title: Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli.
abstract: We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.
authors.affiliation: Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: S P
authors.initials: SP
authors.lastname: Becerra
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D054303:HIV Reverse Transcriptase; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase