Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F + expression system inducible by lactose and isopropyl-β- d-thiogalactopyranoside

• Published in: Journal of chromatography. B, Biomedical applications Biomedical applications, 1994-06, Vol.656 (1), p.127-133
• Main Authors: Liška, Vladimír, Evangelista Dyr, Jan, Suttnar, Jiří, Hirsch, Ivan, Vonka, Vladimír
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Antibodies, Bacterial - isolation & purification; Biological and medical sciences; Biotechnology; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral - drug effects; Gene Products, gag - biosynthesis; Gene Products, gag - isolation & purification; Genes, Viral - genetics; HIV-1 - genetics; Humans; Isopropyl Thiogalactoside - pharmacology; Lactose - pharmacology; Methods. Procedures. Technologies; Protein engineering; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification
• ISSN: 0378-4347; 1572-6495
• DOI: 10.1016/0378-4347(94)00079-4

The development of the Escherichia coli expression system, which was prepared by transferring the F′ episome from strain 71/18 to a highly transformable F − strain HB101, is described. These new HB101 (F +) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions. A relatively simple two-step purification of part of a protein ( M r 27 000) encoded by the gag gene of HIV-1 in this expression system is described. The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate. The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves. Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0. The purified protein did not cross-react with antibodies to E. coli.