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


We have devised a single-step method that enables purification of ...
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.




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