Rapid purification of homodimer and heterodimer HIV‐1 reverse transcriptase by metal chelate affinity chromatography

Abstract

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease ; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.Reverse transcriptase (deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, RNA-directed) of human immunodeficiency virus, type 1 (HIV-1) is presently under extensive study as a target for antiviral drugs. Reverse transcriptase constitutes one component of the HIV-1 pol open reading frame [l] and is processed, in either virions or heterologous systems expressing this polyprotein, to polypeptides of 66 kDa and 51 kDa, through the action of the polcodcd protease [2 -61. Although expression systems for highlevel synthesis of the enzymatically active 66-kDa reverse transcriptase (p66) have been documented [7, 81, the occurrence, nevertheless, of a 51 -kDa polypeptide (p51) suggests that a p66/p51 heterodimer may be the relevant enzyme form. To elucidate this, large quantities of the p66/p51 enzyme are required to determine whether its enzymatic or physical properties differ from those of p66. Since p51 reverse transcriptase is produced from p66 via C-terminal processing [9], and its C-terminus (and hence p51 /p66 cleavage recognition site) is not clearly defined, it is impossible by conventional cloning technology to produce a p.51 polypeptide with an authentic C-terminus. We have addressed this problem by over-expressing p66 reverse transcriptase and the pol-coded protease as separate proteins on the same vector, under the control of similar transcription and translation signals. In the absence of protease, p66 reverse transcriptase accumulates to approximately 10% of the total cellular protein. In the dual expression system, a 17-kDa precursor HIV-1 protease (p17) is cleaved to the enzymatically active 10-kDa molecule, which Correspondence to S. F. J. Le Grice, Central Research Units, F. Hoffmann-La Roche & Co. Ltd, CH-4002 Basel, SwitzerlandAhhreviutians. HIV-1, human immunodeficiency virus, type 1 ; p66, 66-kDa HIV-1 reverse transcriptase polypeptide; p51, 51-kDa proteolytic product of p66; p17, 17-kDa precursor polypeptide of HIV-1 protease; His-p66, p66 with (His), added at the amino terminus; His-p51, p51 with (His)6 ...

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

“…Enzymes and Nucleic Acids-Heterodimeric reverse transcriptase p66/p51 was expressed and purified essentially as described (28). Mutant enzymes were generated through sitedirected mutagenesis using the Stratagene QuikChange kit according to the manufacturer's protocol.…”

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

“…Full-length sequencing of mutant RTs was performed to confirm the presence of the desired mutations and to exclude adventitious mutations introduced during mutagenesis. WT and mutant recombinant HIV-1 RTs were overexpressed and purified to homogeneity as described previously (19,20). RT concentrations were determined spectrophotometrically at 280 nm using an extinction coefficient (ε 280 ) of 260 450 M Ϫ1 cm Ϫ1 , and the active site concentration was calculated from presteady-state burst experiments (18).…”

Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

“…Purification of HIV RT has been described (18), whereas the EIAV enzyme was prepared essentially as described by Rausch et al (13), with the exception that cultures were harvested and cooled to 4°C 30 min after induction to prevent overdigestion of EIAV RT by protease. Metal chelate (Ni 2ϩ -NTA-Sepharose) and Mono-S ion exchange chromatography (Pharmacia Biotech Inc.) yielded highly pure enzymes free of contaminating nucleases, with a 1:1 stoichiometry of p66 and p51 subunits.…”

“…The resulting EIAV protease expression cassette was introduced to p6H EIAV RT (19) as an XhoI fragment, creating pEIAV PR-6H RT, which controls co-expression of p66 EIAV RT and protease. The analogous HIV vector, p6HRT-PROT, has been described (18). Mutation of HIV and EIAV RT genes at amino acid position 549 (D549N and D549A) was achieved using the USE mutagenesis kit (Pharmacia Biotech Inc.).…”