PCR-based Gene Synthesis, Molecular Cloning, High Level Expression, Purification, and Characterization of Novel Antimicrobial Peptide, Brevinin-2R, in Escherichia Coli

Abstract

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

“…In addition to the easy and fast growth of E. coli cultures, most of the antimicrobial peptides bear no posttranslational modifications which allow for the use of E. coli as a suitable choice for large-scale production (Huang et al 2009). Several AMPs have already been successfully expressed in E. coli such as brevinin-2R (Mehrnejad et al 2008) and palusterin-2CE (Sun et al 2012). A number of fusion partners have been used to express AMPs including maltose-binding protein, thioredoxin, and green fluorescent protein (Kapust and Waugh 1999;Lee et al 1998;Skosyrev et al 2003).…”

“…Among various expression systems, Escherichia coli is a widely used system (Barrell et al 2004;Zhang et al 1998) due to its high expression level. Furthermore, fused tags which are available in several commercially available plasmid vectors helped to overcome (mask) the intrinsic toxicity of AMPs for the producing bacteria (Mehrnejad et al 2008). Xie et al (2009) have produced recombinant ranalexin in E. coli before: ranalexin was cloned with its entire sequence including prepro signal sequences and fused to glutathione Stransferase.…”

An Overview of Brevinin Superfamily: Structure, Function and Clinical Perspectives

“…vector to allow the expression of Brevinin-2R as a Trx fusion protein in E.coli (Mehrnejad et al 2008). Brevinin-2GU, an antimicrobial peptide isolated from skin secretion of the Asian frog Hylarana guntheri possesses insulin-releasing activity.…”

Molecular Cloning, Expression, Purification, and Functional Characterization of Palustrin-2CE, an Antimicrobial Peptide ofRana chensinensis

“…During the past decade, diverse purified recombinant antimicrobial peptides have been produced by various expression systems. 9,10) Among them, Escherichia coli is still the major system 11,12) due to several advantages, (i) high yield and economy, (ii) the antimicrobial peptides are non-glycosylated proteins, (iii) the fused tag can help to neutralize the toxic activities of antimicrobial peptides as to the host and are easy to purify, and (iv) the possibilities of manipulation and alteration at the gene level. 13,14) In this study, prepropalustrin-2CE3 cDNA directing the synthesis of palustrin-2CE was identified in Rana chensinensis tadpoles.…”