| 摘要 |
FIELD: biotechnologies.SUBSTANCE: invention represents plasmide determining synthesis of ?-galactosidase ?-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 ?-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 ?-galactosidase ?-PsGal and sequence specific for enteropeptidase. The invention also refers to a method for obtaining recombinant ?-galactosidase ?-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 |
