甜味蛋白Brazzein在毕赤氏酵母中表达的初步研究

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

目的:采用pGAPZαA为表达载体,SMD1168毕赤酵母为受体系统,构建分泌型的甜味蛋白Brazzein毕赤酵母重组菌。方法:按照毕赤酵母偏爱密码子设计Brazzein基因,共设计4对引物。在上、下游引物分别引入XbaI与XhoI酶切识别位点,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,电转导入毕赤酵母(Pichia. pastoris SMD1168)中,用高浓度的抗生素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。结果: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质量与理论值一致,在诱导72h后目的蛋白表达量达0.12g/L。对纯化后的蛋白进行生物活性测定,经鉴定具有一定的甜味,表达的Brazzein蛋白生物学活性良好。

Objectives： Construct the secretion Pichia pastoris yeast recombinant germ contain Brazzein gene with pGAP Zeta as express vector and SMDl168 Pichia pastoris yeast as host system. Methods： According to the Pichia pastoris preference codon usage, four pairs of primers were designed and synthesized. Xho I＋Xba I cutting sites were introduced at both the N-and C-terminal ends by genetc modification to convenience of clone. Finally the Brazzein gene was amplified by SOE-PCR, then successfully constructed the recombinant cloning plasmid pUC57-Bra and yeast expressing plasmid pGAPZαA-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. Results： SDS-PAGE analysis indicated a molecular weight of 6.5 kDa protein was obtained at inducing 72 hours and showed no difference with theoretical value. The expression quantity of the target protein was reached to 0.2 g/L. The results of sweet taste and biological activity determination on purified protein showed that Brazzein recombinant protein has a definite sweet ness and highly biological activity.