A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1.
A simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described. The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid. A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting. In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C. When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease. Recombinant p24 was purified to near homogeneity by a simple three-step procedure. The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E. coli by the coexpressed protease. The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.
Tanaka
N
N
Department of Experimental Chemotherapy, Osaka University, Japan.
Saitoh
A
A
Nakata
A
A
Shinagawa
H
H
D000090266:Chromobox Protein Homolog 5; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics; D015750:Genes, gag / Q000235:genetics*; D015751:Genes, pol / Q000235:genetics; D015483:HIV Antibodies / Q000032:analysis; D016655:HIV Core Protein p24 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015658:HIV Infections / Q000175:diagnosis; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D019590:Nucleocapsid Proteins; D016364:Reading Frames / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D054301:gag Gene Products, Human Immunodeficiency Virus
C000715594:CBX5 protein, human; D015683:Gene Products, gag; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1; D000090266:Chromobox Protein Homolog 5