Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast

• Published in: Biochemical and biophysical research communications Vol. 171; no. 2; pp. 589 - 595
• Main Authors: Bathurst, Ian C., Moen, Laura K., Lujan, Marc A., Gibson, Helen L., Feucht, Paul H., Pichuantes, Sergio, Craik, Charies S., Santi, Daniel V., Barr, Philip J.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 14.09.1990; Elsevier
• Subjects: AIDS/HIV; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Gel; Cloning, Molecular; DNA, Viral - genetics; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; HIV-1 - enzymology; HIV-1 - genetics; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - isolation & purification; RNA-Directed DNA Polymerase - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Transferases; HIV-1; RT; PR; TCA; IN; YEP; PMSF; GAP; BOG; BME; EDTA; Human; Yeast; Purification; HIV-1 virus; Enzyme; Retroviridae; Gene expression; Genetic transfer; Fungi; Virus; Enzymatic activity; DNA polymerase RNA-dependent; Ascomycetes; Human immunodeficiency virus; Saccharomyces cerevisiae; Thallophyta; Structural analysis
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(90)91187-W

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 66 kD 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.