Purification and characterization of the RNase H domain of HIV‐1 reverse transcriptase expressed in recombinant Escherichia coli

Abstract

The ribonuclease H (RNase H) domain of human immune‐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 λ p_l 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.