Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli.
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.
Becerra
S P
SP
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, MD 20892.
Clore
G M
GM
Gronenborn
A M
AM
Karlström
A R
AR
Stahl
S J
SJ
Wilson
S H
SH
Wingfield
P T
PT
D000595:Amino Acid Sequence; D001483:Base Sequence; D010450:Endopeptidases; D004722:Endoribonucleases / Q000096:biosynthesis / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016914:Ribonuclease H; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H; D010450:Endopeptidases