RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUENFACIENS

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Two proteins, barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, and barstar, its intracellular inhibitor, are used as a model system for the study of protein folding a protein- protein interactions. Barnase is one of an homologous group of ribonucleases occurring in both prokaryotes and eukaryotes. Recombinant DNA techniques are being applied to the project with three major aims: 1) to facilitate production, 2) to examine the structural and control sequences of the genes and 3) to tailor specifically designed modifications in the sequences to test theories of protein folding. The lethal effect of the cloned wild-type barnase gene in either E. coli or B. subtilis can be repressed by expression of the cloned barstar gene placed on the same plasmid. Secretion vectors for both proteins have been devised. Both genes have been sequenced, providing confirmation of the barnase sequence (110 residues) and a derived sequence (89 residues) for barstar which agrees well with previous amino-acid analysis. Effects of several site-directed mutations of barnase on activity and thermal stability have been studied.