Secretory Expression Of Bovine Lactoferricin B Fused With Fluorescent Protein In Saccharomyces Cerevisiae

Recently,the use of antibiotics has led to problems,such as "Superbacteria".Therefore,we are in need of a new drug as a substitute for antibiotics.Bovine lactoferrin(LfcinB)is a new type of antibacterial peptide found in recent years.Though contains 25 amino acids,its antibacterial activity is more than 400 times as much as lactoferrin.LfcinB fused with red fluorescent protein(mApple)and green fluorescent protein(EGFP)respectively to improve stability,.Recombinant plasmid pYES2-a-LfcinB-mApple and pYES2-a-LfcinB-EGFP were constructed and expressed in Saccharomyces cerevisiae.The antibacterial activity of the expressed products against Escherichia coli and Staphylococcus aureus was detected by tube dish method.The fluorescence of recombinant yeast was observed by laser confocal fluorescence microscope,and the existence of target protein was analyzed by SDS-PAGE.The vector of pYES2-a-LfcinB-EGFP were transformed into Saccharomyces cerevisiae W303 and INVSC1,and with glucose and raffinose respectively as the carbon source of culture medium.The results of inhibition experiments showed that the engineered W303 showed higher antimicrobial activity and quicker induction than engineered INVSC1.Compared with raffinose,presence of glucose in culture medium did not delay the expression as expected,but reduced the maximum diameter of inhibition zones.It was found that the obvious fluorescence was observed in the recombinant yeast cells of the experimental group,and was not observed in the control group,and the secretion of recombinant protein had no adverse effect on the growth of recombinant yeast cells.The recombinant protein had obvious antibacterial activity against Escherichia coli DH5 and Staphylococcus aureus.However,it was found that the target protein band was not detected by SDS-PAGE.One of the reasons is that the target protein is hydrolyzed.The another is that there are multiple glycosylation sites in the fusion protein.