WO2004078309A1 - Protein separation method using asynchronous coil planet centrifugal machine - Google Patents

Abstract

The invention relates to a method of separating protein by providing an asynchronous coil planet centrifugal machine not requiring a rotary seal with an aqueous polymer phase system and applying a sample thereto, and provides a substance separating method characterized in that coil planet centrifuge is asynchronously effected using a coil planet centrifugal machine provided with a coil-like column containing a solvent and an object substance to be separated.

Description

 Description Asynchronous Coil

 The present invention relates to an asynchronous coil using an aqueous-aqueous polymer phase system and a method for separating protein by a planetary centrifuge. Background art

 Countercurrent chromatography (CCC) is a chromatography that uses two liquid phases as the separation phase, and has excellent features such as a large sample processing capacity and small denaturation and loss of the sample. . It has the unique feature of not requiring the use of a solid support [1] (Ref. 1, hereinafter the same).

Since the introduction of CCC in 1970, various CCC devices have been developed for the separation and purification of natural and synthetic products [2-6].

 Among such CCC devices, the asynchronous coil-brain centrifuge (CPC) is used for the rotation of the coil holder (rotation about the axis of the holder itself) and the revolution (rotation about the axis of the centrifuge). It is considered to be the most versatile in that a desirable combination can be obtained [7-11]. Previous studies have shown that this device is useful for partitioning cells [7, 9, 11] and plasmid DNA [9] using an aqueous-aqueous polymer phase system, and for separating cells according to size and density. Has been demonstrated [7-10, 12]. In addition, the present inventors have demonstrated in previous studies that successful separation of protein was achieved by using cross-axis CPC [13-20]. Disclosure of the invention

 An object of the present invention is to provide a protein separation method using asynchronous CPC.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and found that aqueous-aqueous poly The inventors have found that proteins can be separated with high accuracy by using a mer phase system, and have completed the present invention.

 That is, the present invention relates to a method for separating a substance, characterized in that a coil / Branette centrifugation is performed asynchronously using a coil / Branet centrifuge equipped with a coiled column containing a solvent and a target substance for separation. is there. Solvents include those that form an aqueous-aqueous phase or an aqueous-oil phase. The substance to be separated includes, for example, protein or cells.

 Further, the aqueous-aqueous phase can be exemplified by an aqueous-aqueous polymer phase, for example, a polyethylene glycol-hydrogen phosphate diammonium phase or a polyethylene glycol-dextran phase. Furthermore, the rotation speed of the non-coiled and planetary centrifuge is, for example, 0 to 60 i'pm, and the orbital speed is, for example, 800 to 1000 rpm.

 The coiled column is preferably formed by a coaxial multilayer coil or an eccentric coil.

 Hereinafter, the present invention will be described in detail.

 The present invention uses a non-synchronous coil planet centrifuge (Rotary seal-free non-synchronous coil planet centrifuge) that is filled with an aqueous-aqueous phase solvent, and the sample is applied to the centrifuge to perform asynchronous centrifugation. The present invention relates to a method for separating proteins by using a method. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a schematic diagram showing a cross-sectional view of an asynchronous CPC manufactured in the laboratory of the present inventor.

 FIG. 2 is a schematic diagram illustrating a revolving mechanism of an asynchronous CPC that does not use a rotary seal.

FIG. 3A shows CCC chromatograms of proteins obtained with three different volumes of coaxial multilayer coils. 0.8mm ID, 1.59mm OD, and llmL capacity. FIG. 3B shows CCC chromatograms of proteins obtained with three different volumes of coaxial multilayer coils. 0.8mm ID, 1.59mm OD, and 24mL capacity. FIG. 3C shows CCC chromatograms of proteins obtained with three different volumes of coaxial multilayer coils. 0.8mm ID, 1.59nm OD, and 39mL capacity. FIG. 4 shows a CCC chromatogram of a protein obtained by an asynchronous CPC with a coaxial multilayer coil.

 FIG. 5 shows a CCC chromatogram of a protein obtained by an asynchronous CPC with a coaxial multilayer coil.

 Figure 6 shows the results of CCC separation of lysozyme and myodarobin by asynchronous CPC using an aqueous-aqueous polymer phase system.

 Figure 7 shows an asynchronous CPC with coaxial multilayer coils revolving in the opposite direction.

Shows the CCC chromatogram of the LO protein. A indicates the lower phase of the mobile phase, and B indicates the upper phase.

 FIG. 8A shows a CCC chromatogram of a protein obtained by an asynchronous CPC with an eccentric coil assembly (mobile phase: lower phase).

 Figure 8B shows an asynchronous CPC with an eccentric coil assembly.

The CCC chromatogram of the protein obtained by L5 is shown (mobile phase: upper phase).

 FIG. 9 shows the results of separating the blood cell components of the sheep blood.

 Figure 10 shows the results of separation of the hedge blood at 800 to 100 rpm, rotation at 0 to 10 rpm, with rotation as clockwise (CW) and revolution as counterclockwise (CCW). FIG. 11 shows the results of separating blood cell components of human blood.

 Ϊ0

 Explanation of reference numerals

 1: Asynchronous CPC device, 2: Rotating frame, 3: Main module,

 4: center axis, 5: intermediate axis, 6: rotating frame, 7: pulley, 8: pulley, 9: pulley, 10: gear, 11: gear, 12: centerpiece,

 5 13: pulley, 14: pulley, 15: pulley, 16: gear, 17: gear,

 18: Secondary motor, 19: Column holder, 20: Pulley, 21: Pulley, 22: Pulley, 23: Flow tube, 24: Tube support frame

201: First system, 202: Second system BEST MODE FOR CARRYING OUT THE INVENTION The asynchronous CPC employed in the present invention was constructed at Nihon University College of Science and Engineering, Institute of Science and Engineering, Machinery Technology Center One (Chiba Prefecture, Japan). Because the design of the device is detailed in the previous report

[9, 10], an outline is described in this specification. The apparatus used in the present invention can freely adjust the rotation speed (range of 0 ± 10 rpm) of the coil-shaped separation column at a predetermined revolution speed, and at the same time, rotates the eluate without using a conventional rotary seal device. It is eluted by force ram.

 FIG. 1 shows a schematic diagram of an asynchronous CPC device 1 used in the present invention.

 In the device 1, the rotating frame 2 is driven by the main motor 3 and rotates around the central axis 4 of the device 1 at an angular velocity coa. The rotating frame 2 is provided with a pair of intermediate shafts 5 for transmitting the movement to the rotating frame 6. Among the intermediate shafts, the intermediate shaft 5 shown at the bottom of the figure rotates the frame 6 via the pulleys 7, 8, 9 and the gears 10 and 11, whereas the intermediate shaft 5 shown at the top of the figure is the center shaft. The pulley 13 on the piece 12 is rotated via the pulleys 14 (fixed) and 15 and the gears 16 and 17.畐 When the ij motor 18 is stationary, these two intermediate shafts 5 rotate synchronously on the rotating frame 2 at -coa, so that the rotating frame 6 and the pulley 1 3 (on the centerpiece 12) ) Both rotate at the same double speed (2 coa). In this case, the column holder 19 only revolves around the centerpiece 12 together with the rotating frame 6, but does not rotate.

 If the sub-motor 18 rotates in cob, this movement is transmitted via the upper intermediate shaft 5, and the rotation speed of the rotating frame 6 changes to 2 coa-c b. Then, the difference in rotational speed between the rotating frame 6 and the pulley 13 on the center piece 12 is transmitted to the axis of the column holder 19 via the pulleys 20, 21 and 22, and the column holder Rotate 1 1 9 with cob. Therefore, the rotation-revolution ratio of the column holder 19 is represented by cob / (2 coa-c b).

The flow tube (ft) 23 containing the sample consists of a set of outgoing and returning tubes, and extends from the end of the device 1 (left end in the figure) along the center axis 4 inside the centerpiece 12. The tube support frame 24 is retracted and secured along the perimeter. Then, the flow tube 23 enters the rotating frame 2, and when it reaches the center axis 4, proceeds in the longitudinal direction of the center piece 12 and connects to the column holder 19. The flow tube 23 in the column holder 19 is wound in a coil shape clockwise or counterclockwise with respect to the traveling direction. When the flow tube 23 is wound up to the end point of the column holder 19, it is folded back to the next holder. Finally, the end of the tube (the return point of the forward and return paths) is fixed to the holder. In other words, in flow tube 23, one tube is folded into two tubes to form a set of tubes for the forward and return paths, and the column is extended by a predetermined length from the tube turning point. It is wound around the holder and fixed. The return route of the tube follows the same return route as the outward route and returns to the exit.

 The flow tube 23 forms a layer by reciprocating in the longitudinal direction (longitudinal direction) of the column holder 19. In this case, the coil formed when the flow tube 23 is wound around the column holder 19 in both the forward direction and the return direction in the longitudinal direction is referred to as a “coaxial multilayer coil” in the present invention. Also, the column holder 19 is wound in one direction (for example, from right to left), but in the opposite direction (for example, from left to right) until the next column holder starts winding. A coil that is formed when it is not wound is called an eccentric coil. In the present invention, either the above-described coaxial multilayer coil or eccentric coil may be employed.

 The material of the flow tube 23 is not particularly limited, but is preferably made of Teflon.

 The column holder 19 itself can rotate (rotate) about its axis, and can rotate (revolve) around the centerpiece 12 at a specific speed.

Table 1 summarizes the relationship between the angular velocity of the motor and the rotating frame. A more detailed description of this mechanism is well known [9, 10]. Table l Angular velocities of motor and rotating frame in asynchronous coiled-brain centrifuge

このような装置を用いて、 カラムホルダ一 1 9及びフレーム 6を非同期で回転 させると、 フローチューブ 2 3内を通過させた試料から、 目的の物質 （タンパク 質など） を分離することができる。 「非同期」 とは、上記フレーム 6の公転速度（角 速度） とカラムホルダー 1 9の自転速度 （角速度） とが同一とならないことを意 味する。

By rotating the column holder 19 and the frame 6 asynchronously using such an apparatus, a target substance (protein or the like) can be separated from the sample passed through the flow tube 23. “Asynchronous” means that the revolution speed (angular speed) of the frame 6 and the rotation speed (angular speed) of the column holder 19 are not the same.

 When asynchronous centrifugation is performed using a column in which the flow tube 23 is a coaxial multilayer coil or an eccentric coil assembly, the target protein can be separated with extremely high precision. The separation accuracy can be further improved by adjusting the direction of revolution and rotation, or changing the concentration of the solvent.

Figure 2 shows the revolving mechanism of a device that does not require a rotary seal. In FIG. 2, the first system ₂₀₁ corresponds to the movement (speed: _wa ) of the rotating frame 2 in FIG. 1, and the second system ₂₀₂ corresponds to the rotating frame 6 (speed: 2 & m-b) and the column holder. It corresponds to 1 9 (speed: cob) movement. A black circle (〇) indicates that the tube is fixed. Target substance for separation

 In the present invention, the substance to be separated is not particularly limited, and can be arbitrarily selected. Examples include biological substances such as proteins and cells, and physiologically active substances derived from natural products. Separated phase

In the present invention, an aqueous-aqueous phase or an aqueous-oil phase can be used as the separation phase (solvent). Such phases include, for example, aqueous-aqueous polymer phase systems, aqueous- An oil-based polymer phase system may be used, but an aqueous-aqueous polymer phase system is preferred. "Aqueous-aqueous polymer phase system" means a solution of two different polymers or a solution of a polymer and an inorganic salt in water to form a two liquid phase. Examples of the polymer include polyethylene glycol (PEG), dextran, and ficoll.

 The molecular weight of PEG is 1,000-8,000, preferably 1,000-: 500 or 6,000-8,000.

 Dextran has a molecular weight of 15,000 to 500,000, preferably 500,000.

 As the separated phase used in the present invention, those having the following combinations are preferred.

 Phosphate buffer consisting of PEG-1000-dibasic hydrogen phosphate

 PEG-1000- Phosphoric acid consisting of bi-hydrogen phosphate and dihydrogen phosphate

PEG-8000-Dextran T500 (including phosphate buffer and sodium chloride) Separation conditions when the above separated phase was used are as follows.

