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Published on: May 4, 2018
High-level soluble production and characterization of porcine ribonuclease inhibitor
T A Klink1, A M Vicentini, J Hofsteenge
1Department of Biochemistry, University of Wisconsin, Madison 53706, USA.
Abstract:
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.
Insights
Recombinant porcine ribonuclease inhibitor (pRI) was efficiently produced in E. coli, yielding a stable, soluble protein. This improved production method enhances the availability of RI for studying its inhibitory effects on cytotoxic ribonucleases.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Cytotoxic ribonucleases in the cytosol pose a health risk.
- Ribonuclease inhibitor (RI) neutralizes pancreatic-type ribonucleases.
- Current methods for recombinant RI production are inefficient, yielding poorly folded proteins.
Purpose of the Study:
- To develop an efficient expression system for soluble porcine ribonuclease inhibitor (pRI).
- To optimize pRI purification for increased yield and conformational stability.
- To investigate the thermal stability of pRI and its complex with ribonuclease A.
Main Methods:
- Overproduction of pRI in Escherichia coli using a trp promoter and minimal medium.
- Purification of pRI via immobilized ribonuclease A affinity chromatography and anion-exchange chromatography.
- Differential scanning calorimetry to assess thermal denaturation of pRI, RNase A, and their complex.
Main Results:
- A novel expression system yielded soluble pRI, maintaining its native conformation.
- Purification achieved a 60-fold increase in yield (15 mg/L) compared to previous methods.
- The pRI-RNase A complex exhibited enhanced conformational stability over individual components.
Conclusions:
- The developed E. coli expression system provides a highly efficient method for producing soluble, active porcine ribonuclease inhibitor.
- This advancement significantly improves the accessibility of RI for research and potential therapeutic applications.
- The increased conformational stability of the pRI-RNase A complex highlights the effectiveness of RI as a ribonuclease scavenger.
