Chen J H
Department of Pediatrics, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Oncogene Res. 1990;5(4):277-85.
The protooncogene c-ets-1 is preferentially expressed in lymphoid cells. The protein product of this gene has been found to be a phosphorylated nuclear protein. When lymphocytes are stimulated with calcium ionophore, hyperphosphorylation of c-ets-1 occurs. In order to study the biological and biochemical functions of the c-ets-1 protein in detail, it is important to prepare adequate quantity of the c-ets-1 protein. To this end, we have cloned, sequenced, and expressed mouse c-ets-1 cDNA in baculovirus expression vector. Sequence analysis indicated that mouse c-ets-1 cDNA codes for a 50/51-kd protein. Since the mouse c-ets-1 protein in mouse lymphocytes is a 60/62-kd protein, the result obtained indicated that the c-ets-1 protein undergoes posttranslational modification by phosphorylation. When the c-ets-1 cDNA was expressed in the baculovirus expression vector, insect cells infected with a recombinant virus synthesizes a protein of the same size but with 50-100 times more of the c-ets-1 protein than that of mouse lymphocytes. The Staphylococcus aureus V8 protease mapping analysis of mouse c-ets-1 proteins synthesized in mouse and insect cells showed that they are identical. Thus, the c-ets-1 protein synthesized in insect cells will allow us to purify and study the functions of the c-ets-1 protein in detail.
原癌基因c-ets-1在淋巴细胞中优先表达。已发现该基因的蛋白质产物是一种磷酸化核蛋白。当用钙离子载体刺激淋巴细胞时，c-ets-1会发生过度磷酸化。为了详细研究c-ets-1蛋白的生物学和生化功能，制备足够量的c-ets-1蛋白很重要。为此，我们已在杆状病毒表达载体中克隆、测序并表达了小鼠c-ets-1 cDNA。序列分析表明，小鼠c-ets-1 cDNA编码一种50/51-kd的蛋白质。由于小鼠淋巴细胞中的小鼠c-ets-1蛋白是一种60/62-kd的蛋白质，所得结果表明c-ets-1蛋白通过磷酸化进行翻译后修饰。当c-ets-1 cDNA在杆状病毒表达载体中表达时，感染重组病毒的昆虫细胞合成一种大小相同的蛋白质，但c-ets-1蛋白的含量比小鼠淋巴细胞中的多50 - 100倍。对在小鼠和昆虫细胞中合成的小鼠c-ets-1蛋白进行的金黄色葡萄球菌V8蛋白酶图谱分析表明它们是相同的。因此，在昆虫细胞中合成的c-ets-1蛋白将使我们能够详细纯化和研究c-ets-1蛋白的功能。
