High-level soluble production and characterization of porcine ribonuclease inhibitor.
Ribonucleases can be cytotoxic if they retain their ribonucleolytic activity in the cytosol. The cytosolic ribonucleolytic activity of ribonuclease A (RNase A) and other pancreatic-type ribonucleases is limited by the presence of excess ribonuclease inhibitor (RI). RI is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases that competitively inhibits their ribonucleolytic activity. RI had been overproduced as inclusion bodies, but its folding in vitro is inefficient. Here, porcine RI (pRI) was overproduced in Escherichia coli using the trp promoter and minimal medium. This expression system maintains pRI in the soluble fraction of the cytosol. pRI was purified by affinity chromatography using immobilized RNase A and by anion-exchange chromatography. The resulting yield of 15 mg of purified RI per liter of culture represents a 60-fold increase relative to previously reported recombinant DNA systems. Differential scanning calorimetry was used to study the thermal denaturation of pRI, RNase A, and the pRI-RNase A complex. The conformational stability of the complex is greater than that of the individual components.
Klink
T A
TA
Department of Biochemistry, University of Wisconsin, Madison 53706, USA.
Vicentini
A M
AM
Hofsteenge
J
J
Raines
R T
RT
D000818:Animals; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012995:Solubility; D013552:Swine
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011994:Recombinant Proteins; C066953:ribonuclease inhibitor, porcine; D012259:Ribonuclease, Pancreatic