Biotechnologically relevant enzymes and proteins

Applied Microbiology and Biotechnology

, Volume 98, Issue 8, pp 3679-3689

First online:

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

  • Jie-Xian DongAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University
  • , Xi XieAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University
  • , Da-Wei HuAffiliated withEntry-Exit Inspection and Quarantine of Zengcheng
  • , Shu-Chi ChenAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural UniversityShantou Environmental Monitoring Center
  • , Yong-Sheng HeAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural UniversityShenzhen Academy of Metrology and Quality Inspection
  • , Ross C. BeierAffiliated withFood and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture
  • , Yu-Dong ShenAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University
  • , Yuan-Ming SunAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University
  • , Zhen-Lin XuAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University
    • , Hong WangAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University Email author 
    • , Jin-Yi YangAffiliated withGuangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University Email author 

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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 fragment Clenbuterol Codon optimization Expression Pichia pastoris