An efficient system for active bovine pancreatic ribonuclease expression in Escherichia coli.
Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins. It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids. Since this degradation of RNA interferes with normal cell functions, the signal peptide of alkaline phosphatase (phoA, Escherichia coli) was cloned onto the gene coding for RNase A, directing the protein to the periplasm. Several expression systems have been evaluated which use T7, trc, or PR promoters to transcribe the RNase A gene. Also, variation in host strains was tested to optimize the protein yield. It was found that the PR system gave better expression than the two other systems. E. coli strain BL21 was shown to be the strain in which export to the periplasm was most effective and recombinant RNase A could be isolated from the periplasmic fraction of these cells. The system provides a stable yield of active recombinant bovine pancreatic RNase of about 45-50 mg/liter of cell culture.
Okorokov
A L
AL
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Panov
K I
KI
te Poele
R H
RH
Breukelman
H J
HJ
Furia
A
A
Karpeisky MYa
Beintema
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D012259:Ribonuclease, Pancreatic