AN EFFICIENT SYSTEM FOR ACTIVE BOVINE PANCREATIC RIBONUCLEASE EXPRESSION IN ESCHERICHIA-COLI

Bovine pancreatic ribonuclease (RNase A) is a member of a homologous g roup of extensively studied proteins, It is a small, basic protein, co ntaining 124 amino acid residues and four stabilizing disulfide bridge s. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids. Since this degradation of RNA interferes with n ormal cell functions, the signal peptide of alkaline phosphatase (phoA , Escherichia coli) was cloned onto the gene coding for RNase A, direc ting the protein to the periplasm. Several expression systems have bee n evaluated which use T7, trc, or P-R promoters to transcribe the RNas e A gene. Also, variation in host strains was tested to optimize the p rotein yield, It was found that the P-R system gave better expression than the two other systems. E. coli strain BL21 was shown to be the st rain in which export to the periplasm was most effective and recombina nt RNase A could be isolated from the periplasmic fraction of these ce lls. The system provides a stable yield of active recombinant bovine p ancreatic RNase of about 45-50 mg/liter of cell culture. (C) 1995 Acad emic Press, Inc.