甜味蛋白Brazzein基因合成及其在酵母中表达的初步研究

Study on the Synthesis of Sweet Protein Brazzein Gene and the Expression in the Pichia Pastoris Yeast

采用pGAPZαA为表达载体,SMD1168毕赤酵母为受体系统,构建分泌型的Brazzein毕赤酵母重组菌。按照毕赤酵母偏爱密码子设计Brazzein基因,共设计4对引物,采用SOE-PCR法合成Brazzein基因,构建克隆载体pUC57-Bra与酵母表达载体pGAPZαA-Bra。将重组质粒线性化,电转导入Pichia.pastoris SMD1168中,用高浓度的Zeocin筛选高拷贝转化子。将重组菌株接种于YPD培养基诱导表达,进行SDS-PAGE分析。成功构建了分泌型的Brazzein毕赤酵母重组菌,SDS-PAGE表明,目标蛋白的相对分子量与理论值一致,诱导72h后的目的蛋白表达量达0.12g/L。

Construct the secretion Pichia pastoris yeast recombinant germ contain Brazzein gene with pGAP Zeta express vector and SMD1168 Pichia pastoris yeast host systerm. According to the Pichia pastoris preference codon usage, four pairs of primers were designed and synthesized. Finally the Brazzein gene was amplified by SOE-PCR, then successfully constructed the recombinant cloning plasmid pUC57-Bra and yeast expressing plasmid pGAPZ α A-Bra. After linealization of the plasmids with Avr Ⅱ, Brazzein gene was integrated into the genome of host yeast Pichia Pastoris SMD1168 by electroporation. After the optimization of expression conditions the recombination protein was expressed successfully in Pichia yeast. SDS-PAGE analyzing indicated a molecular weight of 6.5 kDa protein was obtained at inducing 72 hours. The expression quantity of the recombinant protein amannted to 0.12 g/L.