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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
The cross-axis coil planet centrifuge (X-axis CPC) performs efficient separation with highly polar two-phase solvent systems, such as aqueous-aqueous polymer phase systems [1, 2]. This apparatus has a unique mode of planetary motion, such that the column holder rotates about its horizontal axis while revolving around the vertical central axis of the centrifuge at the same angular velocity [3, 4]. This motion provides excellent retention of the stationary phase even for the viscous, low-interfacial tension two-phase solvent systems such as aqueous-aqueous polymer phase systems which are generally not efficiently applied to the type-J multilayer CPC. Although our floor model of the type X-1.5L cross-axis CPC was useful for separation of proteins with aqueous-aqueous polymer phase systems . It had some problems such as requirement of a large space, short life of the flow tubes, and difficulty in installing separation columns.
In order to improve the utility of the apparatus, a new small-scale X-axis CPC was designed and fabricated in our laboratory . The down-sizing of the apparatus was done by reducing the scale to half of our original floor model with several other design improvements. In our previous studies, performance of this new X-axis CPC was evaluated on protein separation using an aqueous-aqueous polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate with four multilayer coiled columns. The aqueous-aqueous polymer phase systems are useful for separation of proteins that are denatured by organic-aqueous two-phase solvent systems.
The present paper describes the application of this apparatus to the separation and purification of various types of ribonucleases (RNases) with aqueous-aqueous polymer phase systems.
The small-scale X-axis CPC employed in the present studies was constructed at the Machining Technology Center of Nihon University, Chiba, Japan. The design of the apparatus is described in detail in the previous paper .
Each multilayer coil was prepared by tightly winding a piece of 1.5 mm I.D. and 2.5 mm O.D. PTFE (polytetrafluoroethylene) tubing (Flon Kogyo, Tokyo, Japan) around the holder hub of 3 cm in diameter, forming five tight-coiled layers between a pair of flanges spaced 5 cm apart. Each coiled column was prepared according to the following procedure: The tubing was directly wound onto the holder hub starting on the proximal side (close to the central axis of the apparatus). After each coil layer was completed, the layer was wrapped with an adhesive tape and the tubing was straightly returned to the other side to resume winding in the same direction. This method results in a multilayer coil assembly composed of either entirely right- or left-handed coils, which is different from that used in the type-J multilayer CPC. Left-handed coils were subjected to the forward rotation and right-handed coils, the backward rotation. These two pairs of right- and left-handed coil assemblies were alternately connected in series with flow tubes in such a way that the distal terminal of the first column assembly is connected to the proximal terminal of the second column assembly and so forth. Four coil assemblies at the total capacity of 102 mL were symmetrically mounted on the rotary frame for balancing the centrifuge system.
Polyethylene glycol (REG) 1000 (MW 1000), RNase B from bovine pancreas and RNase T1 from Aspergillus oryzae (MW 11,072) were purchased from Sigma (St. Louis, MO, USA). Dibasic potassium phosphate and monobasic potassium phosphate were obtained from Wako (Osaka, Japan).
Various types of RNase A from bovine pancreas (MW 13,674) were obtained from three different companies, i.e., Sigma (type I-A, I-AS, III-A and XII-A), Wako, Nacalai Tesque (Kyoto, Japan). All other reagents were of reagent grade.
Two types of polymer phase systems used in the present studies were prepared by dissolving PEG 1000 and potassium phosphate salts each at a desired concentration in distilled water: The first polymer phase system composed of 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate was prepared by dissolving 125 g of PEG 1000 and 125 g of dibasic potassium phosphate (anhydrous) in 750 g of distilled water. The second system composed of 16% (w/w) PEG 1000, 6.3% (w/w) dibasic potassium phosphate and 6.3% (w/w) monobasic potassium phosphate was prepared by dissolving 160 g of PEG 1000, 63 g of dibasic potassium phosphate and 6.3 g of monobasic potassium phosphate in 71.5 g of distilled water, Each solvent mixture was thoroughly equilibrated in a separatory funnel at room temperature and the two phases separated after the two clear layers were formed.
The sample solutions were prepared by dissolving each sample mixture in the desired amounts of the two-phase mixture consisting of equal volumes of each phase. The commercial RNase A sample (30 mg) was dissolved with a two-phase solvent mixture (1.0 mL each of upper and lower phases used for separation). The sample solution for recombinant RNase Po1 purification was prepared by adding the desired amount of PEG 1000, and dibasic and monobasic potassium phosphates in the crude extract solution to make about equal volumes of each phase used for CCC separation.
