Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease

Academic Article

Abstract

  • Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa- histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate- polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (K(m) and V(max)) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.
  • Published In

    Pubmed Id

  • 3123514
  • Author List

  • Chattopadhyay D; Evans DB; Deibel MR; Vosters AF; Eckenrode FM; Einspahr HM; Hui JO; Tomasselli AG; Zurcher-Neely HA; Heinrikson RL
  • Start Page

  • 14227
  • End Page

  • 14232
  • Volume

  • 267
  • Issue

  • 20