title: Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris.
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.
authors.affiliation: Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou, Guangdong Province, 510642, People's Republic of China.
authors.forename: Jie-Xian
authors.initials: JX
authors.lastname: Dong
authors.forename: Xi
authors.initials: X
authors.lastname: Xie
authors.forename: Da-Wei
authors.initials: DW
authors.lastname: Hu
authors.forename: Shu-Chi
authors.initials: SC
authors.lastname: Chen
authors.forename: Yong-Sheng
authors.initials: YS
authors.lastname: He
authors.forename: Ross C
authors.initials: RC
authors.lastname: Beier
authors.forename: Yu-Dong
authors.initials: YD
authors.lastname: Shen
authors.forename: Yuan-Ming
authors.initials: YM
authors.lastname: Sun
authors.forename: Zhen-Lin
authors.initials: ZL
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Jin-Yi
authors.initials: JY
authors.lastname: Yang
mesh_terms: D000818:Animals; D001482:Base Composition; D002846:Chromatography, Affinity; D002976:Clenbuterol / Q000276:immunology*; D003062:Codon; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D002976:Clenbuterol