Purification and characterization of human immunodeficiency virus (HIV) core precursor (p55) expressed in Saccharomyces cerevisiae.
The core structure of retroviruses, including the human immunodeficiency virus (HIV), consists of proteins that are initially synthesized as polyprotein precursors and then processed by a virally encoded protease yielding the mature core polypeptides. To obtain sufficient quantities of the purified HIV core precursor p55 for detailed studies, a segment of HIV DNA encoding the full length core precursor polyprotein p55 was expressed in Saccharomyces cerevisiae using a plasmid containing a constitutive galactose promoter. The expression of this DNA produced a protein with an estimated molecular size of 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this protein was immunoreactive to anti-HIV p24 antisera. Following cell lysis, freezing, and thawing, the expressed protein was an insoluble aggregate that served as the starting material for the purification process. Solubilization of the insoluble p55 with guanidine HCl followed by phenyl-Sepharose column chromatography and high performance liquid chromatography resulted in a preparation of p55 that was greater than 95% pure by SDS-PAGE, immunoreactive to anti-HIV core protein antibodies, and completely soluble in aqueous solution. The expressed p55 appeared to be myristoylated as evidenced by the incorporation of radiolabel following incubation of recombinant yeast cells with [3H]myristic acid; in addition the amino terminus of the final purified protein was blocked. Proteolytic digestion of purified p55 with synthetic HIV protease yielded the predicted amino- and carboxyl-terminal products; these were confirmed by amino acid sequence analysis. In contrast, digestion of purified p55 by the protease derived from the avian myeloblastosis virus resulted in fragments that were different in size from those produced by the HIV protease. The availability of the purified, full length water-soluble HIV core precursor will be useful in identifying agents that inhibit its processing by the HIV protease.
Vlasuk
G P
GP
Department of Biological Chemistry, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Waxman
L
L
Davis
L J
LJ
Dixon
R A
RA
Schultz
L D
LD
Hofmann
K J
KJ
Tung
J S
JS
Schulman
C A
CA
Ellis
R W
RW
Bencen
G H
GH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015683:Gene Products, gag; D006678:HIV / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012191:Retroviridae Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism
D015683:Gene Products, gag; D011498:Protein Precursors; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; D014758:Viral Core Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1