Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli

Abstract

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD-intein-pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.

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

“…Also, any additional amino acids at the N-terminus may inhibit antimicrobial activity of AMPs (Hancock and Sahl 2006). Therefore, the N-terminal fusion tags must be removed enzymatically with specific proteases since nonspecific cleavage by methionine aminopeptidase may lead to unstable and inactive products (Boix et al 1996), while intein self-cleavage systems require intensive pH, temperature, and time optimization (Humphries et al 2002;Wang et al 2012). Factor Xa protease cleaves specifically at the C-terminal side of its recognition site (Ile-Glu-Gly-Arg) and facilitates the successful production of an identical ranalexin without any additional amino acids.…”

Investigating the antimicrobial peptide ‘window of activity’ using cationic lipopeptides with hydrocarbon and fluorinated tails

“…The development and widespread application of CAMPs is further complicated by their long length, as the peptides are impractical to produce synthetically. In vivo production of the 15-30 residue sequences is more cost effective but requires the development of sophisticated production and purification methodology to limit bacterial toxicity [10,11].…”

Recent Advances in Developing Insect Natural Products as Potential Modern Day Medicines

“…An alternative strategy to reduce costs of mass production is to use recombinant technology but this has been hindered by the antibacterial activity of the AMPs and their proteolytic degradation during production [114]. Recent studies, however, have used cost effective, modified recombinant techniques with E.coli or with the methylotrophic yeast Pichia pastoris as vectors, to produce fully functional insect cecropins capable of killing a range of bacteria, including MRSA [115, 116]. Finally, regulatory rules governing the required performance prior to approval for the release of drugs in the USA (FDA) and Europe have all to be navigated at extra cost [3].…”