Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase

Protein Expression and Purification
R S FletcherM A Parniak

Abstract

We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.

Citations

May 1, 2008·PloS One·Scott J GarforthVinayaka R Prasad
Jan 17, 2016·Nucleic Acids Research·Xunhai ZhengRobert E London
Jan 12, 2010·Protein Expression and Purification·Meiqing LuMing-Tain Lai
Aug 22, 2003·Protein Science : a Publication of the Protein Society·Nicolas Sluis-CremerMichael A Parniak
Jun 9, 2004·Chembiochem : a European Journal of Chemical Biology·Rami N HannoushMasad J Damha
Dec 1, 2010·Chemical Biology & Drug Design·Qingguo GongRieko Ishima
Apr 29, 2014·Proteins·Naima G SharafRieko Ishima
Oct 10, 2013·Journal of Medicinal Chemistry·Venkata Ramana SirivoluZhengqiang Wang
Jul 20, 2002·Molecular Pharmacology·Nicolas Sluis-CremerMichael A Parniak
Aug 16, 1996·The Journal of Biological Chemistry·D ArionM A Parniak
Dec 31, 2019·Oxidative Medicine and Cellular Longevity·Ekaterina BayurovaMaria Isaguliants
Feb 10, 2004·Protein Expression and Purification·Esther W HouSamuel H Wilson

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