METHOD OF PREPARING RECOMBINANT URIDINE PHOSPHORYLASE, RECOMBINANT PLASMID DNA PERURPH01 AND STRAIN OF ESCHERICHIA COLI BL 21(DEZ)/PERURPH01 FOR ITS REALIZATION

摘要

FIELD: biotechnology, genetic and protein engineering, biochemistry. SUBSTANCE: recombinant plasmid DNA pERURPH01 encoding amino acid sequence of uridine phosphorylase of E. coli consists of DNA fragment Nde I/Sal I of plasmid pET20b(+) containing promoter and terminator of transcription of T7-RNA-polymerase, enhancer of translation of gene 10 phase T7, β- lactamase gene and DNA fragment Nde I/Sal I containing the sequence of gene encoding uridine phosphorylase of Escherichia coli adapted to this sites. The strain-producer is E. coli 21 (DEZ)/pERURPH01 obtained by transformation of E. coli cells by plasmid DNA pERURPH01 is cultured to accumulation of recombinant uridine phosphorylase in the amount 60-70% of total protein level. Cells are disrupted by ultrasonic oscillation in buffer solution and soluble fraction is separated. The strain-producer E. coli BL 21(DEZ)/pERURPH01 is cultured in rich medium (YT-, LB-broth and others) or induced with isopropylthio-β-D-galactoside and cultured again up to maximal culture density. Invention ensures to obtain uridine phosphorylase of E. coli with high yield (80% of total protein content). EFFECT: improved method of preparing, increased yield of enzyme, simplified technology. 4 cl, 3 ex