An expression vector for RNase Po1 was constructed according to the method by Hang et al.  and transformed into Escherichia coli BL21(DE)pLysS (Novagen, Darmstadt, Germany). The cells were cultured with terrific broth at the temperature of 25°C by adding 100 μ g/mL of ampicillin. The crude extract of recombinant RNase Po1 (rRNase Po1, MW 10,772) was prepared according to the extraction procedure of original RNase Po1 from a mushroom (Pleurotus ostreatus)  as follows: The supernatant (2000 mL) obtained by the centrifugation of the 7d culture medium at 10,000 rpm for 20 min was subjected to the fractionation with ammonium sulfate. The precipitate formed in 90% saturation of ammonium sulfate was collected by the centrifugation at 10,000 rpm for 30 min. This precipitate was suspended in 300 mL of 10 mM acetate buffer (pH 6.0) and dialyzed against deionized water. The dialyzate was concentrated to the volume of 100 mL and applied to the Sephadex G-50 column (180 cm × 3.5 cm I.D.) eluting with 10 mM acetate buffer (pH 6.0). The eluate containing rRNase Po1 was concentrated and dialyzed against deionized water and adjusted at pH 5.0 with 1 mol/L hydrochloric acid. This solution (400 mL) was then subjected to the SP-Toyopearl column (20 cm × 1.5 cm I.D.) using 10 mM acetate buffer (pH 5.0) with a linear gradient of sodium chloride (0 – 0.3 M). The RNase active fractions were concentrated and dialyzed against deionized water, and applied to a column packed with Ultro gel AcA54 (180 cm × 3.5 cm I.D.) using 10 mM acetate buffer (pH 6.0). The eluted fractions (300 mL) were collected, concentrated and dialyzed against deionized water. After adjusting to pH 7.5, the solution (300 mL) was subjected to the DEAE-Toyopearl column (10 cm × 1.0 cm I.D.) using 10 mM acetate buffer (pH 7.5). The eluate was concentrated and dialyzed against deionized water again and the solution (100 mL) was subjected to the Heparin-Sepharose column (30 cm × 1.5 cm I.D.) using 50 mM acetate buffer (pH4.5). The eluate was concentrated and dialyzed against deionized water. The yield was calculated at 56.7% by measuring the RNase activity. Aliquots of this solution was subjected to the CCC separation after adding PEG 1000 and potassium phosphates to make the desired two phase composition with about equal volumes of each phase used for separation.
Each separation was initiated by completely filling the column with the stationary phase, followed by injection of the sample solution into the head terminal of the column using a syringe. Then, the mobile phase was pumped into the column using a reciprocating pump (Model LC-6A, Shimadzu, Kyoto, Japan), while the column was rotated at 1000 rpm. The effluent from the column outlet was collected into test tubes at 1.6 mL/tube using a fraction collector (Model CHF100AA, Advantec, Tokyo, Japan).
Each collected protein fraction was diluted with 1 mL of distilled water and the absorbance was measured at 280 nm with a spectrophotometer (Model UV-1600, Shimadzu).
An aliquot of the CCC fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The equipment of PAGE was obtained from ATTO (Tokyo, Japan). SDS-PAGE was carried out with 15% polyacrylamide gel and at 100 V constant according to Leammli’s method .
Assay of RNase activity was carried out as described by Irie  using RNA as a substrate at pH 4.25 and 37°C, and the protein concentration was colorimetrically determined according to the method described by Smith et al. , using bovine serum albumin as a standard.
In order to establish the suitable experimental condition for the small-scale X-axis CPC, the CCC separation between RNase A (type XII-A, Sigma) and RNase B was carried out using an aqueous-aqueous polymer phase system composed of 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate. As illustrated in Fig. 1, these RNases were well separated from each other at the peak resolution of 1.1 with retention of the stationary phase at 24.7%. This separation may be based on the difference in hydrophobicity between RNases B and A. It is well known that RNase B contains a sugar chain composed of 2 N-acetyl glucosamines and 5 – 9 mannoses connected to the 34th asparagine residue while having same amino acid sequences of RNase A. Therefore, more hydrophilic RNase B was first eluted followed by RNase A, when the lower phase was used as the mobile phase.
In our previous studies , a small amount of impurity was found as a single peak in the CCC separation of the commercial RNase sample (type I-A produced by Sigma). This result indicates that the commercial RNase A sample may contain some impurity. Figure 2 illustrates CCC chromatograms of various commercial RNase A samples obtained from Sigma. When using an aqueous-aqueous polymer phase system composed of 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate (pH 6.6) with upper phase mobile, the type I-A sample gave a small impurity peak near the solvent front (Fig. 2A) as observed in the previous study . The purity of RNase A in the type I-A sample was calculated at almost 88.0% from the chromatogram. Both of type III-A and XII-A samples were showed a single RNase A peak at 280 nm, as illustrated in Figs. 2B and 2C, respectively. Among these RNase A samples examined in the present studies, RNase A in the type I-AS sample was sufficiently separated from a relatively large amount of impurities (Fig. 2D). The peak resolution between the impurity and RNase A peaks was 1.9. An amount of RNase A present in the type I-AS sample was calculated from the chromatogram at almost 69.3%. The RNase activities of RNase A fractions were well preserved during the CCC separation while no activities were found in the impurity fractions.
Fig. 3 illustrates the SDS-PAGE electrophoretograms of the CCC fractions separated from various commercial RNase A samples of Sigma shown in Fig. 2. In the type I-A sample (Fig. 3A), several different impurity proteins were detected at the molecular weight of near 18 kDa and under 10 kDa (fraction No. 67 – 74), while RNase A was found at the molecular weight of around 13 kDa (MW 13,680) (fraction No. 77 – 87). Especially, the impurity protein corresponding to the fraction No. 90 and 92 was clearly found as a single band at about 16kDa while no peak was shown in the CCC chromatogram (Fig. 2A). This impurity protein was also found in the type III-A sample (fraction No. 90 and 92) as illustrated in Fig. 3B. The type XII-A sample was most highly purified among the commercial RNase A samples used in the present studies because no impurity proteins were detected in the CCC fractions by SDS-PAGE (Fig. 3C). In the type I-AS sample illustrated in Fig. 3D, a large amount of impurity proteins were found in the CCC fractions except the highly purified fractions (No. 77 and 82) having the RNase A activities as shown in Fig. 2D. These results indicate that commercial RNase A can be highly purified by the CCC separation with an aqueous-aqueous polymer phase system.
Other commercial products of RNase A were also examined using the same experimental conditions as applied to the Sigma samples. As illustrated in Fig. 4, both Wako and Nacalai samples produced a single peak in each CCC separation. However, an impurity with a molecular weight close to that of RNase was detected in the fractions after eluting RNase A (Fig. 5). This result was similar to that obtained from the type III-A Sigma sample.
As described above, it is demonstrated that the impurities present in the commercial RNase A sample can be easily eliminated using the present CCC system with the small-scale X-axis CPC.
When using the solvent system composed of 16.0% (w/w) PEG 1000 – 6.3% (w/w) monobasic potassium phosphate — 6.3% (w/w) dibasic potassium phosphate (pH 6.6) with the lower phase mobile, RNase T1 (MW 11,072) was eluted as illustrated in Fig. 6. The recombinant RNase Po1 (rRNase Po1, MW 10,772), one of RNase T1 family RNases (guanylic acid — specific RNases), was also successfully separated from the crude extract under the same experimental conditions. In Fig. 7, numerous peaks were found in the CCC chromatogram by absorbance at 230 and 280 nm while the RNase activities were detected in a single peak eluted at 400 – 500 ml retention volumes. Fig. 8 also illustrates a phoretogram of these fractions. Most of impurities present in the crude extract were clearly separated and eluted earlier. As the result, rRNase Po1 was highly purified by the CCC procedure. In general, the time-consuming and complicated HPLC method is required to achieve high purification of rRNase Po1 after the separation by Heparin-Sepharose column chromatography. In our laboratory the separation of rRNase Po1 often resulted in a total loss of the enzymatic activities during the HPLC separation, whereas the recovery rate of rRNase Po1 in the present CCC separation was 60%, indicating that RNase activity was well preserved during the CCC separation. The similar results can be achieved with present CCC method simply in one step operation.
Table 1 summarizes the analytical data obtained from the CCC separations examined in the present studies. The difference of elution time between RNase T1 and Po1 may be caused by the difference of the amino acid composition, where RNase T1 was eluted much earlier than RNase Po1. As shown in Table 2, the amount of phenylalanine, the hydrophobic aromatic amino acid in RNase Po1 is much greater that that in RNase T1. The difference of hydrophobicity between these enzymes may affect their elution times.
The overall results indicate that the small-scale X-axis CPC is useful for the separation of various types of RNases with aqueous-aqueous polymer phase systems composed of PEG 1000 and potassium phosphates. Commercial RNase A was highly purified by eluting the upper phase inwards, while RNase T1 and rRNase Po1, by eluting the lower phase outwards. The enzymatic activities were well preserved during the CCC separation. These results demonstrated that the present small-scale X-axis CPC can be efficiently used for the purification of the biologically active proteins from the crude extract.
This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.