| 摘要 |
molecular biology, biotechnology, genetic engineering. SUBSTANCE: recombinant plasmid DNA pERPUPHO1 encoding amino acid sequence of purine nucleoside phosphorylase of E. coli consists of: Nco I/Eco RI-fragment of plasmid pET23d DNA containing promoter and transcription terminator of T7 RNA- polymerase, translation enhancer of gene 10 of phage T7, beta-lactamase gene and Nco I/Eco RI-fragment of DNA containing Escherichia coli purine nucleoside phosphorylase gene sequence adapted to these sites. Strain-producer E. coli BL21(DE3)/pERPUPHO1 obtained by E. coli cells transformation with plasmid DNA pERPUPHO1 is cultured up to accumulation of recombinant purine nucleoside phosphorylase in the amount 60-70% of total protein content. Cells are disrupted by ultrasonic oscillation in buffer solution and soluble fraction is separated. Strain-producer E. coli BL21(DE3)/pERPUPHO1 is cultured in rich medium (YT-, LB-broth and others) or induced with isopropylthio-beta-D-galactoside and cultured again up to attainment of maximal density of culture. Invention decides the problem of producing high-productive recombinant bacterial strain-producer of purine nucleoside phosphorylase. EFFECT: increased yield of Escherichia coli purine nucleoside phosphorylase, simplified technology. 3 cl, 3 ex |
