Secreted Expression And Activity Detection Of Two Hybrid Antimicrobial Peptides In Pichia Pastoris

Anti-bacterial peptide is a kind short peptide which characterized by high antimicrobial activities, wide antimicrobial sectrum and no side effects on animals. It as ideal substitute for antibiotics has aroused people's more interests. Hybrid antimicrobial peptides are a new kind of defence active peptides, which characterized by higher antimicrobial activities and shorter peptide chain, composed by part peptide chain of two or more anti-bacterial peptides. The design of anti-bacterial peptide is a hotspot which designed by acid replacement or sequence recombination with other peptides. The new designed anti-bacterial peptides are more excellent than the original. There were some problems: isolation and purification of the anti-bacterial peptides are difficult, the hybrid gene produces little amount of peptides may kill its hosts cells, the hybrid peptides which express in E.coli may lose activity and so on. In order to solve these problems, we chose the Pichia pastoris expression system.Exploring and designing new hybrid antimicrobial peptides in order to improve the activity and reduce the hemolytic effects is very important and worthy, as the high level expression in pratical application. The study of hybrid antimicrobial peptides from different prototype is hot which reach or approach the aim; David reported the CA(1-7)M(4-11) was a peptide with high activity and short size from some hybrid peptides made up from dofferent segments of Cecropin A and Melittin. As the CB(1-7)M(4-11) has never been reported the paper carried out the following research:1,Designing of the hybrid antimicrobial peptides and reconstructing of the recombinant expression vector. Based on the structure of CA(1-7)M(4-11), the new peptide CB(1-7)M(4-11) was designed which composed by the N-terminus 1-7 amino acid of Cecropin B and 4-11 amino acid of Melittin. Compared with CA(1-7)M(4-11), the fourth amino acid of CB(1-7)M(4-11) was Val in place of Leu; the structure-function relationships are initially studied by bioinformatics. According to the artiality codon of Pichia pastoris, genes of two hybrid antimicrobial peptides were reconstructed, synthetized by SOEing and cloned into pPICZ-a. The recombinant expression vectors were identified by restriction enzyme analysis and sequencing.2,Expression of the hybrid antimicrobial peptides CA(1-7)M(4-11) & CB(1-7)M(4-11) and optimization of the electrotransformation and expression. The recombinant expression vectors were transformed into host strain X-33 and the positive clones were identified. Genetically-engineered strains were fermented and the products were isolated by SDS-PAGE gel electrophoresis which presented with the same molecular weight 2.0kD as forecast. The optimal protocol for electrotransformation was as followings: 7μg plasmid, 2mm cup, P.p X-33 OD₆₀₀=1.0, 2000 votage and 2h recovery. The best expression level 126μg/mL and 127μg/mL could be gained when the seed yeast OD₆₀₀= 6.0, 0.5% methanol, 26°C for 48h. The results showed that the recombinant strains can keep stable consistence.3,Bioactivities, MIC, stability, antibacterial spectrum and the hemolytic effect of the two hybrid antimicrobial peptides were studied and analyzed. The experimental results have demonstrated that both CA-M and CB-M have broad spectrum of antibacterial activity; the inhibitory zone of CA-M were bigger than CB-M for E.coli, P.aeruginosa and B.subtilis; the opposite result occurred for S. aureus,Salmonelladerby; both CA-M and CB-M activity were not affected by different temperature; they have higher activity when pH<7 and CB-M was more endurable in alkaline environment; no hemolytic effects were observed both in vitro and in vivo, the hemolytic effects of Melittin were disappeared in CB-M. We found that the secondary structure a-helix is very important; cation property and amphipathic property are also contributing to the activity; these factors are combineing action which any factor has no linear relationship with activity.