 Revolution: 600 ~: 1000rpm, preferably 800 ~: lOOOi'pm

 Say? s: 0-60 rpm, preferably 0-10 i'pm

 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1> Separation of evening protein

Separation of proteins by countercurrent chromatography was performed using an asynchronous coil 'Brannet centrifuge which does not require a single seal and is manufactured in the laboratory of the present inventor. This device has the unique feature that the rotation speed of the coiled separation column can be freely adjusted at a predetermined revolution speed. Separation uses stable proteins (including cytochrome C, myoglobin and lysozyme) and two different aqueous-aqueous polymer phase systems: PEG (polyethylene glycol) -1000-hydrogen phosphate And PEG-8000-dextran T500 (in sodium chloride and 5 mM phosphate buffer). Using multi-layer coiled columns with different volumes (11, 24, 39 mL) prepared from Teflon tubing material with an inner diameter of 0.8 nm, the effect of column volume on the distribution efficiency was examined under given experimental conditions.

Preparation of two types of coiled columns

 In this example, two types of coiled columns were used. One is a coaxial multilayer coil and the other is an eccentric coil assembly.

 The multilayer coil is prepared by tightly winding a Teflon tube material (Fron Industries, Tokyo, Japan) with an inner diameter of 0.8 mm and an outer diameter of 1.59 mm around a 2.2 cm diameter holder and hub, with a spacing of 23 cm. A tight coil-shaped layer was formed between the pair of installed flanges. Three types of force rams with different volumes, llmL (two layers), 24 mL (four layers) and 39 mL (six layers) were used to study the effect of force ram capacity on distribution efficiency. The eccentric coil assembly was prepared by winding a Teflon tubing material with an inner diameter of 0.8 mm around a plurality of aluminum pipes with a length of 20 cm and an outer diameter of 6 mm to make a series of tight left-handed coils. Eleven coil units were placed symmetrically around a holder hub with an outer diameter of 6 cm so that the axis of each coil unit was parallel to the axis of the holder. The total volume of the column was 20 mL. -

PEG (Polyethylenedalecol) 1000 (Molecular weight: 1,000), PEG 8000 (Molecular weight: 8,000), Cytochrome C (derived from ゥ heart), Myoglobin (derived from ゥ skeletal muscle) and lysozyme (derived from chicken eggs) are available from Sigma Chemical Co. ., St. Louis, MO (USA). Dextran T500 (molecular weight: 500,000) was purchased from Pharmacia, Sollentuna (Sweden). Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium chloride were obtained from Wako Pure Chemical (Osaka, Japan). All other chemicals were reagent grade. Preparation of aqueous-aqueous polymer phase systems and sample solutions

 The following three types of polymer phase systems were prepared [21].

 12.5% (w / w) PEG 1000-12.5% (w / w) dibasic hydrogen phosphate

 4.4% (w / w) PEG 8000-7.0% (w / w) Dextran T500 (5 mM potassium phosphate buffer containing 2 M sodium chloride, pH 7.0)

 4.0% (w / w) PEG 8000-5.0% (w / w) Dextran T500 (5 mM potassium phosphate buffer containing sodium chloride, pH 7.0)

 Each solvent mixture was allowed to completely equilibrate in a separatory funnel at room temperature and the two phases were separated after two clear layers had formed.

 Sample solutions were prepared by dissolving each sample mixture in 1 mL of a solvent containing equal amounts of each phase.

CCC separation method

 Each separation test was started by completely filling the column with the stationary phase and injecting a sample solution (about 1 mL) into the inlet of the column. Next, the mobile phase was injected into the column using a reciprocating pump (Model LC-6A, Shimadzu Corporation, Kyoto, Japan), and at the same time, the column was rotated at a predetermined rotation-revolution speed ratio. The eluate from the column outlet was collected in 0.4 mL per tube using Fraction Collection Yuichi (model SF-20C Avantec, Tokyo, Japan).

