Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector.
The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.
D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D006163:Ribonuclease T1 / Q000096:biosynthesis* / Q000235:genetics; D013329:Structure-Activity Relationship
D021382:Protein Sorting Signals; D004722:Endoribonucleases; D006163:Ribonuclease T1