Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase.
HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule.
Chattopadhyay
D
D
Upjohn Company, Kalamazoo, Michigan 49001.
Einspahr
H M
HM
Brunner
D P
DP
Strakalaitis
N A
NA
Tarpley
W G
WG
Deibel
M R
MR
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase