JP2004073993A - Solution concentrating apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a solution concentration apparatus which can enhance the operational efficiency, enables short-time processing, even when a plurality of kinds of protein solutions are concentrated at the same time, can appropriately grasp circumstances such as a concentrated state by means of a sensor and can perform a concentration operation. <P>SOLUTION: The solution concentrating apparatus which concentrates a solution stored in a solution storing means by a centrifugal operation is provided with a liquid surface detecting means which detects the liquid surface level of the solution stored in the solution storing means, a solution supply amount control means which controls the amount of the solution to be supplied to the solution storing means and a solution supplying means which supplies the solution of the amount determined by the solution supply amount controlling means to the solution storing means. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
[Industrial applications]
The present invention relates to a solution concentration device using a centrifugal separation operation. In particular, the present invention relates to a protein concentrator that can continuously work by measuring the liquid level in a tube to be centrifuged and automatically replenishing an appropriate amount of a solution, and a method for analyzing the three-dimensional structure of a protein using the same. And so on.
[0002]
[Prior art]
In recent years, the decoding of DNA nucleotide sequences of many species including humans has been rapidly progressing. For a huge number of genes extracted from a large amount of genome sequence information, it has become an important issue to clarify the three-dimensional structure of the protein encoded by each gene and elucidate the relationship with the function. .
[0003]
In this "structural genomics", the number of proteins targeted for structural analysis is as large as about 100,000 human genes. The three-dimensional structure of a protein is formed from a combination of 1,000 to several thousand types of basic structural units, and it is considered that the diversity of functions is realized by the combination. Therefore, studies are being conducted with the aim of elucidating all of these basic structural units to clarify the relationship between protein structure and function.
In such a systematic and comprehensive structural analysis, it is urgently necessary to speed up and automate the entire analysis process.
[0004]
On the other hand, proteins are inactivated or lost due to denaturation, decomposition, adsorption and the like. Therefore, when preparing a target protein sample, it is particularly desirable to avoid these losses and perform a series of operations efficiently in a short time as possible.
In particular, when a nuclear magnetic resonance (NMR) spectrum is used in protein structural analysis, a target protein solution is purified and then concentrated to obtain a measurement sample, and improvement in workability in this concentration step is required.
[0005]
As a means for concentration when preparing a sample protein for NMR measurement, a protein solution is put into a centrifuge tube equipped with a filter having a predetermined molecular weight cut-off, and a motor is rotated to perform centrifugation. An external filtration method is common.
[0006]
Conventionally, in this centrifugal ultrafiltration method, a concentrated solution of protein has been obtained as follows. That is, first, the tube into which the protein solution is injected together with the buffer is inserted into the centrifuge. Centrifuge for a certain time. Remove the tube and visually check the condition inside the tube (whether the filter has come off or the filter is not clogged, etc.). Then, the protein solution reduced by the concentration is manually replenished. Return tube to centrifuge. Centrifuge. The above operation is repeated until the amount of the concentrate reaches the concentration and amount required for measurement.
[0007]
In this conventional method, it is necessary to stop the tube until the amount of the concentrate reaches the required amount for measurement, and to perform visual confirmation many times, and it takes a long time to obtain the target concentrate. there were. For example, a normal protein solution concentration operation requires a time of about 8 hours, and has a problem that a great deal of labor is required.
In particular, when multiple types of protein solutions are concentrated by the same centrifuge, the difference in the filtration speed of each solution causes a different level of protein level in each tube, which requires further visual confirmation. There was a problem.
[0008]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel solution concentrating device that improves the working efficiency in such a step of concentrating proteins and the like and enables short-time processing.
Another object of the present invention is to obtain a concentrated solution such as a protein in a good state.
Another object of the present invention is to provide a solution concentrating device that can perform a concentration operation by appropriately grasping the state by a sensor or the like, even when a plurality of types of protein solutions are simultaneously concentrated. Aim.
The present invention provides a rapid and accurate concentration of a protein solution using the solution concentrating device of the present invention, and performs an NMR analysis, an X-ray analysis after crystallization, and the like using the obtained protein concentrate to obtain a protein protein. Another purpose is to perform three-dimensional structure and function analysis.
Another object of the present invention is to carry out drug discovery based on the above analysis.
[0009]
[Means for Solving the Problems]
The present invention solves at least one of the above-mentioned problems. The present invention provides a novel solution concentrating device that is effective in achieving high-throughput structural analysis by automating steps conventionally performed manually. Note that the solution concentrating device is a device that normally rotates a rotor or the like loaded with a tube containing a sample at high speed, and concentrates and separates the sample by centrifugal force generated by the rotation.
[0010]
More specifically, the present invention relates to a solution concentration apparatus for concentrating a solution contained in a solution containing means by a centrifugal operation, and a liquid level detecting means for detecting a liquid level of the solution contained in the solution containing means. Solution supply amount control means for controlling the amount of the solution to be supplied to the solution storage means based on the liquid level information detected by the liquid level detection means, and the amount obtained by the solution supply amount control means And a solution supply means for supplying the solution to the solution storage means. This solution concentrating device detects the liquid level contained in the solution containing means by the liquid level detecting means while centrifuging the solution containing means, and detects the solution level based on the liquid level information detected by the liquid level detecting means. The supply amount control means controls the amount of the solution to be supplied to the solution storage means, and the solution supply means supplies the amount of the solution determined by the solution supply amount control means to the solution storage means, thereby continuously and efficiently supplying the solution. Can be concentrated.
[0011]
With this novel solution concentrator, the protein solution reduced by concentration can be automatically replenished without stopping the rotation of the solution concentrator (centrifuge). Therefore, according to such an apparatus, the workability of the solution concentration is dramatically improved, and the time required for sample preparation can be significantly reduced. Hereinafter, the present invention will be described specifically based on its embodiments.
[0012]
DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 1 is a partial sectional view schematically showing an example of the solution concentrating device of the present invention. As shown in FIG. 1 (a), the solution concentrating device of the present invention comprises a plurality of solution containing means (1), a rotor (2) for holding the solution containing means, and a drive motor (3) for rotating the rotor. A liquid level detecting means for detecting a liquid level of the solution stored in the solution storing means, and a liquid level to be supplied to the solution storing means based on the liquid level information detected by the liquid level detecting means. The apparatus includes a solution supply amount control means (7) for controlling the amount of the solution, and a solution supply means (9) for supplying the amount of the solution determined by the solution supply amount control means to the solution storage means. FIG. 1B is a schematic view of an example of the solution concentrating device of the present invention as viewed from above.
[0013]
(Solution storage means)
The solution storage means is not particularly limited as long as it can store a solution. As the solution accommodating means, a tube or the like can be mentioned, and preferably, a tube of a material (for example, made of polycarbonate or the like) to which a solute is not easily adsorbed on the surface inside the tube can be mentioned. The number of the solution storage means such as a tube is not particularly limited, but may be 1 to 100. The optimal number is determined from the number and amount of the types of the solution to be concentrated, the working efficiency, and the like. Since it is necessary to maintain a rotational balance, it is preferable that the number of tubes is large, and examples thereof include multiples of 4 and multiples of 8, such as 32, 24, 12, 8, and 4. Preferably, the plurality of solution storage units are symmetrically arranged with respect to the drive shaft (10) of the drive motor.
[0014]
An example of the preferred solution storage means (1) of the present invention will be described with reference to FIG. The solution accommodating means includes a filtrate accommodating section (11) for collecting a filtrate when the solution is concentrated by the solution concentrating device of the present invention, a body (13), and a cap (17) connectable to the body. ). The body includes a filter membrane (15) having a predetermined molecular weight cutoff, and is configured such that the filtrate container and the cap can be hermetically connected. In order to collect the concentrated liquid from the solution storage means having such a configuration, the concentrated liquid accumulated at the bottom of the body may be collected with a pipette or the like. As shown in FIG. 2 (b), the concentrated liquid is concentrated by inverting and centrifuging the concentrated liquid collecting section (19) between the filtrate storage section (11) and the inverted body (13). It can be collected in the liquid collecting section. In order to avoid contamination with impurities, the filtrate and the concentrated solution are preferably sealed with a cap (21) as shown in FIG. 2 (c).
[0015]
As the filtration membrane (15), a known filtration membrane used for concentration, centrifugal filtration, or the like is appropriately selected according to the target solution, and is installed according to a known method. As the filtration membrane, a filtration membrane suitable for a target solution (for example, a protein solution) can be selected, and specific examples include a polyethersulfone membrane, a triacetyl cellulose membrane, and a regenerated cellulose membrane. The polyethersulfone membrane does not exhibit either hydrophilic or hydrophobic interactions. The polyethersulfone membrane has characteristics such as low fouling, a high flow rate, and a wide range of pH. In addition, the triacetyl cellulose membrane is characterized by having high hydrophilicity, high chemical resistance, and low nonspecific adsorption. The regenerated cellulose membrane has a feature that it is highly hydrophilic, can be sterilized by autoclave, and can be easily washed.
[0016]
(solution)
Examples of the solution that can be treated by the solution concentrating device of the present invention include a solution or suspension containing proteins, enzymes, hormones, nucleic acids, antibodies, immunoglobulins, biomolecules, cells, antibiotics, and the like. A mixed solution of a purified solution and a buffer may be used. The solution may be a mixture of a plurality of proteins. In the case of simultaneously concentrating a plurality of protein solutions, since filtration speeds of the respective solutions are different, rotation had to be stopped to grasp the amount of concentration by conventional visual inspection. Accurate and quick judgment can be made by directly detecting with the sensor.
[0017]
(Gas pressurization means)
It is preferable that the solution concentrating device of the present invention further has a gas pressurizing unit in which the solution containing unit is pressurized and centrifuged by gas. The gas pressure centrifugation means a method of centrifuging while pressurizing with gas. For example, gas-pressure centrifugation can be realized by using a combination of Vivaspin 20 and Vivasell 70 manufactured by Viva Science. According to the gas pressure centrifugation, the filtration (concentration) speed is increased by about 30% to 50% as compared with the operation of only centrifugation.
[0018]
(Drive means)
A driving motor may be used as a driving unit for driving the solution containing unit. As the drive motor, a known drive motor such as a swing rotor or an angle rotor can be used. Examples of the swing rotor include those described in JP-A-9-155235.
[0019]
(Liquid level detection means)
Examples of the liquid level detecting means include a CCD sensor and the like. However, the liquid level detecting means is not particularly limited as long as it can detect image information of the liquid level in the tube and a change in state such as light transmission, light absorption, and light scattering. Absent. As the CCD sensor, a color CCD sensor is preferable, and a two-dimensional color CCD sensor having a larger amount of information is more preferable. According to such a liquid level detecting means, the liquid level can be accurately and quickly observed even while the solution containing means is rotating.
[0020]
As the liquid level detection means, a light source for a CCD sensor or the like may be further provided. It is preferable that the light emitted from the light source is irradiated to the solution storage means, and the liquid level is detected as image information or the like by a change in the reflectance. Therefore, preferably, the position and focus of the color CCD camera lens are adjusted so that the level of the liquid level in the tube can be observed from a full level to a small level below the filter membrane in order to obtain at least appropriate image information. The light source is not particularly limited as long as it is a light source suitable for the liquid level detecting means. The light source may be, for example, a light source integrated with the CCD camera, a light source provided near the CCD camera, or a light source opposed to the CCD camera with a tube interposed therebetween. When the tube is installed obliquely, the light source may be installed at an angle such that light is directly applied to the liquid surface. Note that there is preferably no obstacle between the light source and the solution accommodating means to block the light, and the light introduction path and the light exit path are clearly defined.
The liquid level detecting means preferably detects the liquid level of the solution at a timing synchronized with the rotation cycle of the solution containing means. In this case, the synchronization with the rotation cycle means that the rotation cycle of the solution storage means is completely the same as the detection (imaging) timing cycle, and the rotation cycle of the solution storage means is the detection (imaging) timing (period). ) It also means the case where it is an integral multiple. Such an integer includes, for example, the number of solution storage means included in the apparatus. Such a liquid level detecting means is preferably fixed at a specific position.
[0021]
(Solution supply amount control means)
The solution supply amount control means (for example, PC) obtains information on the liquid level (the height of the liquid level in the tube) from the liquid level detection means and the liquid level storage means for storing information on the solution supply amount in advance. A solution amount calculating means in the solution storing means for calculating an amount of the solution stored in the solution storing means (for example, a tube); a solution amount in the solution storing means calculated by the solution amount calculating means; Comparison means for comparing the amount of solution stored in the storage means, and solution supply amount calculation for calculating the amount of solution supplied to the solution storage means by the solution supply means according to the amount of solution compared by the comparison means Means and a solution supply amount information transmitting means for transmitting to the solution supply means the amount of the solution supplied to the solution storage means by the solution supply means calculated by the solution supply amount calculating means.
The solution supply amount control means can be constructed using a known personal computer or server.
[0022]
When a plurality of types of solutions are centrifuged, if the centrifugation is performed for a certain period of time, the respective solution accommodating means will have different filtration rates due to different filtration speeds, and all the solution accommodating means will have a predetermined solution amount. It will not be the same timing. The reasons for the difference in the filtration speed include insolubilization due to association of proteins and the properties of the protein solution itself.
Therefore, in the present invention, the protein is preferably concentrated as follows. First, after a certain period of concentration, the solution is separated into a non-concentrable solution and a condensable solution. The non-concentrable solution corresponds to a solution in which a fixed amount of the solution has not been reduced for a certain period of time. Next, the solution having the slowest concentration rate is selected from the solutions that can be concentrated, and the time required for the solution to be concentrated to a predetermined solution amount is calculated. From the required time, the solution injection amount is calculated so that all the solutions have the same amount of solution at the same time. The amount of the solution obtained here is injected into each solution storage means. By doing so, it is possible to obtain a predetermined solution amount for all the solutions at the same timing.
As described above, it is preferable to simultaneously control the amount of the solution stored in the plurality of solution storage units, and to synchronize the control signal to the solution supply unit so that each solution storage unit can be replenished with an appropriate amount of the solution. Is preferred.
[0023]
With reference to FIG. 3, an example of the liquid level adjusting mechanism of the solution containing means in the present invention will be described. In FIG. 3, A is the liquid level when the protein solution in the tube (solution storage means) is full. X is the liquid level of the liquid surface of the solution during concentration. F is the liquid level of the desired concentrated protein concentrate. A liquid level detecting means (32) such as a CCD camera detects liquid level information on the height of the liquid surface irradiated by the light source (31). Information on the detected liquid level is transmitted to the solution supply amount calculation means (33) of the control means, and the solution supply amount calculation section (33a) of the control means calculates the liquid level X and the desired liquid level. Compare with F. If X and F match, a control signal for terminating the concentration operation is transmitted to the solution supply means (34), a motor control mechanism, and the like. If X and F do not match, information about the appropriate amount of solution to be supplied (for example, AX or AF amount) is transmitted to the solution supply means (34). The solution supply means (34) includes a protein solution reservoir (34a) and a liquid replenishment / injection automatic dispenser (34b), and replenishes an appropriate amount of solution to the solution storage means based on information from the solution supply means. . The concentration operation is repeatedly performed until X and F match.
