AN IMPROVED SYSTEM FOR RIBONUCLEASE BA EXPRESSION

The extracellular ribonuclease from Bacillus amyloliquefaciens (barnas e, RNase Ba) is a well-characterized enzyme extensively used in struct ure-function studies. A new system for efficient expression and purifi cation of barnase has been developed. The strong regulated expression cassette with the Pr promoter of lambda phage and the cooperative expr ession of barnase and barstar under its control have been applied to e xpression of these proteins in Escherichia coli. The expression casset te containing the Pr promoter of E. coli lambda phage under cI repress or regulation and the nucleotide sequence coding for barnase and barst ar structural genes were merged into the plasmid pTN441, which was use d for large-scale barnase production. The phoA signal peptide was used to express the target protein into cell periplasm. The purification o f RNase Pa was carried out in two steps: the initial sample was concen trated followed by RP-HPLC. The system provides a stable yield of homo geneous protein of about 100-150 mg per liter of culture medium. (C) 1 994 Academic Press, Inc.