High-level soluble production and characterization of porcine ribonucleaseinhibitor

Ribonucleases can be cytotoxic if they retain their ribonucleolytic activit y in the cytosol. The cytosolic ribonucleolytic activity of ribonuclease A (RNase A) and other pancreatic-type ribonucleases is limited by the presenc e of excess ribonuclease inhibitor (RI). RI is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases that competitively inhibits their ribonuc leolytic activity. RI had been overproduced as inclusion bodies; but its fo lding in vitro is inefficient. Here, porcine RI (pRI) was over-produced in Escherichia coli using the trp promoter and minimal medium. This expression system maintains pRI in the soluble fraction of the cytosol. pRI was purif ied by affinity chromatography using immobilized RNase A and by anion-excha nge chromatography. The resulting yield of 15 mg of purified RI per liter o f culture represents a 60-fold increase relative to previously reported rec ombinant DNA systems. Differential scanning calorimetry was used to study t he thermal denaturation of pRI, RNase A, and the pRI-RNase A complex. The c onformational stability of the complex is greater than that of the individu al components. (C) 2001 Academic Press.