JP4066250B2 - Protein crystal and protein structure analysis method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention provides a novel protein that can be purified, crystallized, and analyzed for three-dimensional structure by X-ray diffraction under uniform conditions after any protein is produced in a soluble fraction. The present invention relates to a structural analysis method.
[0002]
[Prior art]
Each protein has a unique three-dimensional structure, but in recent years, information on the three-dimensional structure of proteins has become important for drug development and the like. For example, if the three-dimensional structure of a protein can be known, a substance that interacts with the protein can be designed with certainty, which can make an important contribution to the manufacture of pharmaceutical products. In addition, these findings can be used to elucidate the mechanism of diseases, increase food production, and preserve the environment.
[0003]
Conventionally, in order to analyze the X-ray crystal structure of a protein, it has been necessary to examine in detail purification conditions and crystallization conditions for each target protein. Therefore, much effort and time are required for purification and crystallization to obtain a high-purity target protein. In addition, when the target protein is a recombinant protein, the expressed target protein cannot correctly form a three-dimensional structure, becomes an inclusion body, shows toxicity to the host, and the target protein is a soluble protein. In many cases, the productivity was extremely low due to degradation to host protease.
For this reason, it has been desired to develop a technology that speeds up a series of steps such as expression, purification, crystallization, and structural analysis of a recombinant protein.
[0004]
[Problems to be solved by the invention]
In view of the above situation, the present invention can perform purification, crystallization, and analysis of a three-dimensional structure by X-ray diffraction under uniform conditions after producing any protein in a soluble fraction. An object of the present invention is to provide a new protein structure analysis method.
[0005]
[Means for Solving the Problems]
The present invention is a protein crystal obtained by crystallizing a fusion protein in which one or two or more chaperonin subunits and a target protein are linked via a peptide bond. The present invention is described in detail below.
[0006]
The fusion protein crystallized in the present invention is one in which one or two or more chaperonin subunits and a target protein are linked via a peptide bond. When two or more chaperonin subunits and the target protein are linked via peptide bonds, the two or more chaperonin subunits are linked in one direction via peptide bonds.
[0007]
The chaperonin is generally called a molecular chaperone that assists in protein folding or contributes to structural stabilization in the presence or absence of the energy substance ATP when stress such as heat shock is applied to cells. The expression of the protein group is induced. Among these molecular chaperones, the subunit molecular weight is about 60 kDa, which is present in all organisms of bacteria, archaea, and eukaryotes. It has folding support and degeneration protection functions.
[0008]
Chaperonin is a structure consisting of two layers of rings consisting of 10 to 20 subunits, that is, two layers of chaperonin rings consisting of 5 to 10 chaperonin subunits associated non-covalently via the ring surface. A ring structure (hereinafter, the ring is referred to as a chaperonin ring) is formed. For example, E. coli chaperonin has a cavity having an inner diameter of 4.5 nm and a height of 14.5 nm (see FIG. 1). The cavity of one layer of chaperonin ring has enough space for one globular protein of 60 kDa. Chaperonin has a function of temporarily storing various protein folding intermediates and denatured proteins in this cavity. When a protein folding structure is formed, it is coupled with the decomposition of ATP, and the stored protein is cavityd. To release from. Bacterial and archaeal chaperonins can be easily mass-produced in the E. coli cytosolic soluble fraction while maintaining the ring structure. This indicates that various chaperonins of different origins can self-assemble in E. coli and can take a two-layer ring structure consisting of 10 to 20 mers.
[0009]
The three-dimensional structure of chaperonin has already been clarified by X-ray crystal structure analysis, and according to the analysis result, the N-terminal and C-terminal of chaperonin are both located on the cavity side and have a highly flexible structure. In particular, at least 20 amino acids at the C-terminal show a highly flexible structure (George et al., 2000, Cell, 100, P.561-573).
[0010]
The chaperonin used in the present invention is not particularly limited, and any chaperonin derived from bacteria, archaea, and eukaryotes can be used. However, the structure of the chaperonin varies depending on the organism from which it is derived. For example, in the case of chaperonin derived from bacteria, the number of chaperonin subunits constituting the chaperonin ring is 7, 8 to 9 from archaebacteria, and 8 from eukaryotes. In the present invention, it is preferable to select the ratio of the number of chaperonin subunits and the target protein in the fusion protein depending on the origin of the chaperonin used. The ratio of the chaperonin subunit to the target protein (chaperonin subunit number: target protein number) can be 1: 1 to 12: 1, but preferably 1: 1 to 9: 1. If the number of chaperonin subunits per protein of interest exceeds 9, it may become difficult to form a chaperonin ring.
