1. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris.
- Author
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Dong, Jie-Xian, Xie, Xi, Hu, Da-Wei, Chen, Shu-Chi, He, Yong-Sheng, Beier, Ross, Shen, Yu-Dong, Sun, Yuan-Ming, Xu, Zhen-Lin, Wang, Hong, and Yang, Jin-Yi
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GENETIC code , *NUCLEOTIDE sequence , *CLENBUTEROL , *PERFORMANCE-enhancing drugs , *PICHIA pastoris , *ENZYME-linked immunosorbent assay - 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 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, respectively. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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