WO2004029079A1

WO2004029079A1 - Method of purifying protein complex, assay method, purification device and assay device

Info

Publication number: WO2004029079A1
Application number: PCT/JP2003/012063
Authority: WO
Inventors: Nobuhiro Takahashi; Toshiya Hayano
Original assignee: Tokyo University Of Agriculture And Technology Tlo Co., Ltd.
Priority date: 2002-09-25
Filing date: 2003-09-22
Publication date: 2004-04-08
Also published as: JPWO2004029079A1; AU2003268651A1

Abstract

A method of purifying a protein complex interacting with a target protein by using microfluidics having a two-stage trap. This method comprises the following steps: (a) binding a fused protein containing a target protein, a first tag and a second tag to a protein complex; (b) capturing the target protein and the protein complex by a first trap containing a first affinity carrier capable of binding to the first tag; (c) separating the target protein and the protein complex from the first affinity carrier; (d) capturing the target protein and the protein complex by a second trap containing a second affinity carrier capable of binding to the second tag; and (e) separating the protein complex from the second affinity carrier.

Description

^

Specification

Technical field

TECHNICAL FIELD The present invention relates to a method for purifying a protein complex using microfluidics or nanofluidics, or an Atsuy method. The present invention also relates to an apparatus for purifying a protein complex using microfluidics or nanofluidics or an atsey apparatus. Background art

In order to know how proteins interact with each other in vivo using proteomics techniques, it is necessary to use a known target protein and extract it from a sample such as a cell extract. It is effective to purify the interacting unknown protein complex and identify it by mass spectrometry or the like. As a method for purifying the protein complex for this purpose, affinity chromatography using a column is known.However, since a relatively large amount of a sample is required, only a small amount of a sample can be obtained. Is inappropriate.

As a method for increasing the efficiency of protein complex purification, a tandem affinity purification (TAP) method is known (Rigaut, et al "Nature Biotechnology, 17, 1030-1032). Is expressed as a fusion protein with two types of affinity tags, and the protein complex bound to the target protein is recovered by two-step affinity purification, but the expression level of the fusion protein is controlled. And the use of calcium ions in the second-stage affinity purification has a limited range of applications. Another problem is that the sample loss is large due to adsorption to the container or the wall of the instrument, and the recovery rate is low. Since the affinity carrier itself is lost due to the repeated separation process of the carrier and the solution, there is a problem in the reproducibility of the experimental results. ^

There is a disadvantage that purification cannot be performed while monitoring the action.

On the other hand, using a sensor chip that can monitor the interaction between molecules by surface plasmon resonance, the target analyte is captured by the interaction surface layer, and then the mass spectrum of this analyte is measured. (Natsume, et al., Ana Chem. 2000, 72, 4193-4198). This method can handle a very small amount of sample, but when handling a cell extract, non-specific capture of impurities in the cell extract together with the analyte of interest. As a result, there was a drawback that good results could not be obtained for a subject such as a protein complex that had to be highly purified before analysis. Unlike column chromatography with a relatively large capacity, when microfluidics is used to purify a small amount of a sample, impurities that are non-specifically adsorbed on the flow channel / carrier or in the system There is a unique problem that it is difficult to sufficiently wash away the contaminants remaining in the gap. In this regard, the authors of this document state that this contaminant separates in reversed-phase HPLC before performing mass spectral measurements, but does not provide a solution for the removal of proteinaceous contaminants. It is an object of the present invention to provide a method for purifying a small amount of a protein complex by using the method and performing an assay, and a purification device or an assay device for use in these methods. Disclosure of the invention

We have used two-step traps on microfluidics or nanofluidics to affinity purify the protein complex that interacts with the target protein. The present inventors have found that it is possible to easily purify a desired minute amount of protein complex, and have completed the present invention.

The present invention is particularly useful in the field of proteomics for elucidating the interaction of proteins in vivo. In order to know how multiple proteins interact in vivo, a known target protein is used to interact with the known target protein from a sample such as a cell extract. Unknown protein complex It is effective to purify and identify this by mass spectrometry or the like. For that purpose, it is necessary to purify the protein complex. The present invention provides a method for purifying the protein complex. The method of the present invention is easily applicable to a sensor chip for a real-time BIA core (registered trademark).

