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Protein Eng. 1995 Mar;8(3):261-73.

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11.

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  • 1Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.

Abstract

Bovine pancreatic ribonuclease A (RNase A) has been the object of much landmark work in biological chemistry. Yet the application of the techniques 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 Saccharomyces cerevisiae (fused to a modified alpha-factor leader sequence) or Escherichia coli (fused to the pelB signal sequence). RNase A secreted into the medium by S.cerevisiae was an active but highly glycosylated enzyme that was recoverable at 1 mg/l of culture. RNase A produced by E.coli was in an insoluble fraction of the cell lysate. Oxidation of the reduced and denatured protein produced active enzyme which 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 system was used to substitute alanine, asparagine or histidine for Gln11, a conserved residue that donates a hydrogen bond to the reactive phosphoryl group of bound substrate. Analysis of the binding and turnover of natural and synthetic substrates by the wild-type and mutant enzymes shows that the primary role of Gln11 is to prevent the non-productive binding of substrate.

PMID:
7479688
[PubMed - indexed for MEDLINE]
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