Expression and purification of retroviral HIV-1 reverse transcriptase.
Modern molecular biology techniques have provided valuable tools which allow for the expression of large amounts of enzyme in E. coli. For potential therapeutic targets such as HIV-1 reverse transcriptase, it is desirable that the enzyme studied is pure and correlates to the active form of the enzyme found in vivo. This poses a particular challenge for those researchers studying HIV-RT since a significant degree of heterogeneity is introduced by nonspecific proteolytic cleavage of the p66 subunit by E. coli proteases. The advantage of the purification protocol presented here is that the association of monomers is facilitated by mixing an excess of p51 subunit, which is truncated at a site that is N-terminal to known bacterial cleavage sites, with p66 protein. This avoids enzymatic processing of the larger subunit since the formation of heterodimeric RT is rapid and the dimer is stable against proteolytic cleavage. Therefore, it is possible to isolate a pure homogeneous p66/p51 heterodimer. An enzyme prepared in this manner yields crystals that defract to a 3.2-A resolution. It has also been used to study both sensitivity of HIV-1 RT mutants to azidothymidine triphosphate and the kinetics of a potent nonnucleoside RT inhibitor (L-743,726). Finally, it is interesting to note the similarity of HIV-1 RT with reverse transcriptases from other lentiviruses (FIV and EIAV RT). Both of these enzymes consist of heterodimers of p66 and p51 subunits and share other biophysical characteristics. Purification of these reverse transcriptases can, in all likelihood, be optimized by using methods similar to those described in this chapter.
Stahlhut
M W
MW
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Olsen
D B
DB
D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D019281:Dimerization; D004926:Escherichia coli; D054303:HIV Reverse Transcriptase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase