Codon Optimization of SMAP-29 Gene and Its Expression in Pichia pastoris

作     者：Yaojun REN Enpeng HE Xinhua WANG Xinwen BO 

作者机构：Assets DepartmentXinjiang Normal University The Breed & Biotechnology Key Laboratory of Sheep in Reproduction & Construction Group of Xinjiang Key Laboratory of Sports Human ScienceXinjiang Normal University 

出 版 物：《Agricultural Biotechnology》 (农业生物技术（英文版）)

年 卷 期：2012年第1卷第4期

页      面：49-53页

学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

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) 

主　　题：SMAP-29 Pichia pastoris Intracellular expression Antibacterial activity 

摘      要：[ 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.