甜味蛋白Brazzein基因酵母表达系统的建立

根据甜味蛋白Brazzein基因成熟区的氨基酸序列,结合毕赤酵母的密码子偏爱性以及Brazzein蛋白质结构的相关研究,对甜味蛋白Brazzein基因进行改造,使表达的目的蛋白中包含3个突变氨基酸(29Asp→Lys,31His→Ala,41Glu→Lys)。采用重叠PCR(SOE-PCR)合成目的基因并克隆到pMD18-T载体,经测序鉴定,序列正确的质粒用EcoRⅠ和XbaⅠ双酶切。将回收得到的目的基因连接到经同样双酶切处理的pGAPZαA载体上,经PCR、酶切和测序鉴定,证明已成功构建pGAPZαA-Bra真核表达载体。将构建好的重组载体pGAPZαA-Bra用AvrⅡ线性化,电转进入巴斯德毕赤酵母中。筛选ZeocinTM阳性克隆菌,进行蛋白表达,SDS-PAGE结果表明蛋白表达成功。

Construction of Yeast Expression System of Sweet Protein Brazzein

According to amino acid sequences of the sweet protein Brazzein, the biased codons of Pichia pastoris yeast and the latest research advances about the protein structure and mechanism, the gene of Brazzein, which includes 3 sites of mutation(29Asp→Lys,31His→Ala,41Glu→Lys) , was reformed. The Brazzein geneamplified by SOE-PCR was inserted into the plasmid pMD18-T, then sequenced. The recombinant plasmid pMD18-T-Bra with the right sequence was digested by EcoRⅠ and XbaⅠ, the retained gene was connected to pGAPZαA which was also digested by EcoRⅠ and XbaⅠ. The recombinant plasmid pGAPZαA-Bra was obtained. The pGAPZαA-Bra was linearized by AvrⅡ,then introduced into Pichia pastoris yeast with the Gene Pulser System. The ZeocinTM positive strains were screened,then cultured in YPD medium. After 4 days, the protein was analyzed with SDS-PAGE.The results proved the expectation.