Protein Expression and Purification.

BL21(DE3)-RIL cells containing two vectors carrying the p66 and p51 subunits of HIV-1 RT were expressed in LB and induced with 1 mM isopropyl β-d-1-thiogalactopyranoside at OD₆₀₀ of ∼0.6–0.8, followed by expression at 37 °C for 3 h. Cell pellets were treated with lysozyme, homogenization, and sonication. RT was purified by nickel-affinity chromatography (GE Healthcare), followed by Mono Q anion exchange (GE Healthcare), and size-exclusion chromatography (Shodex) as previously described (15). Fractions with pure RT were concentrated and stored at −80 °C.