NUCLEASE A IN BACILLUS SUBTILIS INTERACTIVE EXPERT EDITING SUBSYSTEM LOW-COST HRPT DIRECT READOUT STATION

BOVINE PANCREATIC RIBONUCLEASE A (RNASE A) IS A MODEL SYSTEMFOR ESTABLISHING RULES THAT GOVERN PROTEIN STABILITY AND FOLDING. BY LEARNING AND APPLYING THESE RULES, MORE ROBUST ENZYMES OF VARIOUS TYPES COULD BE DESIGNED FOR COMMERCIAL APPLICATIONS. SYNTHESIS, CLONING AND EXPRESSION IN BACILLUSSUBTILIS OF A GENE ENCODING RNASE A WILL ALLOW FOR THE ECONOMICAL PRODUCTION OF VARIANT PROTEINS WHICH ARE GENERATED BY OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS (ODM). THTHESE VARIANT PROTEINS WILL THEN BE USED FOR STRUCTURE/FUNCTION AND/OR FOLDING PATTERN STUDIES. THE EASE AND EFFICIECY OF PRODUCING SPECIFIED PROTEIN VARIANTS IN THIS SYSTEM COUPLED WITHT HE WEALTH OF PHYSICAL DATA AVAILABLE ON RNASE A SHOULD RESULT IN THE RAPID ACCUMULATION OF PERTINENT DATA ON THE FOLDING BEHAVIOR OF PROTEINS. IN PHASE I A STRAIN OFB. SUBTILIS THAT SECRETES RNASE A WILL BE DEVELOPED BY FUSING A SYNTHETIC GENE TO EACH OF THREE PLASMID VECTOR SYSTEMS DESIGNED TO PROMOTE EXPRESSION AND SECRETION OF FOREIGN PROTEINS. IN PHASE II, THE CLONED GENE WILL BE USED TO PRODUCE SPECIFIC PROTEIN VARIANTS BY ODM FOR STRUCTURE/ FUNCTION AND FOLDING PATTERN STUDIES. THE POTENTIAL CO COMMERCIAL APPLICATION AS DESCRIBED BY THE AWARDEE: BY ESTABLISHING THE RULES THAT GOVERN THE STABILITY OF A PROTEIN RELATIVE TO EXTREMES OF PH, HEAT, AND DENATURANTS MORE ROBUST ENZYMES CAN BE DESIGNED FOR USE IN INDUSTRIAL SYSTEMS.