Braspenning J, Manetti R, Zumbach K, Meschede W, Gissmann L, Tommasino M
Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Protein Expr Purif. 1997 Jul;10(2):192-201. doi: 10.1006/prep.1997.0731.
A purification protocol was developed to obtain human papillomavirus (HPV) type 16 E7 protein expressed in the yeast Schizosaccharomyces pombe. Only three chromatographic steps were necessary to purify the unfused HPV 16 E7 protein to homogeneity (95-99%) as shown by silver staining after polyacrylamide gel electrophoresis. Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells. The purified HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality. Binding to the proteins Rb1 and p107 in vitro and induction of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological activities. Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV types such as HPV 18 and 11.
已开发出一种纯化方案，用于获取在粟酒裂殖酵母中表达的人乳头瘤病毒16型（HPV - 16）E7蛋白。通过聚丙烯酰胺凝胶电泳后的银染显示，仅需三步色谱步骤就能将未融合的HPV - 16 E7蛋白纯化至同质状态（95 - 99%）。从5×10¹⁰个酵母细胞中可获得约0.8毫克高度纯化的E7蛋白。对纯化后的HPV - 16 E7磷蛋白（Ser 31/32）进行重折叠并检测其功能。体外与Rb1和p107蛋白结合以及显微注射到血清饥饿的NIH 3T3细胞后诱导DNA合成，表明E7蛋白保留了一些生物学活性。最重要的是，该纯化策略也适用于不同的HPV - 16 E7突变体以及来自其他HPV类型（如HPV - 18和11）的E7蛋白。
