Countercurrent chromatographic separation and purification of various ribonucleases using a small-scale cross-axis coil planet centrifuge with aqueous–aqueous polymer phase systems

Abstract

Countercurrent chromatographic separation and purification of various ribonucleases (RNases) was performed using small-scale cross-axis coil planet centrifuge (X-axis CPC) with aqueous-aqueous polymer phase systems. RNases B and A were well resolved from each other with an aqueous-aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate (pH 9) as the lower phase mobile. The commercial RNase A samples obtained from three different companies were also highly purified using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) using the upper phase as the mobile phase. Recombinant RNase Po1, an RNase T1 family enzyme, was further successfully separated from the crude extract using the same solvent system with the lower phase used as the mobile phase. The RNase activities were well preserved during the CCC separation. The overall results demonstrate that small-scale X-axis CPC is useful for a simple and rapid purification of various RNases in a preparative-scale

Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid–liquid phase separation of cellular biomolecules

“…CCC is also useful for the separation of a protein and its sugar conjugate due to the difference in K values. In our laboratory, glycosylated RNase B was eluted earlier than nonglycosylated RNase A in CCC separation using the 12.5% (w/w) PEG 1000/12.5% (w/w) dibasic potassium phosphate system (pH 9.2), as described earlier (Shinomiya et al, 2009). Sugar moiety increases the hydrophilicity of the glycoprotein structure.…”

“…In further studies, RNase B and RNase A (type XII‐A) were well separated from each other using the lower mobile phase of the 12.5% (w/w) PEG 1000/12.5% (w/w) dibasic potassium phosphate system (pH 9.2). In particular, the type I‐AS RNase A was highly purified from relatively large amounts of hydrophobic impurities with the upper mobile phase of the 16.0% PEG 1000/6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system where the RNase activity was well retained during the CCC separation (Shinomiya et al, 2009).…”

Countercurrent Chromatographic Separation of Proteins Using an Eccentric Coiled Column with Synchronous and Nonsynchronous Type-J Planetary Motions

“…The type-J multilayer CPC is mainly used for separation with organic-aqueous two-phase solvent systems, whereas the cross-axis CPC is used for separation with aqueous-aqueous polymer phase systems. In our laboratory, the improved small-scale model of cross-axis CPC has been developed and applied to separation of standard stable protein samples 5 and enzymes such as several types of ribonucleases 6 and collagenases 7 with aqueous-aqueous polymer phase systems. In the type-J multilayer CPC, the separation column rotates about its axis synchronously in the same direction to the revolution whereas in the cross-axis CPC, the column revolves around the vertical central axis of the centrifuge while rotating about its horizontal axis at the same angular velocity.…”

Partition efficiencies of newly fabricated universal high-speed counter-current chromatograph for separation of two different types of sugar derivatives with organic–aqueous two-phase solvent systems

“…In general, type-J multilayer CPC is useful for separation with organic-aqueous two-phase solvent systems and cross-axis CPC with aqueous-aqueous polymer phase systems. In our laboratory, the improved small-scale model of cross-axis CPC has been developed and applied to separation of stable proteins [5] and enzymes such as several types of ribonucleases [6] and collagenases [7] with aqueous-aqueous polymer phase systems.…”