Cx31相互作用蛋白:p11在酵母中表达可能存在翻译移码  

Translational Frameshift may be Occur in p11, an Interaction Protein of Cx31, in Yeast

作　　者：黄亮群[1] 施小六[1] 谭志平[1] 房海燕[1] 郑多[1] 夏家辉[1] 夏昆[1] 

机构地区：[1]中国医学遗传学国家重点实验室,长沙410078

出　　处：《生物化学与生物物理学报》2002年第2期219-224,共6页

基　　金：国家重点基础研究发展规划 ( 973)项目 (No .G19980 5 10 0 2 );国家自然科学基金资助项目 (No .39970 372 );国家杰出青年科学基金资助项目 (No .3992 80 16 );教育部全国优秀博士学位论文作者专项资金 (No .2 0 0 0 2 4)~~

摘　　要：间隙连接蛋白 31(connexin 31,Cx31)是间隙连接蛋白 (connexin)家族中的一员 ,关于Cx31的功能及其调节知之甚少。Cx31胞内羧基端包含几个可能的磷酸化位点。为对其功能有所了解 ,利用酵母双杂交技术 ,筛选与Cx31羧基端 (2 0 6～ 2 70位密码子之间 )相互作用的蛋白质 ,分离到了 p11蛋白 [依钙蛋白I(calpactinI) ,轻链 ],即 :S10 0家族的独特成员 ,膜联蛋白II(annexinII)四聚体的两个亚单位之一。有意思的是按照酵母双杂交Gal4AD质粒 (编码Gal4激活域 )的编码顺序 ,出现 3个不同的阅读框。经证实p11编码区序列编码的蛋白质与Cx31存在相互作用 ,而 3种不同的 5′UTR区均不存在与Cx31的相互作用 ,推测 p11融合蛋白可能存在翻译移码。且通过分段构建诱饵质粒 ,将Cx31与p11相互作用区段缩短至 2 0 6～ 2 37位密码子之间。Connexin 31 is a member of connexins family. The carboxy-terminal cytosolic domain of connexin 31 contains several potential phosphorylation sites. In this work, a yeast two-hybrid protein interaction screen have been used to identify proteins that bind to the carboxy-terminus of connexin 31, and the p11 protein, an unique member of S100 protein family, and one of the two subunits of the annexin II tetramer was isolated. Interestingly, from yeast two-hybrid AD's coding sequence, three different reading frames of p11 DNA sequence were found, which come from different AD plasmids. By constructing AD plasmids using p11 ORF or 5′UTR, the protein coding by p11 ORF bind to connexin 31, while polypeptides coding by three kinds of 5′ UTR did not bind to connexin 31, suggesting a translational frameshift of p11 fusion protein. To construct baits by deviding connexin 31 C-terminus into two domain, the p11 binding domain of connexin 31 was found located between 206—237 codons. The plasmid Cx31CT-pGEX-4T-2 was constructed for expression and purification of GST-Cx31CT; and p11-pQE30 for expression and purification of 6×His-p11. In vitro binding assay showed that recombinant Cx31 interacted with recombinant p11.

关 键 词：酵母双杂交 间隙连接蛋白31 依钙结合蛋白Ⅰ 轻链 移码 体外结合 Cx311相互作用蛋白 表达 P11 

分 类 号：Q78[生物学—分子生物学]