Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris
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.
KeywordsSingle-chain variable fragment Clenbuterol Codon optimization Expression Pichia pastoris
J.-X. Dong and X. Xie contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant 30871755, 31271866), Science and Technology Plan Projects in Guangdong Province (2012A020100002, 2010A032000001–4), and the Doctoral Innovation Program of Hopson Zhujiang Education Fund (H2011001).
- Chan CP-y, Cheung Y-c, Renneberg R, Seydack M (2008) New trends in immunoassays. In: Advances in biochemical engineering/biotechnology: biosensing for the 21st century, vol 109. Springer, Berlin, pp 123–154Google Scholar
- Chen S-C, He Y-S, Xu X-Y, Lu T-Z, Wang H (2011) Expression of a single chain Fv antibody against clenbuterol in Pichia pastoris. Chin Sci Technol Food Ind 7:217–219Google Scholar
- Koganesawa N, Aizawa T, Shimojo H, Miura K, Ohnishi A, Demura M, Hayakawa Y, Nitta K, Kawano K (2002) Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris. Protein Expr Purif 25:416–425PubMedCrossRefGoogle Scholar
- Maertens B, Spriestersbach A, von Groll U, Roth U, Kubicek J, Gerrits M, Graf M, Liss M, Daubert D, Wagner R, Schäfer F (2010) Gene optimization mechanisms: a multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli. Protein Sci 19:1312–1326PubMedCentralPubMedCrossRefGoogle Scholar
- País-Chanfrau JM, García Y, Licor L, Besada V, Castellanos-Serra L, Cabello CI, Hernández L, Mansur M, Plana L, Hidalgo A, Támbara Y, del Abrahantes-Pérez CM, del Toro Y, Valdés J, Martínez E (2004) Improving the expression of mini-proinsulin in Pichia pastoris. Biotechnol Lett 26:1269–1272PubMedCrossRefGoogle Scholar
- Rao NH, Balou BP, Rajendra L, Sriraman R, Pang Y-YS, Schiller JT, Srinivasan VA (2011) Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles. Vaccine 29:7326–7334PubMedCentralCrossRefGoogle Scholar