Increased yield of homogeneous HIV-1 reverse transcriptase (p66/p51) using a slow purification approach.
A chromatographic procedure to purify recombinant reverse transcriptase (RT) from human immunodeficiency virus-1 is reported. A bacterial system which expressed large amounts of p66 RT polypeptide was used. The purification scheme was optimized for high-yield production of homogeneous p66/p51 RT using a combination of chromatographic matrices in the following order: Q-Sepharose, heparin-Sepharose, phenyl-Sepharose, S-Sepharose, Poly(A)-Sepharose and Q-Sepharose. The p66 polypeptide remained intact after the first chromatographic step on Q-Sepharose, where it was recovered in the non-adsorbed fraction. A high yield of p66/p51 RT was obtained when the time from application to elution of heparin-Sepharose in the second chromatographic step was prolonged. Phenyl-Sepharose was used in the next chromatographic step to separate the heterodimeric 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 done by affinity chromatography on Poly(A)-Sepharose followed by anion-exchange chromatography on Q-Sepharose. Amounts of 25-35 mg of the pure heterodimer p66/p51 RT were recovered from 50 g of bacterial cells.
Bhikhabhai
R
R
Department of Molecular Biology, University of Uppsala, Sweden.
Carlsson
T
T
Unge
T
T
Lövgren
S
S
Strandberg
B
B
D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase