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.
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 (Km and Vmax) 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.
Chattopadhyay
D
D
Upjohn Company, Kalamazoo, Michigan 49001.
Evans
D B
DB
Deibel
M R
MR
Vosters
A F
AF
Eckenrode
F M
FM
Einspahr
H M
HM
Hui
J O
JO
Tomasselli
A G
AG
Zurcher-Neely
H A
HA
Heinrikson
R L
RL
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D016333:HIV Protease / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D016333:HIV Protease