Sundaram P, Brandsma J L
Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520-8016, USA.
J Virol Methods. 1996 Mar;57(1):61-70. doi: 10.1016/0166-0934(95)01965-0.
Cottontail rabbit papillomavirus (CRPV) E7 protein is one of the :high risk' papillomavirus E7 oncoproteins that are partially insoluble in aqueous solution. An Escherichia coli expression system was used for purification of CRPV E7 protein in quantities sufficient for immunologic studies and structural analysis. A glutathione S-transferase (GST)-CRPV E7 fusion protein was solubilized in the presence of non-ionic and ionic detergents, and isolated on an affinity column of glutathione Sepharose beads. The CRPV E7 portion was cleaved from the column with thrombin at a thrombin cleavage site between the fused partners. Thrombin was removed subsequently by adsorption to benzamidine. This method is rapid, requiring just one week, and efficient, yielding 3 mg of pure CRPV E7 protein per liter of bacterial culture. It produced a protein product that was about 95% pure. High-titered polyclonal antisera generated to the product recognized CRPV E7 but not GST. Purified CRPV E7 protein exhibited the ability to bind pRB, making it the first unfused, non-denatured CRPV E7 product reported to do so. This attribute could facilitate structure-function studies of CRPV E7-pRB interactions.
棉尾兔乳头瘤病毒（CRPV）E7蛋白是“高危”乳头瘤病毒E7癌蛋白之一，该蛋白在水溶液中部分不溶。利用大肠杆菌表达系统大量纯化CRPV E7蛋白，以满足免疫学研究和结构分析的需求。谷胱甘肽S-转移酶（GST）-CRPV E7融合蛋白在非离子和离子去污剂存在的情况下溶解，并在谷胱甘肽琼脂糖珠亲和柱上分离。CRPV E7部分在融合伙伴之间的凝血酶切割位点用凝血酶从柱上切割下来。随后通过吸附到苯甲脒上去除凝血酶。该方法快速，仅需一周时间，且效率高，每升细菌培养物可产生3毫克纯CRPV E7蛋白。它产生的蛋白质产物纯度约为95%。针对该产物产生的高滴度多克隆抗血清可识别CRPV E7，但不能识别GST。纯化的CRPV E7蛋白表现出结合pRB的能力，使其成为首个报道的具有这种能力的未融合、未变性的CRPV E7产物。这一特性有助于CRPV E7与pRB相互作用的结构-功能研究。
