RU2000105214A - METHOD FOR PRODUCING RECOMBINANT PURINNUCLEOSIDE-PHOSPHORILASE, RECOMBINANT PLASMID DNA PERPUPHO1 AND STRAIN ESCHERICHIA COLI BL21 (DE3) / PERPUPHO1 FOR ITS IMPLEMENTATION - Google Patents

Claims (3)

1. A method of producing purine nucleoside phosphorylase of E. coli, comprising cultivating a producer strain obtained by transformation of Escherichia coli cells with plasmid DNA followed by cell disruption and a buffer solution, characterized in that a specially designed pERPUPHOl DNA is used as plasmid DNA, the producer uses the Escherichia coli strain BL21 (DE3) / pERPUPHOl, which is cultured in a rich medium, and the destruction of the cells is carried out by ultrasound and the soluble fraction is separated.  2. Recombinant plasmid DNA pERPUPHOl encoding the amino acid sequence of E. coli purine nucleoside phosphorylase, having a mol. wt. 2.90 MDa, consisting of an NcoI / EcoRI DNA fragment of plasmid pET23d (+), containing a promoter and transcription terminator of a T7 RNA polymerase, a translation enhancer of the T7 phage 10 gene, β-lactamase gene, and an NcoI / EcoRI DNA fragment, containing the Escherichia coli purine nucleoside phosphorylase gene sequence adapted to these sites; containing, as a genetic marker, a β-lactamase gene that determines the resistance of penicillin antibiotics transformed with plasmid pERPUPHOl to E. coli; unique recognition sites of restriction endonucleases located at the following distance to the right of the NcoI site: XbaI - 38 bp , BglII - 96 bp , PνuII - 741 bp, BglI - 2163 bp , PνuI - 2413 bp , EcoRI - 4389 bp  3. The strain Escherichia coli BL21 (DE3), containing recombinant plasmid DNA pERPUPHOl, superproducer purine nucleoside phosphorylase (VKPM B-7888).