[0011]
Specifically, when a chaperonin derived from bacteria is used, a fusion protein in which the number of chaperonin subunits: the number of target proteins is 1: 1 or 7: 1 is preferable because of the ease of forming a ring composed of chaperonin subunits. In the case of using an archaeal chaperonin having 8 subunits constituting the chaperonin ring, the number of chaperonin subunits: the number of target proteins is 1: 1 because of the ease of forming a ring composed of chaperonin subunits. Those of 2: 1, 4: 1 or 8: 1 are preferred. However, other number ratios may be suitable depending on the shape and molecular weight of the target protein.
[0012]
The target protein used in the present invention is not particularly limited. For example, it is important for the development of drugs derived from animals such as humans, mice, nematodes, pathogenic bacteria, infectious viruses, etc., and the structure has not been analyzed. Examples include proteins. Among them, 7-transmembrane receptors, G protein-coupled receptors, transmembrane proteins such as cell adhesion factors; membrane-bound proteins, protein-related proteins such as protein kinases, virus-derived proteases, intranuclear Hormone receptor proteins, reverse transcriptases and the like are preferred, and transmembrane proteins, membrane-bound proteins, and nuclear hormone receptor proteins are more preferred.
[0013]
As a linking pattern between the chaperonin subunit of the fusion protein and the target protein in the present invention, the N-terminal, C-terminal, or chaperonin subunits of the chaperonin subunits are linked so that the target protein fits in the cavity of the chaperonin ring. It is preferable to arrange the target protein between them.
[0014]
In the present invention, the target protein expressed as a fusion protein is stored in the cavity of the chaperonin ring, so that it is protected from the in vivo environment and is not easily digested by proteases.
[0015]
In the present invention, the target protein is preferably stored in a ring composed of four or more chaperonin subunits. If the number of chaperonin subunits is less than 4, it may protrude from the chaperonin ring depending on the size of the target protein.
[0016]
Even if the target protein has the property of inhibiting natural mechanisms important to the host organism, the target protein is isolated from the in vivo environment by the chaperonin ring, so that it may also exert an inhibitory effect on the host physiological mechanism. Absent. In addition, many protein folding intermediates, such as those observed when expression is induced by a strong promoter, are not associated with each other and are individually fixed in the cavity of the chaperonin ring. And inclusion body formation seen in cell-free translation systems can also be suppressed. Since chaperonin is expressed in the soluble fraction of the host organism's cytoplasm or body fluid, even if the protein stored in the chaperonin ring is a membrane-bound protein, it cannot be transferred to the membrane and destroy the membrane structure of the host organism. And no toxicity to the host organism. Moreover, if any protein is stored in the same chaperonin ring, it can be purified as a fusion protein under the same purification conditions.
[0017]
The fusion protein obtained in the present invention is purified and then crystallized.
The method for purifying the fusion protein is not particularly limited, and for example, methods such as salting out, hydrophobic chromatography, ion exchange chromatography, gel filtration, and affinity chromatography can be used. Further, by adding a histidine sequence of about 6 residues to the N-terminal or C-terminal of the fusion protein, it is possible to purify the fusion protein also by chelate chromatography using a nickel chelate carrier.
[0018]
When chaperonin is heat-resistant, most of the other contaminating proteins are precipitated by heat-treating the crude product and can be easily removed by centrifugation. At this time, since chaperonin has an action of suppressing heat denaturation of the protein, the target protein stored in the chaperonin ring is soluble without being heat denatured even if it is not itself a heat-resistant protein. It can be obtained as a fusion protein.
Since the target protein is stored inside the chaperonin ring, it can be purified as a fusion protein by an established uniform method regardless of the type of protein.
[0019]
The above fusion protein can be crystallized under uniform conditions because the surface properties of the fusion protein are the same regardless of what target protein is stored inside the chaperonin ring.
Crystallization of the fusion protein can be performed by the hanging drop method. Add a precipitating agent such as ammonium sulfate or polyethylene glycol (PEG) to 10 μl of a fusion protein solution of about 10 mg / ml dissolved in buffer so that the concentration is lower than saturation, and place it on a thin glass plate. Put on a small container (reservoir) and seal. About 1 ml of precipitant solution is placed in the container. Vapor diffusion slowly equilibrates the fusion protein solution and the container. The salt concentration of the fusion protein solution increases as water escapes into the reservoir. After the saturation point, the fusion protein slowly precipitates out. When pH and salt concentration conditions are appropriate, crystals of the fusion protein are produced in the fusion protein solution. For example, when the chaperonin subunit used for the fusion protein is GroEL derived from E. coli, an equal volume of 3.6% PEG6K, 200 mM MgCl ₂ , 50 mM KCl is added to a 10 mg / ml fusion protein solution containing 2.5 mM ADP. Crystallization can be carried out under the condition of adding 100 mM Tris-HCl (pH 8.0) buffer containing.
[0020]
The protein crystal of the present invention preferably has a quality capable of elucidating the three-dimensional structure and / or the total atomic space arrangement of the fusion protein by X-ray crystal structure analysis. The quality that can elucidate the three-dimensional structure and / or all-atom space arrangement of the fusion protein by X-ray crystal structure analysis means, for example, the quality that the fusion protein is detected as a single band by SDS-PAGE. To do.
[0021]
The present invention is based on the idea of using chaperonin as a molecular container for structural analysis of a target protein. After the target protein is crystallized as a fusion protein without releasing it from the chaperonin, the X-ray crystal structure Use for analysis.
[0022]
By irradiating the protein crystal of the present invention with X-rays, the three-dimensional structure and / or the total atomic space arrangement of the fusion protein can be analyzed.
As an X-ray source, characteristic X-rays generated when electrons collide with copper or molybdenum are used at the laboratory level. When using particularly intense X-rays or X-rays of any wavelength, synchrotron radiation is used. In Japan, the use of SPring-8, which is a third-generation synchrotron radiation facility, provides clearer diffraction spots than before and enables measurement with less error. In general, the intensity of X-ray diffraction is measured by a two-dimensional detector such as an X-ray film or an imaging plate (IP). Many diffraction lines are generated by rotating the crystal while applying X-rays. When using IP, the recorded diffraction pattern can be scanned and digitized and stored in a computer.
[0023]
Prepare multiple heavy atom isomorphous substitute crystals of the obtained crystal, apply X-rays of the same wavelength as the original crystal to these, and use the change in diffraction intensity due to incorporation of heavy atoms to reflect the reflection phase of the original crystal To decide. If the reflection phase is determined, the electron density can be obtained. However, a resolution of at least 2.8 mm is required to perform the analysis up to the atomic position determination. If there is an electron density map with a resolution of 2 to 2.8 cm, a molecular model can be assembled.
[0024]
By comparing the three-dimensional structure and / or all-atom space configuration of the fusion protein analyzed by X-ray crystal structure analysis with the three-dimensional structure and / or all-atom space configuration of the chaperonin ring, the three-dimensional structure of the target protein And / or the total atomic space configuration can be analyzed.
[0025]
By using the usual series of X-ray crystal structure analysis techniques as described above for the protein crystal of the present invention, it is stored inside the ring composed of chaperonin subunits without the need to isolate the target protein. In this state, it is possible to directly analyze the structure of the target protein.
A protein structure analysis method for analyzing the three-dimensional structure and / or the total atomic space arrangement of a fusion protein by irradiating the protein crystal of the present invention with X-rays, and irradiating the protein crystal of the present invention with X-rays Then, the three-dimensional structure and / or all-atom space arrangement of the fusion protein is analyzed, and the analyzed three-dimensional structure and / or all-atom space arrangement of the fusion protein and the three-dimensional structure of the ring composed of chaperonin subunits are analyzed. A protein structural analysis method for analyzing the three-dimensional structure of the target protein and / or the total atomic space configuration by comparing the total atomic space configuration with the total atomic space configuration is also one aspect of the present invention.
[0026]
According to the present invention, the target protein is expressed as a fusion protein with a chaperonin subunit, and the target protein is surely placed inside the cavity of the chaperonin ring, thereby expressing the toxicity of the target protein to the host, degradation by the protease, and It solves the problem of inclusion body formation and can be expressed in large quantities as a soluble protein. In addition, according to the present invention, any protein can be stored in the same chaperonin ring, so that purification, crystallization, and structural analysis can be performed under the same conditions. It can be carried out.
[0027]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
[0028]
[Example 1]
(Preparation of expression vector)
From the hyperthermophilic archaeon Thermococcus KS-1 chaperonin α subunit (TCPα) gene, a gene having a BglII cleavage site on the 5 ′ side and a BamHI site on the 3 ′ side is amplified by PCR, and this is amplified into a pT7blueT vector. TA cloning was performed on Novagen and the inserted sequence was confirmed.
From this, the insert fragment was recovered by cleavage with BglII and BamHI. The recovered DNA fragment is subjected to a ligation reaction with T4 DNA ligase, the reaction purified product is again cleaved with BglII and BamHI, and two TCP-α genes linked in the same direction (TCPα) 2 are separated by electrophoresis. And recovered. This (TCPα) 2 was cloned into a pT7 blue-T vector that had been digested with BglII-BamHI and treated with alkaline phosphatase to prepare a large amount of plasmid pT7- (TCPα) 2. The insert fragment was recovered from pT7- (TCPα) 2 by cleavage with BglII and BamHI. The recovered DNA fragment was ligated with T4 DNA ligase, the reaction purified product was again cleaved with BglII and BamHI, and (TCPα) 4 in which two (TCPα) 2 genes were ligated in the same direction was separated by electrophoresis. It was collected. This was again cloned into the pT7 blue-T vector, and a large amount of plasmid pT7- (TCPα) 4 was prepared. The insert fragment was recovered from pT7- (TCPα) 4 by cleavage with BglII and BamHI. The recovered DNA fragment was subjected to ligation reaction with T4 DNA ligase, the reaction purified product was again cleaved with BglII and BamHI, and (TCPα) 8 in which two (TCPα) 4 genes were ligated in the same direction was separated by electrophoresis. Recovered. After the introduction of the BglII and BamHI sites, (TCPα) 8 was cloned into an expression vector having an alkaline phosphatase-treated T7 promoter to construct pT (TCPα) 8.
After treating pT (TCPα) 8 with BamHI, a synthetic DNA serving as a cloning site for a foreign gene was ligated, cleaved with BglII, and then circularized by a ligation reaction to construct pTF8TCP-α.
pTF (TCPα) 8 is a vector for expressing a fusion protein in which a foreign protein is connected to eight linked chaperonin α subunits through a thrombin cleavage sequence.
[0029]
(Expression of 8TCPα-hCyP)
A gene encoding human cyclophilin (cyclosporin-binding protein, hCyP) as a foreign protein was recovered from the pT7blue-T vector into which the gene had been introduced by cleavage with NheI and SpeI and subjected to the same restriction enzyme treatment. pT (TCPα) 8-hCyP was constructed by ligating to pTF (TCPα) 8 to express a fusion protein (8TCPα-hCyP) of eight chaperonin α subunits and hCyP. Escherichia coli was transformed with pT (TCPα) 8-hCyP, 10 colonies were inoculated into 2XYT liquid medium (bactotryptone 16 g, yeast extract 10 g, NaCl 5 g / L), and 35 ° C. in the presence of ampicillin (100 μg / ml). And 1 mM IPTG was added at an OD ₆₀₀ of 0.6 to 1.0. The cells were further cultured for 10 hours to induce the expression of 8TCPα-hCyP. After completion of the culture, the cells were collected.
[0030]
(Expression of 8TCPα-5HT)
In the same manner as 8TCPα-hCyP described above, a fusion protein (8TCPα-5HT) of 8TCPα and a human serotonin receptor (5-HT7 receptor) was expressed in E. coli.
[0031]
(Purification / crystallization / analysis)
Each cell was collected and purified by heat treatment at 65 ° C. for 30 minutes, twice anion exchange chromatography and gel filtration. The purified sample was uniform by SDS-PAGE. Each purified sample was concentrated by ultrafiltration to a concentration of 10 mg / ml, and then ATP having a final concentration of 2.5 mM was added. Thereafter, crystallization was performed under the conditions of 100 mM Tris-HCl (pH 8.0), 3.6% PEG6K, 200 mM MgCl ₂ , and 50 mM KCl. As a result, all the octahedral crystals of about 0.1 mm square were obtained. Was obtained.
Further, when an X-ray diffraction experiment was performed on the obtained crystal using an X-ray generator, a diffraction point with a resolution of 2.5 mm was obtained.
[0032]
[Example 2]
(Preparation of expression vector)
From the E. coli chaperonin subunit (GroEL) gene, a gene having SpeI on the 5 ′ side and an XbaI site introduced on the 3 ′ side was amplified by PCR, and this was TA-cloned into a pT7blueT vector (manufactured by Novagen). It was confirmed.
From this, the insert fragment was recovered by cleavage with SpeI and XbaI. The recovered DNA fragment is ligated with T4 DNA ligase, the reaction purified product is again cleaved with SpeI and XbaI, and two GroEL genes linked in the same direction (GroEL) 2 are separated by electrophoresis, It was collected. This (GroEL) 2 was cloned into a pT7 blue-T vector that had been digested with SpeI-XbaI and treated with alkaline phosphatase to prepare a large amount of plasmid pT7- (GroEL) 2. The insert fragment was recovered from pT7- (GroEL) 2 by cleavage with SpeI-XbaI. The recovered DNA fragment is subjected to a ligation reaction with T4 DNA ligase, the reaction purified product is again cleaved with SpeI-XbaI, and two (GroEL) 2 genes linked in the same direction (GroEL) 4 are electrophoresed. Separated and recovered. This was again cloned into the pT7 blue-T vector to prepare a large amount of plasmid pT7- (GroEL) 4. After cleaving pT7- (GroEL) 4 with XbaI, alkaline phosphatase treatment was performed, and (GroEL) 3 was ligated to obtain pT7- (GroEL) 7 in which seven chaperonin subunits were linked. The insert fragment was recovered from pT7- (GroEL) 7 by cleavage with SpeI-XbaI. After the introduction of the SpeI and XbaI sites, (GroEL) 7 was cloned into an expression vector having an alkaline phosphatase-treated T7 promoter to construct pT (GroEL) 7.
After treating pT (GroEL) 7 with XbaI, a synthetic DNA serving as a cloning site for a foreign gene was ligated, then cleaved with SpeI, and then circularized by a ligation reaction to construct pTF (GroEL) 7.
pTF (GroEL) 7 is a vector that expresses a fusion protein in which a foreign protein is linked to a 7-fold linked body (7GL) in which 7 chaperonin subunits are linked via a thrombin cleavage sequence.
[0033]
(Expression of 7GL)
pTF (GroEL) 7 is transformed into E. coli, 10 colonies are inoculated into 2XYT liquid medium (bactotryptone 16 g, yeast extract 10 g, NaCl 5 g / L) and cultured at 35 ° C. in the presence of ampicillin (100 μg / ml). Then, 1 mM IPTG was added at an OD ₆₀₀ of 0.6 to 1.0. The culture was further continued for 10 hours to induce the expression of 7GL. After completion of the culture, the cells were collected.
[0034]
(Expression of 7GL-GFP)
A gene encoding jellyfish-derived green fluorescent protein (GFP) as a foreign protein is recovered from the pT7blue-T vector into which the gene has been introduced by digestion with NheI and SpeI, and pTF (GroEL) 7 subjected to the same restriction enzyme treatment. And pTF (GroEL) 7-GFP that expresses a fusion protein of seven chaperonin subunits and GFP was constructed.
Next, a 7GL-GFP fusion protein (7GL-GFP) was expressed in E. coli in the same manner as 7GL described above.
[0035]
Both 7GL and 7GL-GFP were designed so that a histidine tag (6-residue histidine sequence: 6His) was added to the N-terminal side, and a FLAG tag (manufactured by Sigma) was added to the C-terminal side.
[0036]
(Purification / crystallization / analysis)
Each cell was collected and purified by affinity chromatography using anti-FLAG antibody, nickel chelating chromatography and gel filtration. Both purified samples were uniform by SDS-PAGE. 7GL and 7GL-GFP at a concentration of 2.9 mg / ml were crystallized with Tris-acetate buffer at pH 8.0 containing 2.0 M ammonium sulfate, 1% PEG8000 and 4 mM CaCl _2. A fine crystal of about ˜20 μm square was formed.
7GL-GFP microcrystals are shown in FIGS.
[0037]
[Example 3]
(Preparation of expression vector)
In the same manner as in Example 1, a large amount of plasmid pT7- (TCPα) 4 was prepared.
After treating pT (TCPα) 4 with BamHI, a synthetic DNA serving as a cloning site for a foreign gene was ligated, cut with BglII, and then circularized by a ligation reaction to construct pTF4TCP-α.
pTF (TCPα) 4 is a vector for expressing a fusion protein in which a foreign protein is connected to four linked chaperonin α subunits through a thrombin cleavage sequence.
[0038]
(Expression of 4TCPα)
pTF (TCPα) 4 was transformed into E. coli, 10 colonies were inoculated into 2XYT liquid medium (16 g bactotryptone, 10 g yeast extract, 5 g NaCl / L) and cultured at 35 ° C. in the presence of ampicillin (100 μg / ml). Then, 1 mM IPTG was added at an OD ₆₀₀ of 0.6 to 1.0. The culture was further continued for 10 hours to induce the expression of 4TCPα. After completion of the culture, the cells were collected.
[0039]
(Expression of 4TCPα-GFP)
A gene encoding jellyfish-derived green fluorescent protein (GFP) as a foreign protein is recovered from the pT7blue-T vector into which the gene has been introduced by NheI and SpeI digestion, and pTF (TCPα) subjected to the same restriction enzyme treatment 4 was ligated to pT (TCPα) 4-GFP, which expresses a fusion protein (4TCPα-GFP) of four chaperonin α subunits and GFP.
Subsequently, a fusion protein of 4TCPα and GFP (4TCPα-GFP) was expressed in E. coli in the same manner as 4TCPα described above.
[0040]
(Expression of 4TCPα-hCyP)
In the same manner as 4TCPα-GFP described above, pT (TCPα) 4-hCyP that expresses a fusion protein (4TCPα-hCyP) of 4TCPα and human cyclophilin (cyclosporin-binding protein, hCyP) was constructed, and 4TCPα-hCyP was It was expressed in E. coli.
[0041]
It was designed so that 6His was added to the C-terminus of 4TCPα. The two fusion proteins were designed from the N-terminal side in the order of 4TCPα, the target protein, and 6His.
[0042]
(Purification / crystallization / analysis)
Each cell was collected and purified by nickel chelate chromatography, hydrophobic chromatography and gel filtration. The purified sample was uniform by SDS-PAGE. Each purified sample was concentrated by ultrafiltration to a concentration of 10 mg / ml, and then ATP having a final concentration of 2.5 mM was added. Thereafter, crystallization was performed under the conditions of 100 mM Tris-HCl (pH 8.0), 3.6% PEG6K, 200 mM MgCl ₂ , and 50 mM KCl. Was obtained.
Further, when an X-ray diffraction experiment was performed on the obtained crystal using an X-ray generator, a diffraction point with a resolution of 2.8Å was obtained.
[0043]
【The invention's effect】
According to the present invention, since any protein can be stored in the same chaperonin ring, the three-dimensional structure can be analyzed by purification, crystallization and X-ray diffraction under the same conditions. Can be done much more quickly.
[Brief description of the drawings]
FIG. 1 is a diagram schematically showing the three-dimensional structure of E. coli chaperonin (GroEL).
2 is a view showing 7GL-GFP microcrystals obtained in Example 2. FIG.
3 is a view showing 7GL-GFP microcrystals obtained in Example 2. FIG.

Claims (1)

A three-dimensional structure and / or an all-atom space arrangement of a crystal of a fusion protein having a structure in which a target protein is linked to a chaperonin through a peptide bond and is contained in a ring structure composed of chaperonin subunits; By comparing the three-dimensional structure and / or all-atom space arrangement of a protein crystal consisting of only a ring structure composed of chaperonin subunits and not linked to the target protein, the three-dimensional structure of the target protein and A method for analyzing the structure of a protein, comprising analyzing the total atomic space configuration.

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