[0024]
(Solution supply means)
The solution supply means accommodates the solution, and supplies the amount of the solution determined by the solution supply amount control means to the solution accommodation means. The solution supply means is rotatable, for example, in synchronization with the rotation cycle of the solution storage means, and is connected so that the solution can be supplied to the solution storage means (tube). It is preferable that the solution storage means, the solution supply path, and the solution supply means rotate integrally. A known solution supply path can be used as the solution supply path connecting the solution storage means and the solution supply means. One that can supply the solution to the solution supply means even when the solution storage means is rotationally driven is exemplified. For example, a solution supply passage and a solution supply unit that rotates together with the solution storage unit may be used. It is preferable that the solution supply means and the solution storage means be connected and rotate together. Another preferred example is one in which the solution supply means also rotates in synchronization with the rotation cycle of the solution storage means. With such a configuration, the relative speed of the solution containing unit and the solution supplying unit with respect to the specific position can be kept constant, and the solution is supplied to the solution supplying unit even when the solution containing unit is rotationally driven. You can do it.
Alternatively, the solution supply means is not connected to each of the solution storage means, and is synchronized with the rotation of each of the solution storage means to release the solution so that the specific solution storage means stores the solution. To supply the solution.
In this case, in order to prevent the solution to be supplied from being shaken off by the rotation of the solution storage means, a wall may be provided, or a tube may be extended from the solution supply means to the edge of the mouth of the solution storage means.
Preferably, the solution supply means is electrically connected to the solution supply amount control means (PC). It is preferable that the solution contained in the solution supply means is not affected by vibration or the like. Therefore, a reservoir or the like that constitutes the solution supply means and contains a component (eg, protein) of the solution to be supplied to the solution storage means is installed, for example, on the same axis as the central axis of the plurality of solution storage means or on the concentric axis of the rotor. It is preferred that In addition, components of a solution (such as a buffer solution) that is less likely to be affected by vibration or the like may be provided around the rotor.
[0025]
(Contrast member)
In the concentration device of the present invention, it is preferable that a contrast section is provided on the side of the solution containing means (tube) opposite to the liquid level detecting means (CCD). That is, it is preferable that the arrangement is such that the solution storage means is located between the contrast member and the liquid level detection means. The contrast member preferably has a surface having a complementary color relationship with the color of the object such as a protein. For example, the contrast member has a black surface, and the liquid level detecting means (CCD) is contained in a solution containing means (tube). For detecting the liquid level more clearly. In addition, the thing which colored the hole part of the rotor which accommodates a tube in black etc. may be used as a contrast member, and it is good also as a contrast member provided with a lining.
[0026]
(Abnormal vibration detection device)
It is preferable that the solution concentrating device of the present invention can prevent abnormal vibration by the abnormal vibration detecting device. As the abnormal vibration detecting device, for example, the device described in Japanese Patent Application Laid-Open No. 5-104030 can be used. That is, as a means for detecting abnormal vibration such as when the rotational balance is not maintained, the rotor is determined by a mechanism for determining the rotating body of the centrifuge, and after passing through the critical speed, the drive shaft unit or the motor unit or the rotor is determined. Centrifugal separation with the function of measuring the vibration of the rotor, comparing the permissible vibration value of each rotor that has been input in advance with the measured vibration value, and stopping the rotor if the permissible value of the rotor is exceeded. It is preferable to use an abnormal vibration detection device of the machine. According to this abnormal vibration detection device, when the rotor starts to rotate, the rotor is determined by the adapter attached to the bottom of the rotor, and the rotation sensor is used to determine that the vehicle has passed the critical speed, and then the deflection is measured by the distance meter Then, it is compared with an allowable run-out amount for the rotor that has been measured and input in advance. As a result of the comparison, if the value exceeds the allowable value of the rotor previously input, the rotor is decelerated. In this way, abnormal vibration can be prevented.
[0027]
(Solution concentration method)
Hereinafter, an example of a solution concentration method using the solution concentration device of the present invention will be described with reference to FIG. Note that “S” in FIG. 4 means a step.
[0028]
First, a tube (solution storage means) is loaded into the solution concentrating device (centrifuge) of the present invention (S101). When using a plurality of tubes, it is preferable to install them while keeping a balance on a diagonal line. More specifically, when four tubes are used as shown in FIG. 5, it is preferable that the tubes are arranged in the order of diagonal lines 1, 2, 3, and 4 in FIG. Even when the number of target solutions is smaller than the number of tubes provided, it is preferable to put a dummy solution in the tubes to achieve balance. For example, in the case of a device for centrifuging four tubes at the same time, even if only three tubes are required, the remaining one is filled with a liquid having the same weight as the solution and four tubes are filled. It is preferable to centrifuge at the same time. By doing so, the rotational balance can be maintained, and the occurrence of abnormal vibration can be prevented.
