Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast.
The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is comprised of two subunits of approximately 66kD and 51kD. We have defined the carboxyl terminus of the 51kD molecule using the 66kD RT and HIV-1 protease (PR) expressed in yeast. Precise constructs encoding the 66kD and 51kD molecules were expressed individually, in yeast, at high levels. The purified recombinant subunits were shown to associate into heterodimers that retained both RT and RNase H activities. Only the 66kD molecule could associate into homodimers. Such homodimers retained approximately 80% of the RT activity of the heterodimers. Our data demonstrates that the 51/66kD heterodimer, analogous to that found in vivo, can be reconstituted in vitro and is more efficient in both RT and RNase H activity than the homodimer.
Chiron Corporation, Emeryville, California 94608.
I C
IC
Bathurst
L K
LK
Moen
M A
MA
Lujan
H L
HL
Gibson
P H
PH
Feucht
S
S
Pichuantes
C S
CS
Craik
D V
DV
Santi
P J
PJ
Barr
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004279:DNA, Viral; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase