Construction Of Prokaryotic And Eukaryotic Expression System For Cationic Antimicrobial Peptide G13

Granulysin is a cationic antimicrobial peptides from cytotoxic T lymphocytes and natural killer cells. Cationic antimicrobial peptide G13contais19amino acid residues and has a-helix and loop structure. It has antibacterial activity against bacteria but is non-toxic to animal cells. Therefore it has important research value.The expression of antimicrobial peptides often use the fusion expression technology to inhibit the antimicrobial peptide’s toxic. The fusion protein is generally a large proportion in the recombinant protein, leads to antimicrobial peptides express in lower yield. In this experiment, base on the pThiohisA-G13vector’s fusion protein, we designed a pair of reverse primer, using the inverse PCR method toremove the fusion protein’s87amino acid residues, we construct the pThiohisA-G13-A plasmid. It has highly expressed antimicrobial peptide and actual production is2.3times higher than everbefore。 It has an important value of application.The fusion protein’s cleaving has been a big problem of antimicrobial peptide fusion expression. Enzyme cleave is high costs, the chemical method, CNBr is extremely toxic.They are difficult to be widely used. In this study, Asp-Pro peptide bond will become unstable under low PH, by adding the acid hydrolysis cleavage site between the fusion protein and antimicrobial peptides, we construct the pThiohisA-G13-C plasmid and obtained the soluble expression of recombinant proteins. At the same time, we studied the reaction conditions of acid hydrolysis, and succeeded in obtaining the cationic antimicrobial peptide G13. We purified the recombinant peptide G13by cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13exhibited antibacterial activity of E. coli DH5a.This will laid the foundation for the application of antimicrobial peptides in the low-cost mass production.We study the cationic antimicrobial peptide G13of eukaryotic expression. Successfully in optimize the G13expression level in Saccharomyces cerevisiae and obtain the activity of cationic antimicrobial peptide G13in order to establish a sets of stable expression method. It provide a basis for subsequent purification and similarexpression of antimicrobial peptides.We also successfully constructed the Saccharomyces cerevisiae pYES2-α-L50A expression vector, and obtained the Enterocin L50A.This laid the foundation for subsequent expression studies.This also preliminary evidence the possibility of the Saccharomyces cerevisiae eukaryotic expression system for expressing antimicrobial substances.