Walter G, Hutchinson M A, Hunter T, Eckhart W
Proc Natl Acad Sci U S A. 1982 Jul;79(13):4025-9. doi: 10.1073/pnas.79.13.4025.
We have used antibodies against the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the six COOH-terminal amino acids of the polyoma virus medium tumor (T) antigen, to purify the medium T antigen by affinity chromatography. Release of the medium T antigen from the anti-peptide antibody was achieved under mild conditions by using a large excess of the peptide in an isotonic buffer at neutral pH containing mixed detergents. This procedure yielded a 2,500-fold purification of the medium T antigen in a single step. The protein kinase activity associated with the medium T antigen was also released and was studied in this active state in solution. Sedimentation analysis showed that the bulk of the purified medium T antigen was in a monomeric form (Mr about 42,000) not associated with protein kinase activity. A small fraction of the medium T antigen was found in a rapidly sedimenting form (Mr about 200,000) that possessed protein kinase activity.
我们使用了针对合成肽Lys-Arg-Ser-Arg-His-Phe的抗体，该肽对应于多瘤病毒中肿瘤（T）抗原的六个COOH末端氨基酸，通过亲和色谱法纯化中肿瘤T抗原。在温和条件下，通过在含有混合去污剂的中性pH等渗缓冲液中使用大量过量的肽，从中抗肽抗体中释放中肿瘤T抗原。此方法在一步中实现了中肿瘤T抗原2500倍的纯化。与中肿瘤T抗原相关的蛋白激酶活性也被释放出来，并在溶液中的这种活性状态下进行了研究。沉降分析表明，大部分纯化的中肿瘤T抗原呈单体形式（分子量约42,000），与蛋白激酶活性无关。发现一小部分中肿瘤T抗原呈快速沉降形式（分子量约200,000），具有蛋白激酶活性。
