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 JX, Xie X, Hu DW, Chen SC, He YS, Beier RC, Shen YD, Sun YM, Xu ZL, Wang H, and Yang JY
- Subjects
- Animals, Base Composition, Chromatography, Affinity, Enzyme-Linked Immunosorbent Assay, Gene Expression, Mice, Molecular Sequence Data, Recombinant Proteins genetics, Recombinant Proteins immunology, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Analysis, DNA, Single-Chain Antibodies genetics, Single-Chain Antibodies isolation & purification, Clenbuterol immunology, Codon, Pichia genetics, Single-Chain Antibodies immunology, Single-Chain Antibodies metabolism
- 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 encoding 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.
- Published
- 2014
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