PLASMID 40Gal DETERMINING SYNTHESIS OF α-GALACTOSIDASE α-PsGal, STRAIN Ecoli Rosetta(DE3)/40Gal - PRODUCER OF CHIMERIC PROTEIN CONTAINING AMINO-ACID SEQUENCE α-PsGal, AND METHOD FOR ITS PRODUCTION Russian patent published in 2014 - IPC C12N15/00 

FIELD: biotechnologies.

SUBSTANCE: invention represents plasmide determining synthesis of α-galactosidase α-PsGal, which includes Ncol/Sall - fragment of plasmid pET-40b(+) (Novagen) and DNA fragment, with the size of 2142 pairs of bases, which contains a chimeric gen consisting of structural part of gene α-PsGal, which is adapted on N-end for expression in E. coli cells, and a nucleotide coding a specific sequence for enteropeptidase. The invention also refers to strain E. coli Rosetta (DE3) transformed with the above plasmid - producer of chimeric protein containing amino-acid sequence of recombinant α-galactosidase α-PsGal and sequence specific for enteropeptidase. The invention also refers to a method for obtaining recombinant α-galactosidase α-PsGal, which involves the following stages: incubation of the above strain-producer in liquid nutritional medium LB during 12 hours at 16°C, deposition of bacterial cells by centrifugation, disintegration of a suspension of cells in a buffer, centrifugation of an extract, chromatography of above-deposit liquid on a column with metal affine resin, elution of protein, concentration of active fractions by means of ion-exchange resin, incubation with enteropeptidase and separation of a target product by gel-filtration.

EFFECT: invention allows obtaining more active and stable recombinant alpha-galactosidase with high efficiency degree.

3 cl, 1 dwg, 3 ex