INCREASED YIELD OF HOMOGENEOUS HIV-1 REVERSE-TRANSCRIPTASE (P66 P51) USING A SLOW PURIFICATION APPROACH/

A chromatographic procedure to purify recombinant reverse transcriptas e (RT) from human immunodeficiency virus-1 is reported. A bacterial sy stem which expressed large amounts of p66 RT polypeptide was used. The purification scheme was optimized for high-yield production of homoge neous p66/p51 RT using a combination of chromatographic matrices in th e following order: Q-Sepharose, heparin-Sepharose, phenyl-Sepharose, S -Sepharose, Poly(A)-Sepharose and Q-Sepharose. The p66 polypeptide rem ained intact after the first chromatographic step on Q-Sepharose, wher e it was recovered in the non-adsorbed fraction. A high yield of p66/p 51 RT was obtained when the time from application to elution of hepari n-Sepharose in the second chromatographic step was prolonged. Phenyl-S epharose was used in the next chromatographic step to separate the het erodimeric forms of RT from p66 RT on the basis of hydrophobicity. The chromatography on S-Sepharose resolved the major heterodimeric form, p66/p51, from other heterodimeric variants. Further purification was d one by affinity chromatography on Poly(A)-Sepharose followed by anion- exchange chromatography on Q-Sepharose. Amounts of 25-35 mg of the pur e heterodimer p66/p51 RT were recovered from 50 g of bacterial cells.