Schaffhausen B, Benjamin T L, Lodge J, Kaplan D, Roberts T M
Nucleic Acids Res. 1985 Jan 25;13(2):501-19. doi: 10.1093/nar/13.2.501.
The three products of the early region of polyoma virus have been cloned for expression in E. coli using the Tac promoter. Although the identical promoter and ribosome binding site are used in each final construction, the observed level of protein expression is different for each protein. While plasmids expressing wild type T antigens as well as a plasmid expressing the truncated Py-1387T middle T antigen lacking the membrane-anchoring sequence give rise to synthesis of proteins readily detectible by 35S-methionine labeling and immunoprecipitation, only small T and the middle T of Py-1387T are made in amounts sufficient for ready detection in total cell protein. Unlike middle T expressed in animal cells, middle T produced in E. coli is not detectibly phosphorylated. Further, the E. coli protein lacks tyrosine kinase activity.
利用Tac启动子,多瘤病毒早期区域的三种产物已被克隆,用于在大肠杆菌中表达。尽管在每个最终构建体中使用了相同的启动子和核糖体结合位点,但观察到的每种蛋白质的表达水平不同。表达野生型T抗原的质粒以及表达缺乏膜锚定序列的截短Py-1387T中间T抗原的质粒,会产生通过35S-甲硫氨酸标记和免疫沉淀易于检测到的蛋白质合成,但只有小T和Py-1387T的中间T以足以在总细胞蛋白中易于检测的量产生。与在动物细胞中表达的中间T不同,在大肠杆菌中产生的中间T没有可检测到的磷酸化。此外,大肠杆菌蛋白缺乏酪氨酸激酶活性。