Nicolet C M, Friedberg E C
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
Yeast. 1987 Sep;3(3):149-60. doi: 10.1002/yea.320030303.
The cloned RAD2 gene of S. cerevisiae was tailored into regulatable expression vectors for overexpression of Rad2 protein in E. coli and in yeast. In E. coli both Rad2/beta-galactosidase fusion protein and native Rad2 protein are insoluble, but are extractable with 1% Sarkosyl. In yeast some of the overexpressed native Rad2 protein is also insoluble; however, soluble protein is readily detected by immunoblotting with Rad2-specific antibodies. All forms of the protein detected in transformed or untransformed yeast cells and the insoluble species in E. coli migrate in denaturing polyacrylamide gels with an apparent molecular weight considerably larger than the size predicted from the sequence of the RAD2 coding region. This property is not the result of post-translational glycosylation detectable by binding of concanavalin A, or of phosphorylation of the protein. Overexpression of the RAD2 gene is toxic to yeast. Transformed yeast cells grow much more slowly than untransformed controls and when yeast transformants are serially propagated cultures show considerably colony heterogeneity and concomitant selection for rapidly growing variants which express less Rad2 protein. Antisera raised against Rad2/beta-galactosidase fusion protein expressed in E. coli do not cross-react with Rad1, Rad3 or Rad10 protein in crude extracts of yeast, nor with purified E. coli UvrA, UvrB or UvrC proteins.
酿酒酵母的克隆RAD2基因被定制到可调控表达载体中，以便在大肠杆菌和酵母中过表达Rad2蛋白。在大肠杆菌中，Rad2/β-半乳糖苷酶融合蛋白和天然Rad2蛋白均不溶，但可用1%十二烷基肌氨酸钠提取。在酵母中，一些过表达的天然Rad2蛋白也不溶；然而，通过用Rad2特异性抗体进行免疫印迹很容易检测到可溶性蛋白。在转化或未转化的酵母细胞中检测到的所有蛋白形式以及大肠杆菌中的不溶物在变性聚丙烯酰胺凝胶中迁移时，其表观分子量明显大于根据RAD2编码区序列预测的大小。这种特性不是伴刀豆球蛋白A结合可检测到的翻译后糖基化的结果，也不是该蛋白磷酸化的结果。RAD2基因的过表达对酵母有毒。转化的酵母细胞比未转化的对照生长慢得多，当酵母转化体连续传代培养时，菌落显示出相当大的异质性，并伴随选择快速生长的变体，这些变体表达较少的Rad2蛋白。针对在大肠杆菌中表达的Rad2/β-半乳糖苷酶融合蛋白产生的抗血清与酵母粗提物中的Rad1、Rad3或Rad10蛋白不发生交叉反应，也与纯化的大肠杆菌UvrA、UvrB或UvrC蛋白不发生交叉反应。
