ENGINEERING RIBONUCLEASE-A - PRODUCTION, PURIFICATION AND CHARACTERIZATION OF WILD-TYPE ENZYME AND MUTANTS AT GLN11

Bovine pancreatic ribonuclease A (RNase A) has been the object of much landmark work in biological chemistry, Yet the application of the tec hniques of protein engineering to RNase A has been limited by problems inherent in the isolation and heterologous expression of its gene. A cDNA library was prepared from cow pancreas, and from this library the cDNA that codes for RNase A was isolated, This cDNA was inserted into expression plasmids that then directed the production of RNase A in S accharomyces cerevisiae (fused to a modified alpha-factor leader seque nce) or Escherichia coli (fused to the pelB signal sequence). RNase A secreted into the medium by S. cerevisiae was an active but highly gly cosylated enzyme that was recoverable at 1 mg/l of culture. RNase A pr oduced by E. coli was in an insoluble fraction of the cell lysate. Oxi dation of the reduced and denatured protein produced active enzyme whi ch was isolated at 50 mg/l of culture. The bacterial expression system is ideal for the large-scale production of mutants of RNase A. This s ystem was used to substitute alanine, asparagine or histidine for Gln1 1, a conserved residue that donates a hydrogen bond to the reactive ph osphoryl group of bound substrate, Analysis of the binding and turnove r of natural and synthetic substrates by the wild-type and mutant enzy mes shows that the primary role of Gln11 is to prevent the non-product ive binding of substrate.