Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening.
Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening.
复制标题
用于药物筛选的 Mg2 依赖性 HIV-1 RNase H 构建体的表达。
DOI：
10.1128/aac.00658-11
复制
发表时间：
2011
影响因子：
4.9
通讯作者：
Leon,Oscar
中科院分区：
文献类型：
--
作者：
Farias,RichardV;Vargas,DeborahA;Castillo,AndresE;Valenzuela,Beatriz;Cote,MarieL;Roth,MonicaJ;Leon,Oscar
A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similarKms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.
