Skip to main content
Log in

Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Backmann N, Zahnd C, Huber F, Bietsch A, Plückthun A, Lang H-P, Güntherodt H-J, Hegner M, Gerber C (2005) A label-free immunosensor array using single-chain antibody fragments. Proc Natl Acad Sci USA 102:14587–14592

    Article  PubMed  CAS  Google Scholar 

  • Byrne MP, Titball RW, Holley J, Smith LA (2000) Fermentation, purification, and efficacy of a recombinant vaccine candidate against Botulinum neurotoxin type F from Pichia pastoris. Protein Expr Purif 18:327–337

    Article  PubMed  CAS  Google Scholar 

  • Chang S-W, Lee G-C, Shaw J-F (2006) Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J Agric Food Chem 54:815–822

    Article  PubMed  CAS  Google Scholar 

  • Cramer B, Bretz M, Humpf H-U (2007) Stable isotope dilution analysis of the Fusarium mycotoxin zearalenone. J Agric Food Chem 55:8353–8358

    Article  PubMed  CAS  Google Scholar 

  • Damasceno LM, Pla I, Chang H–J, Cohen L, Litter G, Old LJ, Batt CA (2004) An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif 37:18–26

    Article  PubMed  CAS  Google Scholar 

  • Hu S, Li L, Qiao J, Guo Y, Cheng L, Liu J (2006) Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expr Purif 47:249–257

    Article  PubMed  CAS  Google Scholar 

  • Hu X, O’Dwyer R, Wall JG (2005) Cloning, expression and characterization of a single-chain Fv antibody fragment against domoic acid in Escherichia coli. J Biotechnol 120:38–45

    Article  PubMed  CAS  Google Scholar 

  • Lee MS, Hur YJ, Kim TI, Kim KH, Kim JH, Choi HJ, Koh KN (2002) Surface plasmon resonance study on the interaction between the fructose and phenylboronic acid monolayer. Microchem J 72:315–321

    Article  CAS  Google Scholar 

  • Ogunjimi AA, Chandler JM, Gooding CM, Recinos A III, Choudary PV (1999) High-level secretory expression of immunologically active intact antibody from the yeast Pichia pastoris. Biotechnol Lett 21:561–567

    Article  CAS  Google Scholar 

  • Pestka JJ, Abouzied MN, Sutikno (1995) Immunological assays for mycotoxin detection. Food Technol 49:120–128

    CAS  Google Scholar 

  • Scorer CA, Clare JJ, McCombie WR, Romanos MA, Sreekfrishna K (1994) Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology 12:181–184

    Article  PubMed  CAS  Google Scholar 

  • Sreekrishna K (1993) Strategies for optimizing protein expression and secretion in the methylotrophic yeast Pichia pastoris. In: Baltz RH, Hegeman GD, Skatrud PL (eds) Industrial microorganisms: basic and applied molecular genetics. American Society for Microbiology, Washington, pp 119–126

    Google Scholar 

  • Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay J-T, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190:55–62

    Article  PubMed  CAS  Google Scholar 

  • Yuan Q, Clarke JR, Linz JE, Pestka JJ, Hart LP (1997) Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone. Appl Environ Microbiol 63:263–269

    PubMed  CAS  Google Scholar 

  • Yuan Q, Hu W, Pestka JJ, He SY, Hart LP (2000) Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants. Appl Environ Microbiol 66:3499–3505

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This work was supported by a research grant from the Korea Food Research Institute.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hyang Sook Chun.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chang, HJ., Choi, SW. & Chun, H.S. Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris . Biotechnol Lett 30, 1801–1806 (2008). https://doi.org/10.1007/s10529-008-9770-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-008-9770-x

Keywords

Navigation