High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli.

Xingjun Feng, Wenshan Xu, Pei Qu, Xiaochong Li, Liwei Xing, Di Liu, Jian Jiao, Jue Wang, Zhongqiu Li, Chunlong Liu

Biotechnology progress 2015 Mar-Apr

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The antimicrobial peptide fowlicidin-2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin-2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin-2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin-2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET-32a(+), which features fusion protein thioredoxin at the N-terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria-Bertani (LB) medium. After isopropyl-β-D-thiogalactopyranoside (IPTG) induction, the fowlicidin-2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse-phase high-performance liquid chromatography (RP-HPLC), ∼6.0 mg of fowlicidin-2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria, and even drug-resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large-scale production. © 2015 American Institute of Chemical Engineers.

Citation

Xingjun Feng, Wenshan Xu, Pei Qu, Xiaochong Li, Liwei Xing, Di Liu, Jian Jiao, Jue Wang, Zhongqiu Li, Chunlong Liu. High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli. Biotechnology progress. 2015 Mar-Apr;31(2):369-74