[0029]
Next, the solution (purified protein and buffer) is filled into the tube (S103), and the desired amount is automatically set by the liquid level detection means, the solution supply amount control means, and the solution supply means. Preferably it is supplied.
[0030]
Enter the rotation speed of the concentrator. Then, the solution supply amount control means of the present invention waits for input information on the rotation speed (S105). Preferably, the input information is stored by the storage unit of the solution supply amount control unit. It is also preferable that input means for the rotational speed is stored in advance.
[0031]
The drive motor receives the information on the rotation speed stored in the solution supply amount control means, and rotates the rotor holding the tube (S107). The rotation speed includes, for example, 1000 rpm to 10000 rpm, and more specifically, 3000 rpm, but is not limited thereto. The rotation time is not particularly limited, and may be, for example, 10 to 30 minutes.
[0032]
The liquid level detection means detects the liquid level in the tube when the rotation of the rotor is stopped once, the rotation of the rotor is slowed, or the rotor is rotating (S109). At this time, it is preferable that the liquid level detecting means can detect even if there is an abnormality such as clogging of the filter or displacement of the filter. If there is any abnormality, replace the tube or adjust the filter etc., and rotate again (return to S101).
[0033]
The rotor is rotated again (S111). The rotation speed at this time includes, for example, 1000 rpm to 10000 rpm, and more specifically, 5000 rpm for obtaining a predetermined centrifugal acceleration, but is not limited thereto. The rotation time is not particularly limited, and may be, for example, 10 minutes to 30 minutes.
[0034]
When the rotation of the rotor is stopped once, the rotation of the rotor is slowed, or the rotor is rotating, the liquid level detection means detects the liquid level in the tube (S113). At this time, it is preferable that the liquid level detecting means can detect even if there is an abnormality such as clogging of the filter or displacement of the filter. If there is any abnormality, replace the tube or adjust the filter, etc., and rotate again (return to S101).
[0035]
The information on the liquid level of each tube detected by the liquid level detecting means is transmitted to the solution amount controlling means, and the solution amount controlling means sends information on an appropriate amount of the solution to be supplied to the tube to the solution supplying means. (S115).
[0036]
The solution supply means calculates the average value of the liquid level according to the liquid level of each tube based on the information about the appropriate amount of the solution to be supplied to the tubes transmitted from the solution amount control means. Then, an appropriate amount of solution is supplied to each tube (S117).
[0037]
Steps 111 to 117 are repeated until a desired amount of the concentrated solution is obtained.
[0038]
When the desired amount of concentrated solution is obtained, reduce the number of revolutions of the rotor. The number of rotations at this time is, for example, 100 rpm to 3000 rpm, and specifically, 1000 rpm (S119).
[0039]
Further, the liquid level may be checked to confirm that the desired amount has been collected (S121). In addition, it is preferable to manufacture about 300 microliter as a protein concentrate for NMR analysis.
[0040]
The rotation of the tube is stopped (S123). Thereafter, the tube is taken out (S125). The operation of removing the tube may be performed manually or may be automatically performed by a machine.
[0041]
The removed tube is preferably set up on a column and allowed to stand (S127). Thus, a concentrated solution can be obtained. The present invention is particularly useful as a method for concentrating proteins.
[0042]
When a concentrated solution of a protein is obtained through the above steps, the solution can be suitably used for NMR and X-ray analysis, and is greatly utilized for three-dimensional structure analysis of proteins and domains and functional analysis of proteins.
[0043]
(Nuclear magnetic resonance method)
NMR techniques place the material to be examined (usually in a suitable solvent) in a strong magnetic field and align with the magnetic field until excited by radio frequency (rf). They absorb resonance energy and re-emit at a frequency determined by i) the type of nucleus and ii) the atomic environment. In addition, resonance energies transfer from one nucleus to another through binding or three-dimensional space, giving information about the environment of a particular nucleus and its surrounding nuclei.
Examples of the NMR apparatus include DRX-600 available from Bruker, INOVA600 available from Varian, and α-600 available from JEOL.
[0044]
NMR can be measured as follows. First, the mixed solution is poured into the NMR sample tube using a cooled Pasteur pipette or the like. At this time, a concentrated solution of the protein concentrated by the solution concentrating device of the present invention is used. According to the present invention, an appropriate concentrated solution can be obtained in a short time, so that operations including NMR can be performed appropriately and quickly. Furthermore, three-dimensional structure analysis and functional analysis of proteins and domains based on NMR analysis data can be rapidly performed.
[0045]
The Pasteur pipette preferably has a length of 20 cm or more, and can be purchased from, for example, Towa Kagakusha. The NMR sample tube is inserted into an NMR static magnetic field which has reached a target temperature (17 ° C. to 57 ° C.) in advance. The magnitude of the magnetic field is preferably between 90 MHz and 1000 MHz, more preferably between 600 MHz and 900 MHz. Since the degree of the magnetic field orientation is proportional to the square of the magnetic field strength, a higher magnetic field condition is more desirable. For example, as two-dimensional NMR, 2D, DOQ-COSY, TOCSY, NOESY, HSQC, etc., and as multidimensional NMR, HNCO, HCACO, HNCA, HCA (CO) N, HN (CO) CA, HNHB, CBCANH, H ( CA) NH, HBHA (CO) NH, HCCH-COSY, HCANH, HCCH-TOCSY, HCACON, ^Fifteen N-NOESY-HSQC, ¹³ C-NOESY-HSQC and the like are known.
[0046]
(X-ray analysis method)
In X-ray crystallography, an X-ray beam is directed at a crystal (or fiber) of a substance to be examined and is refracted by molecular atoms that align within the crystal. The scattered X-rays are captured by a photosensitive plate and developed using standard techniques. The diffracted X-rays can be visualized as a series of spots on the photosensitive plate, and the molecular structure in the crystal can be determined from this pattern. In X-ray crystallography, it is desired to obtain a pure and large crystal. By using a protein concentrate concentrated by the solution concentrating device of the present invention, a protein crystal with high purity can be obtained, so that a pattern of X-ray crystal analysis with high accuracy can be obtained. Further, according to the present invention, since a concentrated solution can be obtained in a short time, operations including X-ray crystal analysis can be performed appropriately and quickly. Furthermore, the analysis of the three-dimensional structure of the protein and the functional analysis of the protein based on the X-ray crystallographic analysis data can be performed quickly.
[0047]
(Method of analyzing three-dimensional structure of protein)
In the latter half of the 1980's, three-dimensional structure analysis of proteins by NMR rapidly advanced (K. Wuthrich, Yoshimasa Kyogoku and Yuji Kobayashi, "NMR of proteins and nucleic acids-Structural analysis by two-dimensional NMR," Tokyo Chemical Doujin ( 1991)). In particular, use of ultra-high field magnets (currently 800 MHz at the resonance frequency of protons) and stable isotopes of nitrogen atoms and carbon molecules ^Fifteen N and ¹³ A new heteronuclear multidimensional NMR method using C has been developed. Further, from the structural information of the biomolecule obtained by NMR, it is possible to perform three-dimensional structure analysis and functional analysis of the biomolecule. Many programs for performing structural analysis from NMR data are well-known. For example, NMR Pipe, PIPP, Cap, Felix, NMR View, XEASY, and X-PLOR as three-dimensional structure calculation software for chemical shift assignment. CNS, DYANA, DYNAMO and the like are used.
[0048]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, in the process of concentrating proteins etc., the operation efficiency can be improved and the novel solution concentrating apparatus which can process in a short time can be provided.
ADVANTAGE OF THE INVENTION According to this invention, the solution concentrating apparatus which can obtain the concentrated liquid of protein etc. of a favorable state can be provided.
Advantageous Effects of Invention According to the present invention, it is possible to provide a solution concentrating device that can efficiently perform a concentration operation even when a plurality of types of protein solutions are simultaneously concentrated. According to the present invention, protein concentration is performed quickly and accurately using the solution concentrating device of the present invention, and NMR or X-ray analysis after crystallization is performed using the obtained protein concentrate to obtain protein or protein. Analyze the three-dimensional structure and function of the domain.
According to the present invention, drug discovery can be performed based on the above analysis. For this reason, the solution concentrating device of the present invention is particularly effective for drug discovery and the like.
[Brief description of the drawings]
FIG. 1 (a) is a schematic structural diagram showing an example of a solution concentrating device of the present invention. FIG. 1B is a top view of the device.
FIG. 2 is a schematic diagram of a mechanism for adjusting a liquid level according to the present invention.
FIG. 3 is a schematic diagram showing an example of a solution containing means of the present invention.
FIG. 4 is a diagram showing a protocol for concentrating a solution using the solution concentrating device of the present invention.
FIG. 5 is a diagram illustrating an example of an arrangement of a solution storage unit, a solution supply device, and the like.
[Explanation of symbols]
1 Solution storage means
2 rotor
3 Drive motor
5 Liquid level detection means
7 Solution supply amount control means
9 Solution supply device
11 Filtrate storage section
13 Body
15 Filtration membrane
17 Cap part
18 holes
19 Concentrate recovery section
31 light source
32 Liquid level detecting means
33 Solution supply amount calculation means
33a Solution supply amount calculation unit
34 Solution supply means
34a Protein liquid server
34b Liquid replenishment injection automatic dispenser

Claims (12)

A solution concentrator for concentrating the solution contained in the solution containing means by a centrifugal operation,
Liquid level detection means for detecting the liquid level of the solution stored in the solution storage means,
Solution supply amount control means for controlling the amount of the solution to be supplied to the solution storage means, based on the liquid level information detected by the liquid level detection means,
Solution supply means for supplying the amount of solution determined by the solution supply amount control means to the solution storage means,
A solution concentrating device comprising: The centrifugal operation is by rotation drive of the solution storage means,
The solution concentrating device according to claim 1, wherein the liquid level detecting means is a means for detecting a liquid level of the solution at a timing synchronized with a rotation cycle of the solution containing means. A rotation mechanism for rotating the solution supply means in synchronization with a rotation cycle of the solution storage means; and a solution supply path connecting the solution storage means and the solution supply means. The solution concentrating device according to claim 2, wherein the supply of the solution is performed during the rotation of the solution storage means. The solution concentrating device according to claim 1, wherein the solution accommodating unit further includes a gas pressurizing unit that performs gas pressurization and centrifugation. The solution concentrating device according to any one of claims 1 to 4, wherein the solution accommodating unit is held by an angle rotor or a swing rotor. 3. The solution concentrating device according to claim 1, wherein the liquid level detecting means is a CCD sensor. Having a contrast member provided on the side opposite to the liquid level detection means across the solution,
3. The solution concentrating device according to claim 1, wherein the liquid level detecting means can more clearly detect the liquid level of the solution stored in the solution storing means. The solution concentrating device according to any one of claims 1 to 7, wherein the solution accommodating unit, the solution supply path, and the solution supply unit rotate integrally. The solution concentrating device according to any one of claims 1 to 8, wherein the solution contained in the solution containing means contains a protein. A method for producing a sample for nuclear magnetic resonance spectrum measurement, comprising a step of concentrating a protein concentration using the solution concentrating device according to any one of claims 1 to 9. A method for producing a protein crystal, comprising a step of concentrating a concentration of a protein using the solution concentrating device according to claim 1. A three-dimensional structure analysis of a protein by a nuclear magnetic resonance method using a sample produced by the production method according to claim 10 or an X-ray crystal analysis method using a crystal produced by the production method according to claim 11. Method.

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