Analysis of CCC fraction

 Each of the collected protein fractions was diluted with 2.5 mL of distilled water, and the absorbance was measured at 280 nm using a spectrophotometer (model UV-1600, Shimadzu Corporation, Kyoto, Japan). Examination of experimental conditions

 Experimental condition 1:

-Equipment: Asynchronous CPC with coaxial multilayer coil [(A) 0.8 mm ID, .59 mm outer diameter, and llmL capacity; (B) capacity 24 mL; (C) capacity 39 mL] -Samples: cytochrome C (2 rag), myoglobin (8 mg) and lysozyme (lOmg)

 -Solvent system: 12.5% (w / w) PEG 1000-12.5% (w / w) hydrogen oxalate double rim -Mobile phase: Lower phase

 -Flow rate: 0.2mL / min

 SF = solvent front Experimental condition 2:

 -Equipment: Asynchronous CPC with coaxial multilayer coil (ID 0.8mm, OD 1.59mm, and capacity 39mL)

 -Samples: myoglobin (8 mg) and lysozyme (lOmg)

 -Solvent system: 12.5% (w / w) PEG 1000-12.5% (w / w) Hydrogen hydrogenate double rim

-Mobile phase: Upper phase

 -Flow rate: 0.2mL / min

 SF = Solvent tip Experimental condition 3:

 -Equipment: Asynchronous CPC with coaxial multilayer coil (ID 0.8ram, OD 1.59mm, and capacity 39niL)

 Other conditions were the same as those described in Experimental condition 1. SF = Solvent tip Experimental condition 4:

 -Equipment: Asynchronous CPC with coaxial multilayer coil (inner diameter 0.8mm outer diameter 1.59mm, capacity 39mL)

 -Sample: myoglobin (8 mg) and lysozyme aOmg)

 -Solvent system: 4.4% (w / w) PEG 8000-7.0% (w / w) Dextran T500 (in 5 mM potassium phosphate buffer containing 2 M sodium chloride, pH 7.0)

-Mobile phase: Upper phase -Flow rate: 0.2mL / min

 -Revolution: 800rpm

 -Suzuki s ;: lOrpm

 SF = Solvent tip Experimental condition 5:

 -Equipment: Asynchronous CPC with coaxial multilayer coil (0.8 nm ID, 1.59 mm OD, and 39 mL capacity)

 -Samples: cytochrome C (2mg), myoglobin (8mg) and lysozyme (lOmg)

 -Solvent system: 12.5 (w / w) PEG 1000-12.5% (w / w) hydrogen oxalate double-phase-Mobile phase: (A) lower phase, (B) upper phase

 -Flow rate: 0.2mL / min

 -Revolution: 800rpm (reverse direction)

 -Own vehicle · · lOrpm

 SF = Solvent tip Experimental condition 6:

 -Equipment: Ecsen1, Asynchronous CPC with rick coil assembly (ID 0.8mm, OD 1.59mm, left-handed coil, 11 units, and capacity 20niL)

 -Samples: cytochrome C (2mg), myoglobin (8mg) and lysozyme (lOm)

 -Solvent system: 12.5% (w / w) PEG 1000- 12.5% (w / w) dibasic hydrogen hydrogenate -Mobile phase: (A) lower phase, (B) upper phase

 · Flow rate: 0.2mL / min

 -Revolution: 800rpm

 -Rotation: lOrpm

SF = solvent tip Results and Discussion

 The results of experimental conditions 1 to 6 are shown in Figs.

 FIG. 3 shows CCC chromatograms of proteins obtained with three different volumes of coaxial multilayer coils.

 Figure 3A shows the results when the column volume is 11 mL, Figure 3B shows the results when the column volume is 24 mL, and Figure 3C shows the results when the column volume is 39 mL. In FIGS. 3A to 3C, each panel is as follows.

 Left panel: Revolving speed 800i'pm, Revolving speed 10rpm, Elution mode: Head to tail Middle panel: Revolving speed 800i'pm, Revolving speed 0rpm, Elution mode:

 Right panel: revolution speed 800 rpm, rotation speed 10 rpm, elution mode: Tail to head Figure 4 shows the CCC chromatogram of the protein obtained by asynchronous CPC equipped with a coaxial multilayer coil.

