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Secretion of recombinant ribonuclease T 1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase
Author(s) -
Fujimura Takao,
Tanaka Toshiki,
Ohara Kanako,
Morioka Hiroshi,
Uesugi Seiichi,
Ikehara Mono,
Nishikawa Satoshi
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80886-n
Subject(s) - periplasmic space , signal peptide , escherichia coli , rnase p , biochemistry , ribonuclease , alkaline phosphatase , microbiology and biotechnology , mutant , biology , enzyme , recombinant dna , gene , rna
The ribonuclease T 1 (RNase T 1 ) 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 T 1 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 T 1 (Glu‐58 → Ala mutant) in E. coli , the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.