Applied Microbiology and Biotechnology

, Volume 98, Issue 8, pp 3679–3689

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

Authors

  • Jie-Xian Dong
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
  • Xi Xie
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
  • Da-Wei Hu
    • Entry-Exit Inspection and Quarantine of Zengcheng
  • Shu-Chi Chen
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
    • Shantou Environmental Monitoring Center
  • Yong-Sheng He
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
    • Shenzhen Academy of Metrology and Quality Inspection
  • Ross C. Beier
    • Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research ServiceUnited States Department of Agriculture
  • Yu-Dong Shen
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
  • Yuan-Ming Sun
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
  • Zhen-Lin Xu
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
    • Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food ScienceSouth China Agricultural University
Biotechnologically relevant enzymes and proteins

DOI: 10.1007/s00253-013-5324-9

Cite this article as:
Dong, J., Xie, X., Hu, D. et al. Appl Microbiol Biotechnol (2014) 98: 3679. doi:10.1007/s00253-013-5324-9
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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 fragmentClenbuterolCodon optimizationExpressionPichia pastoris

Supplementary material

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

Copyright information

© Springer-Verlag Berlin Heidelberg 2013