Applied Microbiology and Biotechnology

, Volume 98, Issue 8, pp 3679–3689 | Cite as

Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

  • Jie-Xian Dong
  • Xi Xie
  • Da-Wei Hu
  • Shu-Chi Chen
  • Yong-Sheng He
  • Ross C. Beier
  • Yu-Dong Shen
  • Yuan-Ming Sun
  • Zhen-Lin Xu
  • Hong Wang
  • Jin-Yi Yang
Biotechnologically relevant enzymes and proteins

Abstract

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L–1 in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL–1, respectively.

Keywords

Single-chain variable fragment Clenbuterol Codon optimization Expression Pichia pastoris 

Notes

Acknowledgments

J.-X. Dong and X. Xie contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant 30871755, 31271866), Science and Technology Plan Projects in Guangdong Province (2012A020100002, 2010A032000001–4), and the Doctoral Innovation Program of Hopson Zhujiang Education Fund (H2011001).

Supplementary material

253_2013_5324_MOESM1_ESM.pdf (99 kb)
ESM 1 (PDF 98 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Jie-Xian Dong
    • 1
  • Xi Xie
    • 1
  • Da-Wei Hu
    • 2
  • Shu-Chi Chen
    • 1
    • 3
  • Yong-Sheng He
    • 1
    • 4
  • Ross C. Beier
    • 5
  • Yu-Dong Shen
    • 1
  • Yuan-Ming Sun
    • 1
  • Zhen-Lin Xu
    • 1
  • Hong Wang
    • 1
  • Jin-Yi Yang
    • 1
  1. 1.Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural UniversityGuangzhouPeople’s Republic of China
  2. 2.Entry-Exit Inspection and Quarantine of ZengchengZengchengPeople’s Republic of China
  3. 3.Shantou Environmental Monitoring CenterShantouPeople’s Republic of China
  4. 4.Shenzhen Academy of Metrology and Quality InspectionShenzhenPeople’s Republic of China
  5. 5.Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research ServiceUnited States Department of AgricultureCollege StationUSA

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