Chattopadhyay D, Evans D B, Deibel M R, Vosters A F, Eckenrode F M, Einspahr H M, Hui J O, Tomasselli A G, Zurcher-Neely H A, Heinrikson R L
Upjohn Company, Kalamazoo, Michigan 49001.
J Biol Chem. 1992 Jul 15;267(20):14227-32.
Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.
通过固定化金属亲和色谱（IMAC）和HIV-1蛋白酶处理的协同应用，已以毫克量制备了具有含六组氨酸序列的氨基末端延伸的人免疫缺陷病毒1型（HIV-1）活性重组逆转录酶（RT），其为纯的异二聚体（p66/p51）形式。通过IMAC从重组大肠杆菌提取物中分离出的前体蛋白主要以未加工形式（p66）存在，在十二烷基硫酸钠-聚丙烯酰胺凝胶上迁移为一条66 kDa的条带，在较低相对分子质量处有轻微异质性。将该蛋白与重组HIV-1蛋白酶一起孵育产生了一种稳定的异二聚体RT，通过IMAC一步纯化。纯化后的蛋白保留了RT和核糖核酸酶H活性，并通过RNA依赖性DNA聚合反应和核糖核酸酶H活性测定法测量了动力学参数（Km和Vmax）。纯化的异二聚体RT的羧基末端测序表明，一个亚基是完整的p66，而另一个亚基p51是p66的截短形式，在残基Phe440处终止。对HIV-1蛋白酶消化产物的分析揭示了除在Phe440-Tyr441处被切割产生p51的位点外，在Tyr-483-Leu484和Tyr532-Leu533处还有两个切割位点。
