Donaldson L, Capone J P
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
J Biol Chem. 1992 Jan 25;267(3):1411-4.
A glutathione S-transferase fusion to the COOH-terminal acidic transactivation domain of Vmw65 from herpes simplex virus type 1 was overexpressed in Escherichia coli and isolated by affinity chromatography on glutathione-Sepharose. Following cleavage of the fusion protein with thrombin, the transactivation domain was purified to homogeneity by ion exchange chromatography yielding approximately 0.6 mg of protein/liter of bacterial culture. Equilibrium sedimentation analysis showed the purified polypeptide to be monomeric; however, it displayed aberrant electrophoretic and chromatographic properties. Contrary to secondary structure predictions, circular dichroism spectroscopy demonstrated that this transactivation domain was devoid of significant alpha-helical structure at physiological conditions. The polypeptide, however, became notably more structured under hydrophobic conditions or at low pH, suggesting that it was sensitive to its environment. Near-UV circular dichroism suggested that phenylalanyl and tyrosyl residues were under influence from tertiary structure.
一种与单纯疱疹病毒1型的Vmw65羧基末端酸性反式激活结构域融合的谷胱甘肽S-转移酶在大肠杆菌中过表达，并通过谷胱甘肽-琼脂糖亲和层析进行分离。用凝血酶切割融合蛋白后，通过离子交换层析将反式激活结构域纯化至同质，每升细菌培养物产生约0.6毫克蛋白质。平衡沉降分析表明纯化的多肽为单体；然而，它表现出异常的电泳和色谱性质。与二级结构预测相反，圆二色光谱表明该反式激活结构域在生理条件下缺乏明显的α-螺旋结构。然而，该多肽在疏水条件下或低pH值时结构明显更有序，表明它对其环境敏感。近紫外圆二色性表明苯丙氨酰基和酪氨酰基残基受三级结构影响。
