Construction Of Saccharomyces Cerevisiae Expression System For Secretion Of G13Domain Derived From Granulysin And LfcinB

Bovine lactoferricin (LFcin B) is released from the N-terminal domain of bovine lactoferrin by acid pepsin digestion. It contains25acid residues and has bactericidal, antifungal, antiparasitic, antitumor, antiviral, immunomodulatory activities. Because of its limited source and the costly price by chemically synthesis, using the technique of gene engingeering to produce LfcinB to satisfy the need of research and application becomes the research focus. According to the codon usage bias of Saccharomyces cescerevisiae we designed the DNA sequence of LfcinB. and then chemically synthesized and cloned the DNA sequence to construct the recombinant plasmid pYES2-a-LfcinB. The constructed recombinant plasmid pYES2-a-LfcinB was transformed into the competent E.coli DH5a to be multiplied, and then extracted and transformed into the competent Saccharomyces cerevisiae INVScl cells by electroporation. The yeast transformants were screened on selective culture medium plates devoid of Uracil (SC-U). The identified transformants were cultured in shaking flasks containing liquid selective medium and induced with galactose to express LfcinB. By doing antimicrobial activity test to research the characterization of LfcinB and measure the antimicrobial activity of LfcinB with cylinder-plate method. The result showed that:The recombinant plasmids pYES2-a-LfcinB was correctly constructed and transformed into host yeast strains as examined by double enzymatic digestion and sequencing. The expression of the recombinant peptide was induced as shown by Tricine-SDS-PAGE analysis of the condensed culture supernatant. The peptide displayed antimicrobial activity against some E.coli DH5a and Bacillus subtilis strains. The optimum condition for expression induction were as follows:26℃,96h,3%galactose concentration, and the heat stability and the disulfide bond had no influence on its antimicrobial activity. In this study we achieved secretory expression of biologically active LfcinB in Saccharomyces cerevisiae, laying a foundation for further research and development of recombinant LfcinB.G13contains helix2and loop2of granulysin. The positive charges and the stable α-helix are essential for the antimicrobial activity of LfcinB. We successfully optimized the G13expression conditions in Saccharomyces cerevisiae and made research on the characterization of G13.The results showed that the peptide G13had thermostability. G13has strong tolerance to acid and doesn’t have alkali resistance.Because the molecular weight of LfcinB and G13are relatively small,and the output of both are low,we constructed recombinant plasmid pYES2-α-G, and then transformed it into the competent Saccharomyces cerevisiae INVScl cells by electroporation. Then we got peptide LG by induced by galactose. The peptide LG displayed antimicrobial activity against some E.coli DH5a and Bacillus subtilis strains, and its antimicrobial activity is stronger than the LfcinB and G13. That has important application value.Plasmid vectors are used to produce proteins and peptides. Losing the plasmid means the descent of the production. So the phenomenon of plasmid instability is an urging problem. By DES mutagenesis of the INVSc1containing pYES2-α-G13, and sceening with5fluorouracil, we got mutant strains in which the plasmids are more stable when growing in SC medium.