Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase

Academic Article


  • 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. © 1992 Academic Press, Inc.
  • Published In

    Digital Object Identifier (doi)

    Pubmed Id

  • 3661795
  • Author List

  • Chattopadhyay D; Einspahr HM; Brunner DP; Strakalaitis NA; Tarpley WG; Deibel MR
  • Start Page

  • 151
  • End Page

  • 159
  • Volume

  • 3
  • Issue

  • 2