Pekrun K, Petry H, Jentsch K D, Moosmayer D, Hunsmann G, Lüke W
Department for Virology and Immunology, German Primate Centre, Göttingen, Germany.
Eur J Biochem. 1995 Dec 15;234(3):811-8. doi: 10.1111/j.1432-1033.1995.811_a.x.
To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli-expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT.
为了产生研究其结构和功能所需数量的人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT），使用两种不同的杆状病毒载体在Sf158昆虫宿主细胞中表达p66酶亚基。两种载体都能高效表达HIV-1 RT。将所得产物纯化至90%的纯度，进行表征，并研究其对各种蛋白酶消化的敏感性。用pAc373表达载体获得的重组杆状病毒RT被纯化为p66/p60异二聚体。重组His-RT用pBlueBacHis载体表达。因此，该蛋白带有N端六组氨酸肽标签，并被纯化为p70/p70同二聚体。这两种酶在比活性、动力学特性以及病毒和非病毒蛋白酶的体外激活方面存在差异。重组His-RT的比活性低于重组RT。后者产生的酶活性高达大肠杆菌表达的RT。用肠激酶消化从重组His-RT上去除六组氨酸标签导致酶活性完全丧失。因此，六组氨酸标签可能是活性重组His-RT的一个固有部分。
