Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease.
A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library. Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E. coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence. Approx. 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation. When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity. Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene. About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells. Thus this eukaryotic signal sequence is both recognized by the E. coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.
Schein
C H
CH
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (E.T.H.), Zürich.
Boix
E
E
Haugg
M
M
Holliger
K P
KP
Hemmi
S
S
Frank
G
G
Schwalbe
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005813:Genes, Synthetic / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000201:enzymology*
D021382:Protein Sorting Signals; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic