Sharing a Purification Process Scheme for High-Purity RNase A

Ribonuclease A

Ribonuclease A (RNase A), abbreviated as RNase A, is a single-chain polypeptide containing four disulfide bonds, with a molecular weight of approximately 13.7 kDa (amino acid sequence). RNase A functions as an endoribonuclease.

Purification Strategy for Ribonuclease through Chromatography

Extraction and Crude Purification Protocol

We primarily employ the crude extraction of bovine pancreas using ammonium sulfate precipitation, a method modified by McDonald based on the purification approach outlined by Kunitz in ‘Methods in Enzymology,’ Vol. II, Page 429, 1955.

Purification Protocol

We utilize CM Tanrose 6FF as the chromatographic medium, a high-flow agarose-based weak cation exchange resin developed by Monash Technology. This medium, with a 6% cross-linked agarose framework, possesses an average particle size of 90μm and a particle size range of 45-165μm. It offers advantages such as high flow rates and resolution, making it the preferred medium for large-scale purification of biomolecules. Post-elution, the purity of Ribonuclease A can be enhanced to 71-86%. Following further impurity removal steps, the final purity exceeds 95%, with an overall yield of approximately 56%.

Below, we share the chromatography conditions:

Column Packing: CM Tanrose 6FF (Catalog Number: 00062-53002).

Sample: Crude extract from bovine pancreas (containing various impurities and proteins).

Operational Procedure:

Buffer B, 3 column volumes (CV), flow rate 2.5 mL/min

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Equilibration with Buffer A, 13 CV, flow rate 2.5 mL/min

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Sample loading onto the injection loop, flow rate 2.5 mL/min

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Washing with Buffer A to remove impurities, 8 CV, flow rate 2.5 mL/min

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Linear gradient elution with Buffer B, 0-40% B, 10 CV, flow rate 2.5 mL/min

Ion Exchange Chromatography Result Analysis

For the chromatography input sample and elution fractions, SDS-PAGE electrophoresis was performed, followed by analysis using a gel imaging system.

Figure 1: Chromatogram of Ribonuclease A Purified by Ion Exchange Chromatography

Figure 2: Chromatography column loading solution

Figure 3: Chromatography column eluent

After purification through chromatography, SDS-PAGE data analysis reveals that the purity has been enhanced to 82.4% in a single chromatographic step, demonstrating the high resolution of the column packing material.

Conclusion

The processing of crude extract from bovine pancreas through salt precipitation, followed by separation and purification using CM Tanrose 6FF for RNase A, has significantly enhanced its purity. The removal of numerous impurities was achieved, and subsequent processing, including SDS-PAGE gel electrophoresis, demonstrated a single band on gel imaging, meeting the purification criteria.