酵母PHO85结合蛋白PAP1基因的克隆和表达  

Cloning and Expression of a Novel PHO85 Associated Protein PAP1 Gene of Saccharomyces cerevisiae

作　　者：吴健生[1] 夏赞贤[1] 曹铮 敖世洲[1] 

机构地区：[1]中国科学院上海生物化学研究所分子生物学国家重点实验室

出　　处：《生物化学与生物物理学报》1998年第1期14-20,共7页

摘　　要：酵母调控因子ＰＨＯ８５是一个依赖于细胞周期蛋白（ｃｙｃｌｉｎ）的蛋白激酶（ＣＤＫ），参与对细胞周期和酸性磷酸酯酶基因表达的调控。以ＰＨＯ８５为靶分子，利用酵母双杂交（ｔｗｏ－ｈｙｂｒｉｄ）系统从酵母的染色体基因文库中克隆到了一个新的与ＰＨＯ８５相结合的蛋白因子的基因，将此蛋白质命名为ＰＡＰ１（ＰＨＯ８５ａｓｓｏｃｉａｔｅｄｐｒｏｔｅｉｎ）。克隆了包含ＰＡＰ１全部编码区以及上下游调控序列的２．０ｋｂ的基因，并进行了序列测定。ＰＡＰ１基因编码区全长８５５ｂｐ，编码一个２８５个氨基酸的蛋白质。同源性比较表明，ＰＡＰ１蛋白的羧端半分子包含一个细胞周期蛋白保守的结构域（１５０～２６３），与ＰＨＯ８０的保守区（６７～１６８）的同源性为６３％。而ＰＡＰ１蛋白的Ｎ端半分子富含ＰＥＳＴ序列，这种序列使蛋白质的稳定性降低。双杂交实验证明ＰＡＰ１蛋白的全长和Ｃ端（９９～２８５）都能同ＰＨＯ８５相互结合。构建了在ＰＬ启动子控制下的ＰＡＰ１的表达质粒，并在大肠杆菌ＢＬ２１（ＤＥ３）ｐＬｙｓＳ中进行了表达。经４２℃热诱导，得到了分子量约为３２ｋＤ的蛋白质电泳条带，与ＰＡＰ１的计算分子量相符。其Ｎ端氨基酸序列与预测相符。这一表达产物经鉴?Yeast regulatory factor PHO85, which is a cyclindependent kinase (CDK), participates in the regulation of the cell cycle control and the expression of the acid phosphatase gene. Using PHO85 as target, we have cloned from the yeast twohybrid genomic library a novel gene (PAP1) with product which can associate with the PHO85 protein. The PAP1 gene including the 5′ and 3′ flanking sequence was cloned and sequenced. It codes for a protein composed of 285 amino acids, which contains a cyclin conservative region (150-263 aa), and shares 63% similarity with the conservative region of PHO80. The N terminal of the PAP1 protein abounds in PEST sequence, which is believed to be common to rapidly degraded proteins. Using the yeast twohybrid system, we demonstrated that both the fulllength PAP1 protein and the Cterminal (99-285 aa) can interact with PHO85. The coding sequence of the PAP1 gene was cloned and sequenced, then arranged under the control of the PL promoter, and expressed in E.coli BL21 (DE3) pLysS. After heat induction at 42 ℃, protein band of approximately 32 kD was observed in the SDSPAGE, which fits the molecular size of the hypothetical PAP1 protein. The 10 Nterminal amino acids of the 32 kD protein agreed with the deduced PAP1 amino acid sequence. The protein was found as inclusion body in E.coli, and accounted for 50% of the precipitant after purification.

关 键 词：PHO85 结合蛋白 酵母 克隆 表达 

分 类 号：Q513[生物学—生物化学]