983) Rapid and sensitive colorimetrie method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bioblots. Proe. Natl. Acad. Sci. U.S.A. 80, 4045-4049. Lennox, E.S. (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1, 190-206. Maniatis, T., Fritseh, E.F. and Sambrook, J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. Mellor, J., Dobson, M.J., Roberts, N.A., Tuite, M.F., Emtage, J.S., White, S., I.owe, P.A., Patel, T.,, Kingsman, A.J. and Kingsman, S.M. (1983) Efficient synthesis of enzymatieally active calf ehymosin in Saccharomyces cerevisiae. Gene 24, 1-14.
300
Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. Sherman, F., Fink, G.R. and Hicks, J.B. (1986) Methods in Yeast Genetics. A Laboratory Manual. Cold Spring Harbor, NY. Skerra, A. and Pliickthun, A. (1988) Assembly of a immunoglobulin Pv fragment in Escherichia coli. Science 240, 1038-1041. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. Voller, A., Bidwell, D.E. and Bartlett, A. (1979) The enzyme linked immunosorbent assay (ELISA), Vol. 1, Dynatech Europe, U.K. Winter, G. and Milstein, C. (1991) Man-made antibodies. Nature 349, 293-299. Wood, C.R., Boss, M.A., Kenten, J.H., Calvert, J.E., Roberts, N.A. and Emtage, J.S. (1985) The synthesis and in vivo assembly of functional antibodies in yeast. Nature 314, 446-449. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33, 103-119. Zagursky, R.J., Berman, M.L, Baumeister, K. and Lomax, N. (1986) Rapid and easy sequencing of large linear double stranded DNA and supercoiled plasma DNA. Gene Anal. Teeh. 2, 89-94.
