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

Permanent URL:
http://handle.nal.usda.gov/10113/58806
File:
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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.
Author(s):
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
Subject(s):
Pichia pastoris , amino acids , antibodies , chromatography , clenbuterol , codons , detection limit , immunoassays , mice , nucleotides , yeasts
Note:
USDA Scientist Submission
Source:
Applied Microbiology and Biotechnology 2014 v.98
Language:
English
Year:
2014
Collection:
Journal Articles, USDA Authors, Peer-Reviewed
Rights:
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.