HIV nucleocapsid protein. Expression in Escherichia coli, purification, and characterization.
The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Escherichia coli and purified to homogeneity. The mature 55-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E. coli with authentic termini directly, without the need for processing. The protein was purified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis. Sequencing of each terminus of the purified protein indicated that no proteolytic degradation occurred. A molar extinction coefficient (epsilon 280 = 8350 cm-1 M-1) was determined. The purified nucleocapsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay. A binding constant (Kw) of 1 x 10(8) M-1 was calculated. Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined. These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing sequences from its immature precursor. The possible relevance of these findings are discussed.
You
J C
JC
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262.
McHenry
C S
CS
D001483:Base Sequence; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015683:Gene Products, gag / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014364:Tryptophan / Q000737:chemistry; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004279:DNA, Viral; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D014364:Tryptophan