That is, the present invention provides a method for interacting with a target protein using microfluidics or nanofluidics having a first trap, a second trap, and a channel for transporting and discharging a liquid to and from these traps. A method of purifying the acting protein complex, comprising:

(a) binding the fusion protein containing the target protein, the first tag and the second tag to the protein complex,

(b) capturing the target protein and the protein complex by a first trap containing a first affinity carrier capable of binding to the first tag,

(c) separating the target protein and protein complex from the first affinity carrier,

(d) capturing the target protein and the protein complex by a second trap containing a second affinity carrier capable of binding to a second tag; and

(e) separating the protein complex from the second affinity carrier;

Each step.

In the above method, instead of the step (a), a fusion protein containing a first target protein and a first tag; a fusion protein containing a second target protein and a second tag; The method may include a step of binding to a protein complex.

In the above method, the step (c) of separating the target protein and the protein complex from the first affinity carrier preferably comprises dissociating the bond between the first tag and the first affinity carrier, or This is performed by cleaving the bond between the first tag and the target protein. Further, the step (e) of separating the protein complex from the second affinity carrier preferably comprises dissociating the bond between the second tag and the second affinity carrier, or This is accomplished by cleaving the bond between the target tag and the target protein or by degrading the protein complex.

In a preferred embodiment, the fusion protein further comprises a third tag, wherein the target protein and the protein complex are combined before binding to the first affinity carrier. It is enriched by affinity take mouth chromatography using this second tag. More preferably, the method of the present invention comprises the steps of: (d) capturing a target protein and a protein complex with a second trap containing a second affinity carrier capable of binding to a second tag; The step (e) of separating the body from the second affinity carrier further comprises the step of removing contaminants bound to the first trap and the channel. As a result, the purity of the target protein complex can be increased, and the accuracy of the protein complex can be improved.

More preferably, the method of the present invention is performed while monitoring capture and separation in the first trap and / or the second trap. Sensor chips that can monitor interactions between molecules by surface plasmon resonance are known in the art and can be adapted for such purposes.

In another aspect, the present invention uses a microfluidics or nanofluidics having a first trap, a second trap, and a channel for transporting and discharging liquid to and from these traps. Provided is a method of accessing a protein complex that interacts with a target protein, the method comprising:

(a) binding a fusion protein comprising a target protein, a first tag and a second tag to a protein complex,

(b) capturing the target protein and protein complex with a first trap containing a first affinity carrier capable of binding to the first tag;

(c) separating the target protein and protein complex from the first affinity carrier;

(d) capturing the target protein and the protein complex by a second trap containing a second affinity carrier capable of binding to the second tag,

(e) separating the protein complex from the second affinity carrier; and

(f) Attaching the protein complex,

Each step.

In the above method, instead of the step (a), a fusion protein containing a first target protein and a first tag; a fusion protein containing a second target protein and a second tag; The method may include a step of binding to a protein complex. 3 012063

5 Preferably, the step of separating the protein complex from the second affinity carrier (e) is carried out by decomposing the protein complex into peptides with a proteolytic enzyme, and the step of fusing the protein complex (f) This is done by analyzing the peptides produced by the degradation.

In yet another aspect, the present invention provides an apparatus for purifying a protein complex that interacts with a target protein. The device comprises:

A first trap containing a first affinity carrier capable of binding to a first tag in the target protein;

A second trap containing a second affinity carrier capable of binding to a second tag in the target protein;

A channel for conveying and discharging the liquid to these traps,

Means for controlling the flow of liquid in the flow path,

Here, the trap and the channel are arranged on microfluidics or nanofluidics. The control means causes the target protein and the protein complex to be captured by the first trap, separates the target protein and the protein complex from the first affinity carrier, and the target protein and the protein protein are separated by the second trap. The night body flow in the flow path is controlled to capture the complex and separate the protein complex from the second affinity carrier.

Preferably, in the device of the present invention, the control means further comprises means for monitoring the capture and separation of the protein and protein complex in the first and second traps.

The present invention also provides a protein complex assay device comprising the above-described purification device of the present invention and a means for assaying the protein complex separated from the second affinity carrier. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows one example of a microfluidics configuration.

FIG. 2 shows a schematic diagram of the microfluidics used in the method of the present invention. FIG. 3 shows one mode of binding of a fusion protein containing a target protein, a first tag, and a second tag to a protein complex that interacts with the target protein.

FIG. 4 shows one embodiment of the target protein and protein complex captured in the first trap via dextran.

FIG. 5 shows one embodiment of a step of separating a target protein and a protein complex from an affinity carrier using TEV protease in the first trap. FIG. 6 shows one embodiment of a protein complex with a target protein that has been separated from the first trap and captured by the second trap.

FIG. 7 shows one embodiment of a step of separating a target protein and a protein complex from the second trap using a protease.

Figure 8 is an example of the results of monitoring the capture and separation of proteins in a trap.

Figure 9 shows an example of the results of monitoring trapping of evening protein in a trap. FIG. 10 is a schematic diagram showing an apparatus for realizing the method of the present invention.

FIG. 11 is a schematic diagram of a system used for two-step purification and analysis of a protein complex that interacts with a target protein.

FIG. 12 is a schematic diagram of a sensor chip having a first trap (column Fc1 and column Fc2) and a second trap (column Fc3 and column Fc4).

FIG. 13 shows the results of monitoring the process in which the fusion target protein Pin1 and the protein complex interacting therewith bind to amylose in the first trap using the sensor chip of the BIAcore® device.

Figure 14 shows the separation of the protein complex by TEV protease from the first trap (Fc1 and Fc2) and its separation into the second trap (Fc3 and Fc4) by the sensor chip of the BIAcore® device. The result of monitoring the binding is shown.

FIG. 15 shows the results of monitoring the protease degradation in the second trap (Fc3 and Fc4) using the sensor chip of the BIAcore® device. DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The method of the present invention is performed on microfluidics or nanofluidics. Microfluidics is a device in which micro channels, pumps, valves, solution storage units, reaction units, etc., with dimensions on the order of im, are formed on a silicon or glass chip. It is being developed as a chip for micro reaction systems. Nanofluids generally refers to those having a smaller size than microfluidics, but there is no clear division between them, and these terms are used in the present specification without any particular distinction. As an example of a microfluidics, a sensor chip for a biosensor that can monitor the interaction between molecules by surface plasmon resonance has been put into practical use. The product name of BIacore (registered trademark) is Biacor. It is commercially available from eAB (Upp sala, Sweden). Such a chip technology is disclosed in US Pat. No. 5,641,640, US Pat. No. 5,955,729, Japanese Patent No. 3294605, and Japanese Patent Publication No. 07-507865.

Figure 1 shows one example of such a microfluidics configuration. A channel 2, a valve 3, a solution port 4, a reaction section 5, a reaction monitor section 6, and a discharge port 7 are formed on the microphone opening fluidics 1. By controlling the operation of a pump (not shown) and a valve by a control device (not shown), various substances are transported to the reaction section through the flow path, reacted, and discharged. The reaction can be monitored by the reaction monitor 6. The method of the present invention is useful for purifying or assaying a protein complex using such microfluidics. The method of the present invention is readily applicable, especially to sensor chips for real-time BI Acore®.

The present invention provides a method for purifying and accessing a protein complex that interacts with a target protein using microfluidics or nanofluidics. FIG. 2 shows a schematic diagram of the microfluidics used in the method of the present invention. On the microfluidics, a first trap 11, a second trap 12, and a flow path 13 for transferring and discharging a liquid to and from these traps are provided. The flow path 13 includes a liquid inlet 15 for introducing the liquid to be supplied to the trap and a body discharged from the trap in addition to the first and second traps. _g

It communicates with a discharge section 16 for receiving, and the flow of liquid in the flow path 13 can be controlled by a valve 14, a pump, an external computer for controlling the operation of these, and the like.

In the method of the present invention, first, a fusion protein containing a target protein, a first tag, and a second tag is bound to a protein complex that interacts with the target protein. To prepare a vector encoding a fusion protein containing a target protein, a first tag and a second protein by a gene recombination technique, and to express the fusion protein in a host such as Escherichia coli or cultured animal cells. Can be.

Target proteins include, for example, various transport proteins, storage proteins, catalytic proteins, metabolic regulation proteins, various receptors, various site force-in cell growth factors, various transcription factors, various signal transduction factors, cytoskeleton factors, various types Proteins that belong to almost all categories such as on-channels, various G proteins, cell cycle regulators, morphogenesis regulators, protein synthesis-related proteins, etc. Proteins and the like whose presence is predicted only from the base sequence of the genome can be used.

As the first or second tag to be fused with the target protein, various tags capable of binding to an appropriate affinity carrier can be used, such as, but not limited to, chitin-binding protein, maltose-binding protein, Various lectin proteins, darubithion S-transferase, FK506-binding protein, mTOR rapamycin binding domain, pyotinylated protein, lac repressor, various epitope frags (FLAG tag, c_myc tag, histidine tag, HA tag, VSV- G tag etc.) or TAP tag etc. can be used. In addition, a substance capable of electrochemically forming and breaking a chemical bond (for example, a substance described in Japanese Patent Application No. 2002-241072) can also be used as a tag bound to the fusion protein. Methods for producing such fusion proteins by genetic recombination techniques are well known in the art. As an example, a maltose binding protein can be expressed as a first tag, a biotinylated protein as a second tag, and a fusion protein with a target protein in a host cell. Protein complexes that interact with the target protein include one or more proteins that interact strongly or weakly with the target protein by hydrogen bonding, electrostatic bonding, hydrophobic bonding, and the like. NA, RNA), enzymes, sugars, sugar chains, peptides, lipids, vitamins, metal complexes, steroids, terpenoids (terpenes), o-Ichigoids, alkaloids, etc. An active substance or the like may be contained.

Binding of the target protein to the protein complex interacting therewith may occur spontaneously in the host expressing the fusion protein, and may be separated from the sample containing the protein complex, such as a cell extract. It may be formed by mixing the generated fusion protein. Alternatively, both the sample containing the protein complex and the fusion protein may be applied to the first trap.

FIG. 3 is a schematic diagram showing an example of binding of a fusion protein containing a target protein, a first tag, and a second tag to a protein complex that interacts with the target protein. In this example, the fusion protein includes a P5 protein as the target protein 21, a Mal! ^-Binding protein 22 as the first tag, and a biotinylated protein 23 as the second tag. A gene encoding this fusion protein is produced by a gene recombination technique, introduced into Escherichia coli to express the fusion protein, purified, and then mixed with, for example, crushed human 293 EBNA cultured cells. Thus, a cell extract containing the protein complex 31 interacting with the target protein 21 is prepared.

In another embodiment, the method of the present invention comprises, instead of the above-mentioned steps, a fusion protein comprising a first target protein and a first tag, a second target protein and a second tag. The method may comprise a step of binding the containing fusion protein to a protein complex that interacts with both of these target proteins. In such an embodiment, in order to purify a protein complex that interacts with two types of target proteins, two types of fusion proteins in which different tags are respectively bound to two types of target proteins are prepared. These are combined with the protein complex.

In a preferred embodiment of the present invention, the fusion protein used in the method of the present invention further contains a third tag. Target protein and tamper bound to it ^

Before the protein complex is bound to the first affinity carrier, the purification efficiency of the protein complex is further improved by concentrating the protein complex by affinity chromatography using the third tag. Can be. As the third tag to be bound to the target protein, for example, various epitope tags (FLAG tag, c-myc tag, histidine tag, HA tag, VSV-G tag, etc.) can be used. When a histidine tag is used, after preparing a cell extract, the fraction containing the target protein and the protein complex interacting with the target protein can be concentrated using Ni-NTA affinity chromatography. it can.

Next, the target protein and the protein complex are captured by a first trap containing a first affinity carrier capable of binding to a first tag. As the affinity carrier, for example, a sugar such as amylose can be used when using a mal! ^-Binding protein as a tag, and streptavidin or avidin can be used when using a biotinylated protein as a tag. . Methods for selecting affinity carriers and binding conditions that can bind to the first column are well known in the art. The affinity carrier can be bound to the trap, for example, via a dextran layer provided on the wall of the trap.

FIG. 4 is a schematic diagram showing an example of a target protein and protein complex captured in the first trap via dextran. In this example, the affinity carrier amylose 54 is bound via dextran 53 to the first trap wall 52 to capture protein complex 3.1 bound to the target protein. Let me do it. By binding of the first tag, malus-binding protein 22 to amylose 54, the target protein 21 and the protein complex 31 are captured in the first trap. At this time, impurities 55 in the sample are often non-specifically adsorbed to the first trap and the flow path, or stay in gaps in the system.

Preferably, in order to increase the efficiency of capture, the capture step uses an oscillation or a method of stopping the flow of the sample. Oscillation means that when a night body including a sample flows into the trap, the flow direction is switched between forward and reverse in a short time. Oscillation enables traces of carriers in microtubules It is known that the efficiency of capturing on a carrier increases when capturing a sample of

(Abrantes, et al "Anal. Chem" 2001, 73, 2828-2835). It is well known in the art that stopping the flow of the sample increases the capture efficiency. The flow direction can be switched several times a minute to several times a second. Next, the target protein and the protein complex are separated from the first affinity carrier. The separation is performed by dissociating the bond between the first tag and the first affinity carrier, or cleaving the bond between the first tag and the target protein. The mode of dissociation and cleavage will depend on the combination of tag and affinity carrier used, and methods for selecting the appropriate mode are well known in the art. In order to dissociate the bond between the tag and the affinity carrier, for example, the composition of the solution, the ion concentration, and the amount of: H are changed. In order to break the bond between the tag and the target protein, a fusion protein is constructed in advance so as to include a specific protease recognition site between the tag and the target protein, and a tag is attached to the trap when separating. Introduce the enzyme. Examples of such proteolytic enzymes include TEV protease, thrombin, enterokinase, factor Xa, and the like. Alternatively, when the tag is bound by using an electrocleavable affinity carrier, the target protein and the protein complex can be separated from the affinity carrier by electrolytic cleavage.

FIG. 5 is a schematic diagram showing an example of a step of separating a target protein and a protein complex from an affinity carrier using TEV protease in the first trap. In this example, TEV protease 61 is introduced into the first trap after the target protein 21 and the protein complex 31 have been captured by the first trap via the amino acid 54. The fusion protein has a TEV cleavage site between the target protein 21 and the maltose binding protein 22, and the fusion between the target protein 21 and the maltose binding protein 22 is caused by the action of TEV proteinase 61. The peptide bond between them is broken.

Next, the target protein and the protein complex separated from the first trap are captured by a second trap containing a second affinity carrier capable of binding to a second tag. Capture target protein and protein complex in second trap Details of the process are as described for the first trap.

FIG. 6 is a schematic diagram showing an example of a target protein and a protein complex captured by the second trap after being separated from the first trap. The sample cut by TEV protease in the previous step is introduced into a second trap. In this example, the affinity carrier streptavidin 74 is bound to the second trap wall 72 via dextran 73 to capture the protein complex 31 that interacts with the target protein. Let me do it. The binding of the second tag, biotinylated protein 23, to streptavidin 73 causes the target protein 21 and protein complex 31 to be captured in the second trap. In this example, the amount of protein captured in the second trap was 7.3% based on the amount of protein captured in the first trap (100%).

In an embodiment in which two types of fusion proteins each having a different tag are used to purify a protein complex that interacts with two types of target proteins, the first trap and the protein complex are used by a first trap. The second target protein and the protein complex are captured by a second trap.

In a particularly preferred embodiment of the method of the present invention, the target protein and the protein complex are captured by the second trap, and then the first trap and the channel are washed to remove contaminants bound or retained therein. Remove objects. In this removal step, since a washing reagent does not flow into the second trap, it is necessary to use a proteolytic reagent such as an acid, an alkali, a surfactant, urea, or a protease, or a protein denaturant. it can. Preferably, formic acid is used as a washing reagent. By adding this step, it is possible to remove contaminants adsorbed or retained in the channel system used for the liquid sending, so that the content of the contaminants contained in the recovered protein complex is significantly reduced. Can be reduced. Therefore, the purification efficiency of the protein complex can be improved, and the accuracy of the protein complex can be improved. This is an advantage of the method of the invention, which features the use of a two-stage trap.

Next, the protein complex is separated from the second affinity carrier. protein 1 The step of separating the complex from the second affinity carrier is as described for the first trap.

Alternatively, this separation step may be performed by decomposing the protein complex with a protease. As the proteolytic enzyme, trypsin, lysyl endopeptidase and the like can be used. The peptide thus obtained can be subsequently subjected to separation by HPLC, analysis by tandem mass spectrum, and the like. That is, in another aspect, there is provided a method for assaying a protein that interacts with a target protein by assaying the protein complex purified by the above-described method.

FIG. 7 is a schematic diagram showing an example of a step of separating a target protein and a protein complex from a second trap using a protease. By the action of lysyl endopeptidase 81, the captured protein complex 31 is decomposed into peptide molecule 82. The peptide molecules 82 degraded as described above are collected and analyzed by HPLC and Z or mass spectrometry, whereby the protein complex 31 is analyzed. In this example, the method of decomposing and analyzing the protein complex captured by the second trap was described.However, analysis using a laser or the like in the state captured by the second trap is described. Is also possible. Further, similarly to the first trap, after separating the protein complex captured by the second trap, it is also possible to recover the purified protein complex.

More preferably, the method of the present invention is performed while monitoring capture and separation in the first trap and / or the second trap. Sensor chips capable of monitoring the interaction between molecules by surface plasmon resonance are known in the art, and for example, BIA core (registered trademark) can be used. Graphs of the binding reaction obtained by such a monitor are shown in FIG. 8 and FIG. FIG. 8 shows the results of monitoring the capture and separation processes of the target protein and protein complex that bind to the protein via maltose-binding protein in the first trap using the BIA core® system. Show. Amylose was used as the affinity carrier, and 200 ig / ml fusion protein containing maltose-binding protein was injected during capture, and 1 OmM maltose was injected during separation. did. FIG. 9 shows the results of monitoring the capture process of a target protein and a protein complex that binds to streptavidin via a biotinylated protein in the second trap. Although not shown, the manner in which the protein complex is separated by protease degradation can also be monitored in the same manner as the separation monitor portion in FIG. Streptavidin was used as an affinity carrier, and 200 gZm1 of a fusion protein containing a biotinylated protein was injected.

FIG. 10 is a schematic diagram showing an apparatus for realizing the method of the present invention. A first trap 11, a second trap 12, and a flow path 13 for transporting and discharging liquid to these traps are provided on the microphone opening fluidics. The first trap 11 contains a first affinity carrier that can bind to a first tag in the target protein, and the second trap 12 can bind to a second tag in the target protein. Contains a second affinity carrier. The flow path 13 includes, in addition to the first and second traps, a liquid introduction part 15 for holding liquid to be supplied to the trap, a discharge part 16 for receiving liquid discharged from the trap, and It is in communication with the transport unit 17 for sending the sample separated from the trap to the analyzer. Samples, reagents, buffers, and the like necessary for carrying out the method of the present invention are supplied from the liquid introduction unit 15 to the flow path 13 and discharged at the discharge unit 16. The sample purified by the method of the present invention is sent from the trap through the transport section 17 to the protein complex assay device.

The flow of the liquid in the channel 13 is controlled by the control means. The control means includes a valve 14, a pump (not shown), and a computer 18 for controlling these operations. The control means operates the valve and the pump under the control of the computer to cause the first trap to capture the target protein and the protein complex, and to cause the target protein and the protein complex to pass through the first affinity. Separate from the carrier, capture the target protein and protein complex by the second trap, and control the flow of liquid in the flow path so that the protein complex is separated from the second affinity carrier . In the case where the electrolytically cut affinity carrier is applied, the control means further includes an energization control unit for controlling energization to the 11 and 12 trap units.

Preferably, in the device of the present invention, the control means monitors the capture and separation of the labeled protein and protein complex in the first and second traps. 03 012063

15 further includes a monitor section 19. By sending information on capture and separation from the monitor 19 to the computer, it is possible to control the flow of liquid in the flow path more accurately and precisely. That is, the configuration of the device of the present invention including the monitor section 19 is particularly suitable for automation of protein complex purification and analysis.

FIG. 10 shows an example of the configuration of the device of the present invention, and it should be understood that various modifications are possible. For example, those skilled in the art can appropriately change the number and arrangement of the trap, the liquid introduction unit, the discharge unit, and the monitor unit, and the combination and arrangement of the flow path and the valve according to the purpose. Similarly, various modifications can be made to the above-described embodiments of the method and apparatus of the present invention, and these modifications are also within the scope of the present invention.

The contents of all patents and references explicitly cited herein are hereby incorporated by reference. In addition, the entire contents described in the specification and drawings of Japanese Patent Application No. 2002-27874, which is the application on which the priority claim of the present application is based, are incorporated herein by reference. To quote. Example

Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the scope of the present invention.

Two-step purification and enzymatic digestion of a protein complex that interacts with the target protein using the BIA core® device is performed on a BIA core® sensor chip, and the digest is online using nano-LC. The components of the protein complex were identified by analysis with a -MS / MS (micro flow liquid chromatography tandem mass spectrometer) (Natsume et al., Anal. Chem. 74: 4725-4733, 2002). FIG. 11 is a schematic diagram of the system used in this experiment. The BIA core (registered trademark) device and the nano-LC-MS / MS device are connected online, and two-step affinity purification of protein complexes and on-chip protease digestion on the BIA core (registered trademark) sensor chip are performed. The LC-MS / MS analysis was performed online. In this example, a cell cycle regulator Pin1 protein was used as a target protein, a maltose binding protein was used as a first tag, and a biotinylated protein was used as a second tag. Protein and a histidine tag for crude recovery were generated as a target fusion protein. The target fusion protein Ρίη1 has a TEV protease cleavage site introduced between the target protein and the maltose binding protein. A gene encoding this target fusion protein was prepared by a genetic recombination technique, introduced into Escherichia coli to express the fusion protein, and purified, and used in the following experiments.

The fusion target protein Pin1 thus obtained was mixed with a human 293EBNA cell extract, and crudely recovered with a Ni ²⁺ chelate column was transferred to a BIA core (registered trademark) device equipped with the above sensor chip. Introduced. In the BIA core (registered trademark) device, amylose is coupled to two flow paths (column Fc1 and column Fc2) of a commercially available BIA core (registered trademark) sensor chip (research grade CM) to form a first trap, and A sensor chip was attached to the two channels (column Fc3 and column Fc4), which was made by binding streptavidin and used as a second trap (Fig. 12). Figure 13 shows the results of monitoring the process by which the fusion target protein Pin1 and its interacting protein complex bind to the amylose of the first trap (Fc1 and Fc2). After washing Fc1 and Fc2 with the eluent, a solution containing TEV protease is introduced to cleave the protein complex bound to the first trap from the maltose-bound protein, and simultaneously release the protein complex released by this. The body was attached to a second trap (Fc3 and Fc4). To increase the yield of cleavage by TEV protease and binding to the second trap, 2 microliters of the solution containing TEV protease is automatically oscillated between the first and second traps (back and forth). Operation was performed. This oscillation operation was performed once every 30 seconds, and this was repeated for 2 hours. Figure 14 shows the results of monitoring the dissociation of the protein complex from the first trap (Fc1 and Fc2) by TEV protease and its binding to the second trap (Fc3 and Fc4). After binding the target protein complex to the second trap, the first trap and the channel system from the sample introduction part to the discharge part are washed with formic acid, so that non-specific binding derived from the cell extract is performed. Protein was removed from the channel system. After formic acid has been sufficiently removed from the channel system, lysyl endoprotease (LysC) is introduced into the second trap to form a protein complex that interacts with Pin1 The protein was separated into peptides. Figure 15 shows the results of monitoring protease degradation. The protein was identified by sending the degraded peptide to the electromagnetic valve (E-valve) shown in Figure 11 and automatically analyzing it with the nano-LC-MS / MS system. As a result, 58 proteins were identified. When compared with the results of a similar experiment performed with a fusion protein containing only the tag protein without Pin1, there were 24 types of proteins that were considered to be specifically contained in the protein complex that interacts with Pin1.

Claims

The scope of the claims

1. Purify protein complexes that interact with the target protein using microfluidics or nanofluidics, which have a first trap, a second trap, and a channel for transporting and discharging liquids to and from these traps. How to do

(a) binding a fusion protein comprising a target protein, a first tag and a second tag to the protein complex,

(b) capturing the target protein and the protein complex with a first trap containing a first affinity carrier capable of binding to the first tag;

(c) separating the target protein and the protein complex from the first affinity carrier,

(d) capturing the target protein and the protein complex with a second trap containing a second affinity carrier capable of binding to the second tag; and (e) capturing the protein complex with a second trap. Separating from the second affinity carrier.

2. A protein complex that interacts with a target protein using microfluidics or nanofluidics with a first trap, a second trap, and a channel for transporting and discharging liquid to and from these traps. A method for purifying

(a) binding a fusion protein comprising a first target protein and a first tag, a fusion protein comprising a second target protein and a second tag, and the protein complex,

(b) capturing the first target protein and the protein complex by a first trap containing a first affinity carrier capable of binding to the first tag,

(c) separating the first target protein and the protein complex from the first affinity carrier,

(d) capturing the second target protein and the protein complex with a second trap containing a second affinity carrier capable of binding to the second tag; hand

(e) separating the protein complex from a second affinity carrier.

3. In the step (c), the bond 5 between the first tag and the first affinity carrier is dissociated or the bond between the first tag and the target protein is cleaved. The method according to claim 1 or 2, wherein the method is performed.

4. the step (e) dissociates the bond between the second tag and the second affinity carrier, or cleaves the bond between the second tag and the target protein; or 34. The method according to claim 103, wherein the method is performed by degrading the protein complex.

5. The fusion protein further contains a third tag, and the target protein and the protein complex are enriched by affinity mouth chromatography using a third tag. The method described in.

6. The method according to any one of claims 15 to 15, further comprising, between steps (d) and (e), removing a contaminant bound to the first trap and the channel. .

7. The method according to any of claims 16 to 17, wherein capture and separation in the first trap and Z or the second trap is monitored.

8. A protein complex that interacts with the target protein using microfluidics or nanofluidics with a first trap, a second trap, and a channel for transporting and discharging liquid to and from these traps Is a method of learning

(b) capturing the target protein and the protein complex by a ^^ trap containing a first affinity carrier capable of binding to the first tag,

(d) capturing the target protein and the protein complex by a second trap containing a second affinity carrier capable of binding to the second tag, (e) separating the protein complex from a second affinity carrier; and

(f) Attaching the protein complex,

A method comprising the steps of:

9. A protein complex that interacts with the target protein using microfluidics or nanofluidics that have a first trap, a second trap, and a channel for transporting and discharging liquid to and from these traps. Is a way of exercising the body,

(a) a fusion protein comprising a first target protein and a first tag, a fusion protein comprising a second target protein and a second tag, and the protein complex

Join 10 and

15 (d) capturing the second target protein and the protein complex with a second trap containing a second affinity carrier capable of binding to the second tag,

(e) separating the protein complex from the second affinity carrier; and

(f) Attaching the protein complex,

A method comprising the steps of:

20 10. The method according to claim 8, wherein step (e) is performed by decomposing the protein complex into peptides with a protease, and step (f) is performed by analyzing the peptides generated by the decomposition. 9. The method according to 9.

1 1. Equipment for purifying protein complexes that interact with the target protein

'And

25 a first trap containing a first affinity carrier capable of binding to a first tag in the target protein;

A channel for conveying and discharging the liquid to these traps, Means for controlling the flow of the night body in the flow path,

Here, the trap and the channel are arranged on microfluidics or nanofluidics,

The control means captures the target protein and the protein complex with a first trap, separates the target protein and the protein complex from a first affinity carrier, and uses a second trap to trap the target protein. Controlling the flow of a liquid in a flow path so as to capture protein and said protein complex and to separate said protein complex from a second affinity carrier.

12. The protein complex purifying apparatus according to claim 11, wherein the control means further comprises means for monitoring capture and separation of proteins and protein complexes in the first and second traps.

13. A protein complex Assy device comprising: the purification device according to claim 11 or 12; and a means for accessing the protein complex separated from the second affinity carrier.