Abstract
A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of α-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.
Similar content being viewed by others
References
Adam A, Gottschling DE, Kaiser CA, Stearns T (eds) (1997) Isolation of genomic DNA for Southern blot analysis. In: Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., pp 110–111
Boder ET, Wittrup KD (2001) Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol 328:431–444
Boder ET, Midelfort SK, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA 97:10701–10705
Duenas M, Ayala M, Vazquez J, Ohlin M, Soderlind E, Borrenaeck CAK, Gavilondo JV (1995) A point mutation in murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli. Gene 158:61–66
Fujii I, Tanaka F, Miyashita H, Tanimura R, Kinoshita K (1995) Correlation between antigen-combining-site structures and functions within a panel of catalytic antibodies generated against a single transition state analog. J Am Chem Soc 117:6199–6209
Fujii I, Fukuyama S, Iwabuchi Y, Tanimura R (1998) Evolving catalytic antibodies in a phage-displayed combinatorial library. Nat Biotechnol 16:463–467
Horwitz AH, Chamg CP, Better M, Hellstrom KE, Robinson RR (1988) Secretion of functional antibody and Fab fragment from yeast cells. Proc Natl Acad Sci USA 85:8678–8682
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Ito W, Iba Y, Kurosawa Y (1993) Effects of substitution of closely related amino acids at the contact surface in an antigen-antibody complex on thermodynamic parameters. J Biol Chem 268:16639–16647
Keike MC, Cho BK, Boder ET, Kranz DM, Wittrup KD (1997) Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng 10:1303–1310
Keike MC, Shusta EV, Boder ET, Teyton L, Wittrup KD, Kranz DM (1999) Selection of functional T cell receptor mutants from a yeast surface display library. Proc Natl Acad Sci USA 96:5651–5656
Knappik A, Pluckthum A (1995) Engineering turns of a recombinant antibody improve its in vivo folding. Protein Eng 8:81–89
Miyashita H, Hara T, Tanimura R, Tanaka F, Kikuchi M, Fujii I (1994) A common ancestry for multiple catalytic antibodies generated against a single transition-state analog. Proc Natl Acad Sci USA 91:6045–6049
Miyashita H, Hara T, Tanimura R, Fukuyama S, Cagnon C, Kohara A, Fujii I (1997) Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization. J Mol Biol 267:1247–1257
Murai T, Ueda M, Yamamura M, Atomi H, Shibasaki Y, Kamasawa N, Osumi M, Amachi T, Tanaka A (1997) Construction of a starch-utilizing yeast by cell surface engineering. Appl Environ Microbiol 63:1362–1366
Murai T, Ueda M, Kawaguchi T, Arai M, Tanaka A (1998) Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing β-glucosidase and carboxymethylcellulase from Aspergillus aculeatus. Appl Environ Microbiol 64:4857–4861
Pollack SJ, Jacobs JW, Schultz PG (1986) Selective chemical catalysis by an antibody. Science 234:1570–1573
Schoonjans R, Willwms A, Schoonooghe S, Fiers W, Grooten J, Mertens N (2000) Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives. J Immunol 165:7050–7057
Sikorski RS, Hieter PA (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27
Takahashi N, Kakinuma H, Liu L, Nishi Y, Fujii I (2001) In vitro abzyme evolution to optimize antibody recognition for catalysis. Nat Biotechnol 19:563–567
Tramontano A, Janda KD, Lemer RA (1986) Catalytic antibodies. Science 234:1566–1570
Ueda M, Tanaka A (2000a) Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18:121–140
Ueda M, Tanaka A (2000b) Cell surface engineering of yeast—construction of arming yeast with biocatalyst. J Biosci Bioeng 90:125–136
Ulrich HD, Patten PA, Yang PL, Romesberg FE, Schultz PG (1995) Expression studies of catalytic antibodies. Proc Natl Acad Sci USA 92:11907–11911
Zou W, Ueda M, Murai T, Tanaka A (2000) Establishment of a simple system to analyze the molecular interaction in the agglutination of Saccharomyces cerevisiae. Yeast 16:995–1000
Zou W, Ueda M, Tanaka A (2002) Screening of an endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library. Appl Microbiol Biotechnol 58:806–812
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lin, Y., Tsumuraya, T., Wakabayashi, T. et al. Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface. Appl Microbiol Biotechnol 62, 226–232 (2003). https://doi.org/10.1007/s00253-003-1283-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-003-1283-x