Smith J S, Roth M J
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
J Virol. 1993 Jul;67(7):4037-49. doi: 10.1128/JVI.67.7.4037-4049.1993.
We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.
我们已从大肠杆菌中表达并纯化出一种人类免疫缺陷病毒1型（HIV-1）核糖核酸酶H结构域，该结构域由逆转录酶的400至560位氨基酸组成，带有N端或C端多组氨酸标签。HIV-1逆转录酶的天然蛋白酶切割位点在440和441位氨基酸之间。在镍（2+）-次氮基三乙酸琼脂糖上进行纯化，得到了一种高度活跃的核糖核酸酶H结构域，其活性依赖于氯化锰而非氯化镁。通过原位核糖核酸酶H凝胶分析明确了纯化蛋白具有活性。400至426位残基，包括一段色氨酸，对核糖核酸酶H活性没有贡献，且多组氨酸标签对活性至关重要。尽管需要组氨酸标签，但通过检测在几种已知的HIV-1核糖核酸酶H抑制剂（包括核糖核苷钒配合物、dAMP和一种单克隆抗体）存在下的活性，确定重组核糖核酸酶H蛋白保留了野生型异二聚体的特征。重要的是，分离出的核糖核酸酶H结构域在去除tRNA（3Lys）时产生的特异性切割与HIV-1异二聚体相同，使模型tRNA的3'-rA（腺苷5'磷酸）残基与相邻的U5序列相连。这种HIV-1核糖核酸酶H结构域在甘油梯度中以单体形式沉降。
