SINGLE-STEP PURIFICATION OF RECOMBINANT WILD-TYPE AND MUTANT HIV-1 REVERSE-TRANSCRIPTASE

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-m atrix cartridge columns connected in series. The columns are washed wi th low-salt buffer to remove unbound protein, then the Q column is rem oved and reverse transcriptase is eluted from the S column using a sal t gradient. The purification has been carried out with both medium-pre ssure and high-pressure chromatographic systems. Purifications are car ried out at room temperature near neutral pH, providing enzyme with hi gh 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 p51 and p66 homodimer forms of rev erse transcriptase. We have used this method to purify wild-type rever se transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance. (C) 1996 Academic Press, Inc.