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-beta-D-thiogalactopyranoside.
The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a 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.
Liska
V
V
Department of Biochemistry, Institute of Hematology and Blood Transfusion, Prague, Czech Republic.
Dyr
J E
JE
Suttnar
J
J
Hirsch
I
I
Vonka
V
V
D000907:Antibodies, Bacterial / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015683:Gene Products, gag / Q000096:biosynthesis* / Q000302:isolation & purification*; D005814:Genes, Viral / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D007785:Lactose / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D015683:Gene Products, gag; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D007785:Lactose