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A Simple Method for Overproduction and Purification of p24 Gag Protein of Human Immunodeficiency Virus Type 1
Author(s) -
Tanaka Naoto,
Saitoh Atsushi,
Nakata Atsuo,
Shinagawa Hideo
Publication year - 1992
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1992.tb02084.x
Subject(s) - biology , fusion protein , protease , lac operon , group specific antigen , microbiology and biotechnology , recombinant dna , escherichia coli , peptide sequence , plasmid , virus , virology , gene , biochemistry , enzyme
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.