[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding ...[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding SMAP-29 （sheep myeloid antibacterial peptides-29） mature peptide was synthesized and was biased codon usage of Pichia pastoris. The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K, transformed into Pichia pastoris strain GSll5 by electmporation, and screened with G418 for high-copy recombinants. The methanol utility plus phenotype （Mut ＋ ） was selected with MM, MD and PCR. The correctly constructed recombinant strain was induced with methanol. Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine- SDS-PAGE. [ Result] After induced with methanol for 2 d, a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29 ; the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Mo- nilia albican but no significant antibacterial effect against Escherichia coli. [ Conclusion] This study laid the foundation for the application of SMAP-29 in biomedi- cine, agriculture and other fields.展开更多
基金Supported by Basic Research Project from Science and Technology Bureau of Xinjiang Production and Construction Corps (2006JC01)National Natural Science Foundation of China (31160218)
文摘[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding SMAP-29 （sheep myeloid antibacterial peptides-29） mature peptide was synthesized and was biased codon usage of Pichia pastoris. The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K, transformed into Pichia pastoris strain GSll5 by electmporation, and screened with G418 for high-copy recombinants. The methanol utility plus phenotype （Mut ＋ ） was selected with MM, MD and PCR. The correctly constructed recombinant strain was induced with methanol. Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine- SDS-PAGE. [ Result] After induced with methanol for 2 d, a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29 ; the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Mo- nilia albican but no significant antibacterial effect against Escherichia coli. [ Conclusion] This study laid the foundation for the application of SMAP-29 in biomedi- cine, agriculture and other fields.
