Prokaryotic Expression Of The Antimicrobial Peptide CATH PR1-2 From The Cathelicidins Of Paa Robertingeri

The family of cathelicidins is one of the important antimicrobial peptides in the mammalian immune defense system.It has become one of the hot spots of antimicrobial peptides because of its broad spectrum and high antibacterial activity.It has been found that more than 100 kinds of cathelicidins are derived from different organisms,in which amphibians are studied at the latest.The skin of amphibians,the weak physical barrier made them into a dangerous situation.As the skin is the first line of defense which is essential for the survival of amphibians.Antimicrobial peptides as a part of its skin secretion of a large number of bioactive substances is an important part of its innate immune system.Cathelicidins consists of three parts: the N-terminal signal peptide,the cathelin domain and the C-terminal highly mature mature peptide region,which after specific protease cleavage,exposes its mature peptide region to become active antimicrobial peptides and export its biological role.In the previous study,we have obtained two Cathelicidins from the skin of Paa robertingeri by gene cloning,named Cathelicidin-PR1 and Cathelicidin-PR2(referred to as CATH PR1 and CATH PR2);The results showed that: CATH PR1 and CATH PR2 contained 29 amino acid residues(RKCNLFCKAKQKLKSLSSVIGTVVHPPRG)and 25 amino acid residues(KECKDYLCKLLMKLGSSLHIESIDP),respectively.CATH PR1 has a relatively high antimicrobial activity,especially for the drug-resistant strains found in clinical practice;however,no antimicrobial activity has been found in CATH PR2.Besides,natural extraction and chemical synthesis of antimicrobial peptides are on the high cost and technology requirements,which only apply to scientific research,not suitable for mass production of antimicrobial peptides.So this study refers to the recent decades of research on extensive genetic engineering technology,the aim is by E.coli prokaryotic expression system in order to express antimicrobial peptides,The molecular weights of both CATH PR1 and CATH PR2 are relatively low(3195.88 and 2838.34 Da,respectively),so we CATH PR1 and CATH PR2 in series to get a new antimicrobial peptide CATH PR1-2,and addition of restriction sites before and after the CATH PR1-2 sequence respectively,the plasmid pET-32 a treated with the same enzyme,the digestion product was ligated,introducing the recombinant plasmid expressing E.coli BL21,through inducible expression of IPTG,fusion polypeptide CATH PR1-2 was stably expressed;Ni-NTA column and purified by affinity chromatography,the separation of the target protein with heteroaryl;by acid cleavage,the initial recombinant protein CATH PR1-2;The antimicrobial activity assay using Candida albicans 08030102 showed that the antimicrobial activity of CATH PR1-2 was similar to the antimicrobial activity of CATH PR1.This study has a certain biological activity of the recombinant protein CATH PR1-2 by the prokaryotic expression.