title: High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli.
abstract: 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.
authors: College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, China.
authors: Xingjun
authors: X
authors: Feng
authors: Wenshan
authors: W
authors: Xu
authors: Pei
authors: P
authors: Qu
authors: Xiaochong
authors: X
authors: Li
authors: Liwei
authors: L
authors: Xing
authors: Di
authors: D
authors: Liu
authors: Jian
authors: J
authors: Jiao
authors: Jue
authors: J
authors: Wang
authors: Zhongqiu
authors: Z
authors: Li
authors: Chunlong
authors: C
authors: Liu
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics*; D002479:Inclusion Bodies / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C504855:CMAP27 protein, chicken; D011993:Recombinant Fusion Proteins
keywords: Escherichia coli; antibacterial activity; antimicrobial peptides; cyanogen bromide; fowlicidin-2; recombinant expression