A Simple Method for Overproduction and Purification of p24 Gag Protein of Human Immunodeficiency Virus Type 1

Abstract

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 25 C, but not at 37 C. 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 37 C and 25 C, 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.

Overproduction, Purification, and Diagnostic Use of the Recombinant HIV‐1 Gag Proteins, the Precursor Protein p55 and the Processed Products p17, p24, and p15

“…The Gag-Pol proteins, which were synthesized through frameshifting as the minor products, containing the entire protease moiety, had the protease activity and could process the Gag proteins synthesized as major products and themselves in E. coli (39,43). However, the protease synthesized via frameshifting could not process the Gag proteins completely.…”

“…We and others have reported that the gag-to-pol frameshifting occurred during the translation of the gag gene in E. coli, as in the virus-infected cells (38,39,43). The Gag-Pol proteins, which were synthesized through frameshifting as the minor products, containing the entire protease moiety, had the protease activity and could process the Gag proteins synthesized as major products and themselves in E. coli (39,43).…”

“…The Ndel-Pstl fragment (787-1415) derived from the Gag19 (Nde) was cloned into pT7-7 opened by NdeI and Pstl, giving pTG541. The 1.2-kb Pstl fragments containing the Pstl-Ball (1415-2619) regions derived from pPG912 and pPG922 (43) were inserted into the Pstl site of pTG541, giving pTG591 antibody were developed in TBS containing KO, and a chromogenic reagent, 4-chloro-l-naphthol.…”

Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV-1

“…Recombinant p24 (rp24) was produced in Escherichia coli transformed with expression plasmid carrying the corresponding cDNA and was purified as described previously (12). The recombinant proviral clone used was pNL4-3 (13), which contained DNA from HIV-1 isolates NY5 (GenBank accession number HIVNL43) and LAV (14), and the sequences for rp24 derived from NY5.…”

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

“…Recombinant HIV-1 p17 (rp17) and p24 (rp24) antigens were produced in Escherichia coli transformed with expression plasmids carrying the corresponding cDNAs and were purified as described previously (28,31). The recombinant proviral clone used was pNL4-3 (1), which contained DNA from HIV-1 isolates NY5 (GenBank accession no.…”