Applied Microbiology and Biotechnology

, Volume 62, Issue 2–3, pp 226–232 | Cite as

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface

  • Y. Lin
  • T. Tsumuraya
  • T. Wakabayashi
  • S. Shiraga
  • I. Fujii
  • A. Kondo
  • M. Ueda
Original Paper

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.

References

  1. 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 110–111Google Scholar
  2. Boder ET, Wittrup KD (2001) Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol 328:431–444Google Scholar
  3. Boder ET, Midelfort SK, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA 97:10701–10705CrossRefPubMedGoogle Scholar
  4. 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 158:61–66CrossRefPubMedGoogle Scholar
  5. 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 117:6199–6209Google Scholar
  6. Fujii I, Fukuyama S, Iwabuchi Y, Tanimura R (1998) Evolving catalytic antibodies in a phage-displayed combinatorial library. Nat Biotechnol 16:463–467PubMedGoogle Scholar
  7. 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 85:8678–8682PubMedGoogle Scholar
  8. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedGoogle Scholar
  9. 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 268:16639–16647PubMedGoogle Scholar
  10. 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 10:1303–1310CrossRefPubMedGoogle Scholar
  11. 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 96:5651–5656PubMedGoogle Scholar
  12. Knappik A, Pluckthum A (1995) Engineering turns of a recombinant antibody improve its in vivo folding. Protein Eng 8:81–89PubMedGoogle Scholar
  13. 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 91:6045–6049PubMedGoogle Scholar
  14. 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 267:1247–1257CrossRefPubMedGoogle Scholar
  15. 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 63:1362–1366PubMedGoogle Scholar
  16. 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 64:4857–4861PubMedGoogle Scholar
  17. Pollack SJ, Jacobs JW, Schultz PG (1986) Selective chemical catalysis by an antibody. Science 234:1570–1573PubMedGoogle Scholar
  18. 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 165:7050–7057PubMedGoogle Scholar
  19. Sikorski RS, Hieter PA (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  20. Takahashi N, Kakinuma H, Liu L, Nishi Y, Fujii I (2001) In vitro abzyme evolution to optimize antibody recognition for catalysis. Nat Biotechnol 19:563–567CrossRefPubMedGoogle Scholar
  21. Tramontano A, Janda KD, Lemer RA (1986) Catalytic antibodies. Science 234:1566–1570PubMedGoogle Scholar
  22. Ueda M, Tanaka A (2000a) Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18:121–140Google Scholar
  23. Ueda M, Tanaka A (2000b) Cell surface engineering of yeast—construction of arming yeast with biocatalyst. J Biosci Bioeng 90:125–136Google Scholar
  24. Ulrich HD, Patten PA, Yang PL, Romesberg FE, Schultz PG (1995) Expression studies of catalytic antibodies. Proc Natl Acad Sci USA 92:11907–11911PubMedGoogle Scholar
  25. 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 16:995–1000CrossRefPubMedGoogle Scholar
  26. 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 58:806–812CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Y. Lin
    • 1
    • 4
  • T. Tsumuraya
    • 2
  • T. Wakabayashi
    • 2
  • S. Shiraga
    • 1
  • I. Fujii
    • 2
  • A. Kondo
    • 3
  • M. Ueda
    • 1
  1. 1.Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of EngineeringKyoto UniversityKyotoJapan
  2. 2.Biomolecular Engineering Research Institute and Protein Engineering Research InstituteOsakaJapan
  3. 3.Department of Chemical Science and Engineering, Faculty of EngineeringKobe UniversityKobeJapan
  4. 4.Department of BiotechnologySouth China University of TechnologyWushanChina

Personalised recommendations