Overexpression of Escherichia coli genes encoding nucleoside phosphorylases in the pET/Bl21(DE3) system yields active recombinant enzymes.
The Escherichia coli genes encoding purine nucleoside phosphorylase, uridine phosphorylase, and thymidine phosphorylase were cloned into pET plasmids to generate highly effective E. coli BL21(DE3) strains producing each of these enzymes. Optimum conditions for biosynthesis of each enzyme as a soluble protein with intact biological activity were found. The crude preparations are approximately 80% pure and can be used immediately for enzymatic transglycosylation. The enzyme preparations were purified to homogeneity by two steps including fractional precipitation with ammonium sulfate and subsequent chromatography on Sephadex G-100 and DEAE-Sephacel.
Esipov
Roman S
RS
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow 117997, Russia. esipov@ibch.ru
Gurevich
Alexandr I
AI
Chuvikovsky
Dmitry V
DV
Chupova
Larisa A
LA
Muravyova
Tatyana I
TI
Miroshnikov
Anatoly I
AI
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D010957:Plasmids; D011683:Purine-Nucleoside Phosphorylase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013939:Thymidine Phosphorylase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic; D014543:Uridine Phosphorylase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase; D014543:Uridine Phosphorylase; D013939:Thymidine Phosphorylase