Kanner S B, Reynolds A B, Parsons J T
Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
J Immunol Methods. 1989 Jun 2;120(1):115-24. doi: 10.1016/0022-1759(89)90296-2.
Transformation of cells by viral oncogene-encoded tyrosine kinases coincides with the phosphorylation of many cellular proteins on tyrosine. In order to study the potential cellular targets of oncogenic tyrosine kinases, tyrosine phosphoproteins were purified from cells by immunoaffinity chromatography with antibodies to phosphotyrosine. Tyrosine phosphoproteins were purified from both rat-1 cells and primary chicken embryo cells expressing transforming or non-transforming variants of the src oncogene. These proteins were released from anti-phosphotyrosine resins with hapten, and the protein mixtures contained 6-10 highly pure phosphoproteins including the src protein pp60src. The recovered proteins represented approximately 0.03% of total cellular proteins. All of the proteins were shown to contain phosphotyrosine; in addition, virtually all of these proteins were also phosphorylated on serine and threonine. This method thus provides a large-scale, single-step immunoaffinity purification of phosphotyrosine-containing proteins to a purity amenable for immunization protocols and characterization of individual polypeptides.
病毒癌基因编码的酪氨酸激酶对细胞的转化与许多细胞蛋白的酪氨酸磷酸化同时发生。为了研究致癌酪氨酸激酶的潜在细胞靶点，通过用抗磷酸酪氨酸抗体进行免疫亲和层析从细胞中纯化酪氨酸磷酸化蛋白。从表达src癌基因转化或非转化变体的大鼠-1细胞和原代鸡胚细胞中都纯化了酪氨酸磷酸化蛋白。这些蛋白用半抗原从抗磷酸酪氨酸树脂上洗脱下来，蛋白混合物包含6-10种高度纯化的磷酸化蛋白，包括src蛋白pp60src。回收的蛋白约占细胞总蛋白的0.03%。所有蛋白均显示含有磷酸酪氨酸；此外，实际上所有这些蛋白在丝氨酸和苏氨酸上也被磷酸化。因此，该方法提供了一种大规模、单步免疫亲和纯化含磷酸酪氨酸蛋白的方法，其纯度适合用于免疫方案和单个多肽的表征。
