Becerra S P, Clore G M, Gronenborn A M, Karlström A R, Stahl S J, Wilson S H, Wingfield P T
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, MD 20892.
FEBS Lett. 1990 Sep 17;270(1-2):76-80. doi: 10.1016/0014-5793(90)81238-j.
The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the lambda PL promoter. The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion-exchange and size exclusion chromatography. The purified protein appears to be in a native-like homogeneous conformational state as determined by 1H-NMR spectroscopy and circular dichroism measurements. HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441.
