Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits.
Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits.
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具有 1:1 比例的 p66 和 p51 亚基的 HIV-1 逆转录酶的纯化和表征。
DOI：
10.1006/prep.1994.1084
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发表时间：
1994
影响因子：
1.6
通讯作者：
David B. Olsen
中科院分区：
文献类型：
--
作者：
M. Stahlhut;Y. Li;Jon H. Condra;J. Fu;Leah Gotlib;Donald J. Graham;David B. Olsen
野生型和几种突变形式的重组人免疫缺陷病毒1型逆转录酶在大肠杆菌蛋白酶缺陷株中作为p66或p51亚基过表达。在细胞裂解之前，将p51细胞糊与含有相应过表达的p66亚基的细胞糊以一定比例混合，导致较小亚基相对于较大亚基过量。在随后的层析步骤中，将稳定的异源二聚体p66/p51纯化至均一。该蛋白质的特征在于氨基酸分析，变性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，分析型凝胶过滤HPLC，激光解吸质谱，和等电聚焦。此外，我们能够获得晶体的纯化的酶复合的喹唑啉酮类非核苷抑制剂，衍射到3.2 A的分辨率。这种表达/纯化方法的潜在应用是通过定点诱变改变RT-二聚体的仅一个亚基的特定氨基酸残基的能力。
Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.