 Left panel: Revolving speed 800rpm, Revolving speed 10rpm, Elution mode: Tail to head Middle panel: Revolving speed 800i'pm, Revolving speed 0rpm, Elution mode:

 Right panel: revolution speed 800 rpni, rotation speed 10 rpm, elution mode: head to tail Figure 5 shows the CCC chromatogram of the protein obtained by asynchronous CPC equipped with a coaxial multilayer coil.

 A: Revolution speed 600ι'ριη, rotation speed 10 rpm, flow rate: 0.2 mL / min, elution mode: Head

B: Revolution speed 1000i'pm, rotation speed 0rpm, Flow rate: 0.2mL / min, Elution mode: Head to ta

C: Revolution speed: 1000 rpm, rotation speed: 10 rpm, Flow rate: 0.4 mL / min, Elution mode: Head to tail Figure 6 shows lysozyme and lysozyme by asynchronous CPC using an aqueous-aqueous polymer one-phase system. The results of CCC separation of odarobin are shown. Figure 7 shows a CCC chromatogram of a protein obtained by revolving the asynchronous CPC type equipped with a coaxial multilayer coil in the direction opposite to the rotation direction.

 Left panel (lower phase): Revolution speed 800rpni (reverse rotation), rotation speed 10rpm, Elution mode: Head to tail

 Right panel (upper phase): Revolution speed 800 rpm (reverse rotation), rotation speed 10 i'pm, Elution mode: Head to tail Figure 8A shows the protein obtained by asynchronous CPC equipped with an eccentric coil assembly. The CCC chromatogram (mobile phase: lower phase) is shown.

 Left panel: Revolving speed 800rpm, Revolving speed 10rpm, Elution mode: Head to tail Right panel: Revolving speed 800i'pm (reverse rotation), Revolving speed 10i'pm, Elution mode: Head to tail

 FIG. 8B shows the CCC mouth mattogram (mobile phase: upper phase) of the protein obtained by the asynchronous CPC with the eccentric coil assembly.

 Left panel: Revolution speed 800 rpm, rotation speed lOi'pmu Elution mode: Head to tail Right panel: Revolution speed 800 rpm (reverse rotation), rotation speed 10 i'pm, Elution mode: Head to

Of these experiments, the best protein separation was achieved by combining a 39 mL column with a 12.5% (w / w) PEG-1000-12.5% (w / w) dipotassium hydrogen phosphate system. , At a rotational speed of the coil of 10 rpm at 800 rpm. When the lower phase was moved at 0.2 mL / min during elution from the beginning to the end (Head to tail), the resolution between cytochrome C and myoglobin was 1.6, and the resolution between myoglobin and lysozyme was 1.9. Was. The resolution between the lysozyme and myoglopine peaks was 1.5 when the upper phase was shifted during the elution from the beginning to the end. For these two separations, the retention of the stationary phase was 35.0% and 33.3%, respectively. (Fig. 3C, Fig. 4).

 Furthermore, protein separation was performed using a pair of extrinsic coil assemblies (total volume: 20 mL) using a teflon tube material with an inner diameter of 0.8ιιιηι. When both the lower and upper phases were used as mobile phases for elution from the beginning to the end (Head to tail), equivalent resolution was obtained.

 The results of the present invention show that asynchronous CPC is useful for protein separation using an aqueous-aqueous polymer one-phase system.

 Asynchronous CPC offers a unique separation method for cells and macromolecules by allowing the rotation of the coil to be freely adjusted at a given centrifugal field. In the present invention, a series of experiments were performed to evaluate the instrument's ability to separate proteins using an aqueous-aqueous polymer phase system.

 The acceleration caused by the asynchronous net-like motion fluctuates in a plane perpendicular to the axis of the holder. The system of the present invention, which uses low-speed coil rotation (0 to 10 Oi'pm) and high-speed revolution, works very well with the basic hydrodynamic equilibrium system (low-speed rotating coil at unit gravity) [22, 23]. Similar, but different in that unit gravity is replaced by a strong centrifugal force field. Obviously, in the system of the present invention, the two phases are distributed in the rotating coil in approximately equal amounts from the starting end, while any excess of either phase accumulates at the end. In this case, the directions of the coil start and end are determined by the Archimedean screw action, under which all objects of various densities in the rotating coil move toward the coil start. Therefore, a sufficient retention (approximately 50%) of the stationary phase is obtained only when one of the phases is fed at a low flow rate from the beginning of the coil, and the introduction of the mobile phase from the end terminates the stationary phase. This leads to continuous feed, which unnecessarily reduces peak resolution.

 Figure 3 shows three different capacities of coaxial multilayer coils (inner diameter) using a polymer phase system composed of 12.5% (w / w) PEG 1000-12.5% (w / w) hydrogen

0.8 mm). When the lower phase was used as the mobile phase, the best separation was obtained when the 39 mL coil was rotated at 10 rpm to elute from the beginning to the end, with cytochrome C and The resolution between the peaks for myoglobin and myoglobin was 1.6, the resolution between the peaks for myoglobin and lysozyme was 1.9, and the retention of the stationary phase was 35.0%. Unexpectedly, if the rotation was reversed (Tail to head), the stationary phase was significantly lost (3.6% retention) and the peak resolution was reduced. It was not enough.

 When the upper phase was moved (Fig. 4), the best separation was obtained when elution was performed from the beginning to the end at a rotation speed of 10 rpm. Elution from tail to head (Tail to head) reduced retention (2.6%).

 In order to improve the distribution efficiency when the lower phase was moved, the effect of the revolving speed on the peak resolution was investigated using a multilayer coil with a capacity of 39 mL. As shown in Figures 5A and 5B, the peak resolution was effectively improved with increasing orbital speed. When the flow rate was doubled to 0.4 mL / min and the revolving speed was set to 100,000 rpm, the protein peak was completely separated with a shorter separation time (within 2.7 hours) as shown in Figure 5C.

 Another aqueous biphasic polymer phase system, 4.4% (w / w) PEG 8000-7.0% (w / w) Dextran T500 (in 5 ηιΜ potassium phosphate buffer containing 2 M sodium chloride) was used. In some cases, the separation of lysozyme and myoglopine was achieved by moving the upper phase in the start-to-end elution mode. Figure 6 shows the CCC chromatogram obtained using the above polymer phase system. The resolution between the lysozyme and myoglopine peaks was 1.5, while the retention of the stationary phase was reduced to 19.7%. 4.0% (w / w) PEG 8000-5.0% (w / w) Dextran T500 (containing 3% sodium chloride

5 ηιΜ potassium phosphate buffer), the stationary phase was not retained, regardless of elution mode.

Further experiments were performed on the partition efficiency of protein separation using an eccentric coil assembly (0.8 mm id) with a total volume of 20 mL. As shown in Fig. 8, good separation was obtained when the lower phase was eluted in the elution mode from the start to the end. Table 2 summarizes the analysis data calculated from the chromatogram. Table 2 Analytical values obtained from protein separation by asynchronous coil planet centrifugation using two different coiled columns Column Resolution

 u u u U-stage (N) Cyt C / Myo Myo / Lys Stage / column

 p p p P (Lys / Myo) Capacity (N / mL) Coaxial multilayer coil (39mL capacity)

 LP 214 .6 1.9 5.5 Fig. 3

 LP 281.7 2.0 7.2 Figure 7A (reverse revolution)

378 (1.5) 9.7 Figure 4

 380 (1.4) 9.7 Fig. 7B Eccentric coil (20mL volume j

 (35 LP 83 0.6 4.2 Fig.

 LP 97 0.7 4.9 "(reverse orbit)

269 (0.9) 13.5 Figure 8B

 197 (0.9) 9.9 "(reverse orbit) Abbreviations: LP 2 lower phase; UP = upper phase; Cyt C 2 cytochrome C; Myo 2 myoglobin; Lys = lysozyme

The value of the theoretical plate number was calculated from the myoglobin peak when the lower phase was moved and the lysozyme peak when the upper phase was moved in the chromatogram.

