METHOD OF PRODUCING FUNCTIONALLY ACTIVE RECOMBINANT PROTEIN OF LETHAL FACTOR ANTHRAX (LF), RECOMBINANT PLASMID DNA pETGST-LFmin, ENCODING ACTIVE LF PROTEIN AND STRAIN OF Escherichia coli BL-GSTLFmin, PRODUCING ACTIVE PROTEIN OF LETHAL FACTOR ANTHRAX

摘要

FIELD: biology.;SUBSTANCE: designed is a new recombinant plasmid pETGST-LFmin (7704 nucleotide pairs), containing a catalytically active fragment of the gene of lethal factor anthrax (LF) controlled by a bacteriophage T7 promoter, determinant of resistance to ampicillin and a glutathione-S-transferase sequence for efficient purification of the recombinant protein on a sorbent with immobilised glutathione. The plasmid provided for efficient sythensis of the protein of LF anthrax, chimerised with a sequence of glutathione-S-transferase for purification on immobilised glutathione. Escherichia coli BL-LFminGST strain is obtained from transformation of the said plasmid DNA into a E.coli BL21 (DE3) strain, which provides for output of synthesised LF protein of not less than 90 mg/l g of raw biomass. The active LF protein is obtained using a method which involves culturing the said recombinant strain, destruction of bacterial cells in a buffer solution with pH 7.4 in the presence of Triton X-100 and a protease inhibitor, and purification on a sorbent with immobilised glutathione.;EFFECT: output of proteolytic active recombinant chimeric purified protein of LF anthrax in amount of not less than 70 mg/l g of raw biomass.;3 cl, 3 dwg, 3 ex