METHOD FOR OBTAINING OF RECOMBINANT EXOPROTEIN OF A PSEUDOMONAS AERUGINOSA Russian patent published in 2017 - IPC C12N15/31 C12N15/63 C12N15/70 C12N1/19 C07K1/18 C07K14/21 

FIELD: biotechnology.

SUBSTANCE: method includes obtaining of expression plasmid vector pET-rEPA (SEQ NO: 3), containing a promotor sequence of T7 bacteriophage DNA and DNA sequences encoding the N-end region of protein product of translation enhancer (MASMT amino-acid sequence), six histidine residues, site of SUMO protease cleavage and a sequence (SEQ NO: 1) optimized for broadcasting in E. coli encoding recombinant rEPA protein (SEQ NO: 2). Recombinant chimeric precursor protein in the heterological expression system in electro-competent cells of E. coli BL21 (DE3), transformed by the plasmid vector pET-rEPA to obtain a E. coli BL-rEPA strain at 37°C to ensure the maximum amount of accumulation of precursor protein in the soluble fraction. Lysis of the bacterial mass is carried out in the presence of 4% Triton X-100 to preserve the precursor protein in a soluble form. The precursor protein is isolated by metal chelate chromatography on Talon sorbent charged with Co²⁺ ions, followed by hexahistidine and SUMO peptide cleavage with SUMO protease from the polypeptide. The cleaved recombinant protein rEPA is subject to finish purification by anion-exchange chromatography on DEAE-Sepharose sorbent and gel-filtration chromatography on a Superdex 200-filled column, with translation to the target buffer with pH of 7.5, obtaining the recombinant rEPA protein.

EFFECT: production of protein in high yield.

4 dwg, 4 ex