Warren T C, Miglietta J J, Shrutkowski A, Rose J M, Rogers S L, Lubbe K, Shih C K, Caviness G O, Ingraham R, Palladino D E
Department of Biochemistry, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877.
Protein Expr Purif. 1992 Dec;3(6):479-87. doi: 10.1016/1046-5928(92)90065-5.
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.
