Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24).
Capsid protein (p24;CA) of human immunodeficiency virus type 1 (HIV-1) was synthesized in Escherichia coli strain BL21 (DE3) using a plasmid encoding a truncated HIV-1 gag/pol gene. The plasmid, which contained a mutation in the frameshift region, expressed viral proteinase (PR), a pol gene product, in the gag reading frame, resulting in efficient processing of mature CA and other gag-related products. The expressed CA is soluble, recognized by monoclonal antibodies directed against HIV CA and has an N-terminal sequence identical to that of CA purified from HIV. Purification was done under mild conditions where coexpressed HIV PR retained enzymatic activity. Milligram quantities of 90% pure CA protein were obtained after chromatography on DEAE cellulose followed by facilitated aggregation of the CA in the unbound fraction. The precipitated CA was readily dissolved in low ionic strength aqueous buffer. Gel exclusion chromatography results indicated that, in solution, CA existed in oligomeric form.
Ehrlich
L S
LS
Department of Microbiology, State University of New York Stony Brook 11794.
Krausslich
H G
HG
Wimmer
E
E
Carter
C A
CA
D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015683:Gene Products, gag / Q000235:genetics* / Q000302:isolation & purification; D015750:Genes, gag; D015751:Genes, pol; D016655:HIV Core Protein p24; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012995:Solubility; D014758:Viral Core Proteins / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; D015683:Gene Products, gag; D016655:HIV Core Protein p24; D014758:Viral Core Proteins