MOLECULAR CLONING OF THE GENES RESPONSIBLE FOR COLLAGENASE PRODUCTION FROM CLOSTRIDIUM HISTOLYTICUM

ISOLATION OF ISLETS FROM HUMAN PANCREAS TISSUE FOR THE TREATMENT OF TYPE I DIABETES IS HAMPERED BY THE SCARCITY OF THE COLLEGENASE USED IN THE ENZYMATIC DISPERSION PROCESS. CURRENTLY AVAILABLE COMMERCIAL PREPARATIONS OF COLLAGENASE REQUIRES EXTENSIVE SCREENING AND TESTING FOR ITS USEFULNESS.THE UNDERLYING REASON FOR LOT-TO-LOT VARIABILITY IS NOT UNDERSTOOD. THIS PROPOSAL PRESENTS A POTENTIAL SOLUTION TO THESE PROBLEMS BY PROVIDING A PRODUCTION TECHNIQUE WHICH COULD REPRODUCIBLY AND RELIABLY PROVIDE CONSISTENTLY PURE ENZYME WITH COLLAGENASE ACTIVITY IN UNLIMITED QUANTITIES. TO ACHIEVE THIS GOAL WE PLAN TO IDENTIFY AND CLONE THE GENESRESPONSIBLE FOR COLLAGENASE PRODUCTION FROM CLOSTRIDIUM HISTOLYTICUM. CRUDE PREPARATIONS OF COLLAGENASE WILL BE FIRST PURIFIED TO HOMOGENEITY, AN ANTI-COLLAGENASE ANTIBODY RAISED, AND THEN USED AS A PROBE TO IDENTIFY THE CLOSTRIDIUMHISTOLYTICUM GENE. THE RECOMBINANT COLLAGENASE WILL BE EXPRESSED IN ESCHERICHIA COLI HOST AND STUDIED AS TO THE EFFECTIVENESS IN PRODUCING DISPERSED ISLETS.