ALKALINE PHOSPHATASE CmAP SYNTHESIS-DETERMINING 40Ph PLASMID, E. coli rosetta(DE3)/40Ph STRAIN - PRODUCER OF CHIMERIC PROTEIN, CONTAINING AMINO ACID SEQUENCE OF RECOMBINANT ALKALINE PHOSPHATASE CmAP, AND PRODUCTION METHOD THEREOF Russian patent published in 2012 - IPC C12N15/52 C12N15/55 C12N15/72 C12N1/21 C12N9/14 

FIELD: chemistry.

SUBSTANCE: disclosed is a plasmid which determines synthesis of alkaline phosphatase CmAP, containing a NcoI/SacI-fragment of plasmid pET-40b(+) (Novagen) and a DNA fragment of 1530 base pairs, which contains a chimeric gene consisting of a CmAP gene structural part which is adapted at the N-end for expression in E.coli cells, and a nucleotide which codes a specific sequence for TEV protease. Described is an E.coli Rosetta(DE3) strain which is transformed by said plasmid, and is a producer of chimeric protein, which contains an amino acid sequence of recombinant alkaline phosphatase CmAP. Disclosed is a method of obtaining recombinant alkaline phosphatase CmAP, comprising steps for: incubating said producer strain in a liquid broth LB for 12 hours at 20°C, depositing bacterial cells by centrifuging, disintegration of the cell suspension in a buffer, centrifuging the extract, chromatography of the supernatant fluid on a column with a metal affinity resin, elution of the protein, concentrating active fractions by ultrafiltration, incubation with protease TEV, concentrating the protein solution and extracting the end product by gel filtration.

EFFECT: improved method.

3 cl, 1 dwg, 3 ex