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Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E. coli and its use in ELISA.
Sohn MJ; Chong YH; Chang JE; Lee YI; Molecular Genetics Lab, Korea
September 30, 1994
J Biotechnol. 1994 May 15;34(2):149-55. Unique Identifier : AIDSLINE

To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE' gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121-398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.

Cloning, Molecular/METHODS Electrophoresis, Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay/METHODS Escherichia coli Gene Products, gag/*BIOSYNTHESIS/ISOLATION & PURIF *Genes, gag Genetic Vectors HIV-1/GENETICS/*METABOLISM Plasmids Recombinant Proteins/*BIOSYNTHESIS/ISOLATION & PURIF Restriction Mapping JOURNAL ARTICLE