Conclusion

 A series of experiments in the present invention have shown that asynchronous CPC can be used effectively for partitioning proteins using aqueous-aqueous polymer phase systems. The best results are obtained when the coil is rotated at lOrpm and elution is performed from start to end at a revolving speed of 800-1000rpra. Equipped with a long multi-layer coil would further increase the distribution efficiency.

<Example 2>

Blood cell separation

 (1) Preparation of column

 Wrap a Teflon tube (inner diameter 0.8mm, outer diameter 1.59mm) around a 6mm outer diameter metal pipe left-handed to make one unit, attach 11 units to a cylindrical holder (diameter 6cm, length 23cni), and attach each unit to Teflon The tubes were connected in series (20 niL).

 (2) Equipment operating conditions

 Revolution speed 600 ~: 1000rpm, Rotation speed 0 ~: LOrpm

 Separation solvent: isotonic phosphate buffer (pH 7.4)

 Flow rate: 0.4mL / min

 Specimen: 0.5 mL of a solution obtained by mixing the blood of an egg with the same volume of Alsever's solution

 Fractionation: 0.8mL / tube

 (3) Detection

 2.5 mL of water was added to the separated fraction, and after hemolysis, the absorbance was measured at UV230, 260, 280, and 570 nm.

 (4) Result

After examining the direction of rotation of the column using revolving 800 i'pm and lOrpm using hedged blood, it was possible to separate red blood cells and other blood cell components by revolving clockwise (CW) and revolving counterclockwise (CCW). (Figure 9). Considering the separation at 800 revolutions: L000 rpm and the rotation of 0 revolutions: 10 rpm using this direction of rotation, In this case, blood cells other than red blood cells eluted well when spinning Orpin, and red blood cells eluted well when spinning at lOrpm, and both were completely separated (Fig. 10). In addition, as a result of an examination of human blood in the same manner as sheep blood, at a revolution of 700 rpm, blood cell components other than red blood cells were eluted at the rotation of Oi'pm and red blood cells were eluted well at the rotation of lOi'pm. Were completely separated (Fig. 11).

 From the above, it was found that the method of the present invention is also effective for cell separation based on a difference in specific gravity.

Industrial applicability

 According to the present invention, there is provided a method for separating proteins using an asynchronous coil'Branette centrifuge using an aqueous-aqueous polymer phase system. According to the method of the present invention, a protein that is easily denatured can be separated stably and with high precision, and is extremely useful for production of industrial enzymes and pharmaceuticals. References

 1. Y. Ito and R. L. Bowman, Science, 167 (1970) 281.

 2. N. B. Mandava and Y. Ito, editors, Countercurrent Chromatography: Theory and Practice, Marcel Dekker, New York, 1988.

 3. W. D. Conway, Countercurrent Chromatography, Apparatus, Theory and Applications, VCH Publishers, Weinheim, 1990.

 4. Y. Ito and W. D. Conway, editors, High-Speed Countercurrent Chromatography,

 Wiley Interscience, Amsterdam, 1996.

 5. J. M. Menet and D. Thiebaut, editors, Countercurrent Chromatography, Marcel Dekker, New York, 1999.

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Claims

The scope of the claims 1. A method for separating a substance, wherein a coil-Branette centrifugation is asynchronously performed using a coil-Branette centrifuge equipped with a coil-shaped column containing a solvent and a target substance for separation.  2. The method according to claim 1, wherein the substance is a protein or a cell.  3. The method according to claim 1, wherein the solvent forms an aqueous-aqueous phase or an aqueous-oily phase.  4. The method of claim 3, wherein the aqueous-aqueous phase is an aqueous-aqueous polymer phase.  5. The method according to claim 4, wherein the aqueous-aqueous polymer phase is a polyethylene glycol-dipotassium hydrogen phosphate phase or a polyethylene glycol-dextran phase. 6. The method according to claim 1, wherein the rotation speed of the coil and the planet centrifuge is 0 to 60 rpm, and the revolution speed is 800 to 100 000 rpm.  7. The method according to claim 1, wherein the coiled column is formed by a coaxial multilayer coil or an eccentric coil.