Study On The Strategies Of High-level Expression And On The Activity Of The Antimicrobial Peptides

The toxicity of recombinant antimicrobial peptides upon the host cell affects the level of protein expression in genetic engineering. One of the strategies to achieve high expression is changing the fusion partners.In this study, we analyze the OD600 of the engineering bacteria and the effects of different fusion partners on the inhibition of the toxicity of G13. We mutated the gene sequence of the fusion partners of pET28a-G13, the charge of the fusion partners of the mutant was changed in the N-terminal of G13. We designed the mutation primers. A PCR reaction was conducted with the primers and pET28a-G13 as template. The mutated recombinant named pET28a'-G13 and induced by IPTG. The double transformed cells are inoculated to different ratios of antibiotic, to examine the target protein protein expression level and the copy numbers of the plasmids. It was found that the inhibition effect of the fusion partners was greatly affected the net negative charge and the relative location of the acidic amino acid residues.In addition, we tried to improve the method of the purification of G13 domain, to decrease the protein modification of urea through changing the denaturants and their concentrations. Moreover, we obtained purified G13 domain, tested its activity, further to study the hemolytic of it and do the conduct probability analysis.Furthermore, we studied on the expression and activity of antimicrobial peptide vgf-1. We found an antimicrobial peptide (vgf-1) through the protein database of NCBI, that come from cobra venom and has effect on Mycobacterium tuberculosis. According to the protein sequence of vgf-1 and the codon preference of E. coli, we obtained the gene sequences of it. The gene sequence of Vgf-1 was obtained by overlapping PCR, which was amplified by three steps. The cleansed PCR product of three fragments were attach to the expression vector pBAD/TOPO, and transformed into E.coli TOP 10. After sequencing, we named them pBAD-Vgf-1-A, B, C. They were induced by arabinose, treated by ultrasonic treatment, the result of SDS-PAGE electrophoresis detection shows that fusion proteins were in correct position, and the target protein exists in the form of inclusion bodies. The total cell protein measured by Bradford, we analyze the the expression of fusion protein were about the overall protein content of 58.3%,48.7% and 45.5% with Bandscan software. So the projected per liter of fermentation broth produced works about inclusion as 178mg,142mg and 124mg.The enterokinase cutting system of fusion protein pBAD-Vgf-1-B, C was optimization. We used the best system to cut on the fusion protein, after activity detected the purpose of peptide has no antibacterial activity. We used various proportions of GSH/GSSG refolding system of the inclusion body, after concentrated and cutting, the protein has no activity. The other method was tried, after cutting the fusion protein, we treated the protein by the GSH/GSSG refolding system, the result showed that they has no activity.