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ARS Home » Plains Area » College Station, Texas » Southern Plains Agricultural Research Center » Food and Feed Safety Research » Research » Publications at this Location » Publication #294830
Title: Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

Author
item DONG, JIE-XIAN - South China Agricultural University
item XIE, XI - South China Agricultural University
item HU, DA-WEI - South China Agricultural University
item CHEN, SHU-CHI - South China Agricultural University
item HE, YONG-SHENG - South China Agricultural University
item Beier, Ross
item SHEN, YU-DONG - South China Agricultural University
item SUN, YUAN-MING - South China Agricultural University
item XU, ZHEN-LIN - South China Agricultural University
item WANG, HONG - South China Agricultural University
item YANG, JIN-YI - South China Agricultural University

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2013
Publication Date: 4/1/2014
Publication URL: http://handle.nal.usda.gov/10113/58806
Citation: Dong, J., Xie, X., Hu, D., Chen, S., He, Y., Beier, R.C., Shen, Y., Sun, Y., Xu, Z., Wang, H., Yang, J. 2014. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris. Applied Microbiology and Biotechnology. 98:3679-3689.

Interpretive Summary: Clenbuterol can cause the reduction of fat and the increase of lean muscle tissue. Clenbuterol is a beta-agonist bronchodilator and as a result, is not allowed to be used in food animal production. An effective method to analyze for clenbuterol residues can utilize a portion of an antibody, referred to as a single-chain variable fragment (scFv), and was developed against clenbuterol. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once antibodies to a foreign substance are isolated, they can be used in a method to detect the presence of that foreign substance in an enzyme-linked immunosorbent assay (ELISA). An ELISA is an easy-to-use test and is similar to commonly used over-the-counter pregnancy tests. The work described here optimizes a method for production of the scFv antibody portion used in the ELISA for analysis of clenbuterol. The optimized scFv production method has a 2.35-fold higher yield of scFv, which makes it a viable immuno reagent.

Technical Abstract: To improve expression efficiency of the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse in the methylotrophic yeast Pichia pastoris (P. pastoris) GS115, the DNA sequence encoding for CBL-scFv was designed and synthesized 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 recombinant CBL-scFv optimized 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 CBL-recognized activity by the 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 ng mL^–1 and 0.77 ng mL^–1, respectively.