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

FEBS Letters

Karlström, Anders R.²  Clore, G.Marius⁴  Wingfield, Paul T.³  Becerra, S.Patricia¹  Stahl, Stephen J.³  Wilson, Samuel H.¹  Gronenborn, Angela M.⁴ 

[1] Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, MD 20892, USA;Laboratory of Biochemistry, NHLB, National Institutes of Health, Bethesda, MD 20892, USA;Protein Expression Laboratory, National Institutes of Health, Bethesda, MD 20892, USA;Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA

关键词: HIV-1 reverse transcriptase;    HIV-1 RNase H;    HIV protease;    Protein expression;    Protein purification;    Protein conformation;   

DOI  :  10.1016/0014-5793(90)81238-J

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown