Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: preparation of heterodimeric enzyme devoid of unprocessed gene product.
A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification
D011994:Recombinant Proteins; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase