Recombinant Expression In Escherichia Coli Of Antimicrobial Domains Derived From Granulysin

Nowadays, resistance of microbial pathogens to traditional antibiotics is an increasingly severe problem, resulting in urgent demand for new types of antimicrobial drugs, thus the development of new antibiotics has become more and more important. Antimicrobial peptides, a kind of cationic small peptides induced in vivo, have been used to resist infection by pathogenic microbes. Since the discovery of antimicrobial peptides, over a thousand of candidates with different activities have been identified. As a new type of antimicrobial substance, they are widely distributed in many living organisms, from the lowest virus to the higher propagation. And more evidence indicates they are play an essential role in innate immunity. Duing to their special antimicrobial mechanisms, which are different from other traditional antibiotics, antibacterial peptides may become new-type antibacterial drug. And their potential value has been of widespread concern.Granulysin and NK-lysin are homologous cationic antimicrobial peptides produced by human and pig cytolytic lymphocytes, respectively. They are members of the saposin-like protein family SAPLIP with a moderate residue similarity (67%), both having antimycobacterial activity. Their tetiary structures comprise five amphipathic a-helices (H1-H5). G13 peptide, contained 19 amino acids derived from granulysin, shows strong antimicrobial activities against fungus and bacteria, but has no effects on liposome and animal cells. NKLF-2 domain, contained 22 amino acids derived from NK-lysin, could kill Mycobacterium tuberculosis, but do not display haemolytic activity.In this study we used two different types of strategies to express G13 and NKLF-2 domains in E.coli. In order to explore a new approach for high-level expression of the different antimicrobial domains of Granulysin and to avoid the toxic effects on E.coli host cells during its expression, we designed a novel strategy for the production of recombinant cationic antimicrobial peptides by co-expressing an anionic ligand. A segment of DNA sequence encoding a anionic ligand and the cationic antimicrobial peptide gene were separately inserted into the two MCS sites of the plasmid pACYCDuet-1. The recombiant plasmid was transformed into competent E.coli cells and co-expression was induced by IPTG The results demonstrated that a higher expression yield was achieved by using the co-expression method. It will be helpful and meaningful for further study of antibacterial peptides and provides an economical but efficient preparation method.In order to improve the efficiency of translation initiation, the other strategy was that the nucleotides distributing over the translation initiation region of antimicrobial peptide genes was mutated without changing their amion acid sequences. With the purpose of investigating the relationship between the structure of mRNA TIR and gene expression and predicting level of the antimicrobial domains expression in E.coli.We analyzed some experimental data about the expression levels of the domains in vector pACYCDuet-1. The mRNA secondary structures were predicted simultaneously by computer-aided design. Results of the predictive analysis showed the difference between the energy of mRNA secondary structures. According to some references, we suggest a set of judgements. It will be helpful for us to rationally modify these antimicrobial domains for enhancing recombinant expression efficiency.