Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase.
The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase. When this fusion gene was expressed in E. coli under the control of the trp promoter, active RNase T1 having the correct N-terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly. The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture. Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu-58----Ala mutant) in E. coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.
Fujimura
T
T
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Tanaka
T
T
Ohara
K
K
Morioka
H
H
Uesugi
S
S
Ikehara
M
M
Nishikawa
S
S
D000469:Alkaline Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D006163:Ribonuclease T1 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004722:Endoribonucleases; D006163:Ribonuclease T1; D000469:Alkaline Phosphatase