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.

Rational Design and Antimicrobial Potency Assessment of Abaecin Analogues

“…In this study, Abaecin, derived from Bombus pascuorum, was utilized as a fundamental template to reconstitute specific fragments with those from the antimicrobial peptides of Fowlicidin-2, , PEM-2, , Piscidin, , Cecropin A, − or short peptide (RIIRPIIQIIKQKIR) . This resulted in the generation of a series of engineered peptides (AC1 ∼ AC7).…”

Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides

“…The captured AMPs to the long-chain-hydrocarbon resin can then be eluted by increasing concentration of organic solvent such as acetonitrile to weaken the hydrophobic forces. RP-HPLC is also useful as a final polishing step of the AMP after SEC to improve purity, although the purification yield was severely compromised (Feng et al 2015). The levels of purity and yield are always a trade off, and must be considered when designing downstream-processing methods.…”

Enhanced expression of cysteine‐rich antimicrobial peptide snakin‐1 in Escherichia coli using an aggregation‐prone protein coexpression system

“…Among these methods, the E. coli expression system is the most often utilized for AMPs production, largely because E. coli is easy to handle, is inexpensive, and grows rapidly. The formation of inclusion bodies in the E. coli cell cytoplasm is often considered as a suitable technique for heterologous expression of AMPs . Although protein expression in the form of inclusion bodies is generally considered undesirable, it has proven beneficial in a number of biotechnological applications .…”