CN100355949C - Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same - Google Patents
Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same Download PDFInfo
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- CN100355949C CN100355949C CNB2004800295182A CN200480029518A CN100355949C CN 100355949 C CN100355949 C CN 100355949C CN B2004800295182 A CNB2004800295182 A CN B2004800295182A CN 200480029518 A CN200480029518 A CN 200480029518A CN 100355949 C CN100355949 C CN 100355949C
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Abstract
A protein crystallization apparatus with which protein crystallization experiments and crystallization condition screening can be carried out rapidly and economically with high reliability; and a protein crystallizing agent with which the operation of protein crystallization can be carried out in a simple manner. In particular, the protein crystallization apparatus is characterized by including protein crystallization microarray (18) having two or more crystallizing agent holding parts (16) adapted to retain a protein crystallizing agent and, superimposed on the protein crystallization microarray (18), plate (24) wherein the plate (24) has crystallization compartments (32) corresponding to the above crystallizing agent holding parts (16) and adapted to be charged with a protein-containing sample and has recessed parts (34) interposed between crystallization compartments (32). The protein crystallizing agent is characterized in that the crystallizing agent is uniformly held in a gel as a result of gelation of a solution containing a protein precipitating agent and an unsaturated monomer.
Description
Technical field
The present invention relates to be used to carry out the apparatus and method of crystallization of protein or crystallization condition screening, and be used to make protein crystallizing agent that crystallization of protein separates out from proteinaceous sample and preparation method thereof.
Background technology
In recent years, be referred to as the research of structural genomics, this research is attempted by illustrating proteinic space structure and proteinic function relationship, and the function of this proteinic gene of coding is described.
The parsing of proteinic space structure at first will be screened the proteinic crystallization condition that will resolve usually.Then, implement crystallization at the suitableeest crystallization condition.Carry out x-ray structure by the crystallization that will obtain and resolve, carry out the parsing of protein steric structure.Wherein in the process that proteinic crystallization condition is screened, before decision obtains being used to carry out the abundant good crystallization condition of space structure parsing, expensive time and a lot of protein examples.For this reason, the decision crystallization condition becomes the bottleneck in the space structure parsing.In addition, also find out with the various crystallization method headed by the vapor diffusion method.Yet problem such as experimental implementation is miscellaneous is still a lot.So carried out replacing new crystallization method of these existing methods and device exploitation, protein crystallization condition screening or in order to make crystallization rapider and carry out with more micro-sample, the scheme of range protein crystallization apparatus, crystallization method, crystallization condition screening method has been proposed.For example open to have proposed to make in the flat 6-321700 communique and contain precipitation agent and protein in the gel, by with they stacked convection current that suppresses to see in the solution, the method that crystallization is grown in gel the spy.In addition, simplify, carried out utilizing the trial of gelling material in order to make crystallization method.In addition, when carrying out the exploration of crystallization condition, various precipitation agents have been proposed.
Yet, when utilizing gelling material,, maybe can not carry out the problem of gelation reaction because the combination of gelling material and precipitation agent exists the gel gonorrhoea.If the gel gonorrhoea exists the problem that can not have or not crystalline state by microscopic examination.
In addition, the some people among the present inventor with rapidly, the screening of carrying out protein crystallization condition economically with micro-example is purpose, proposed to carry out on the chip of microarray the such crystallization apparatus of crystallization experiment (for example, WO03/053998 brochure).
In the described invention of WO03/053998 brochure, used microarray.This microarray maintains gelling material in each district that forms by communicating pores.And the protein crystallizing agent that in each gelling material, contains many types and concentration.By this microarray is contacted with proteinaceous sample, can once screen a plurality of crystallization conditions.And then can implement with the protein example of trace, efficient is very high.
But, in above-mentioned protein crystallization apparatus, mobile, the mixing (pollution) of proteinaceous sample and/or crystallizing agent can take place between the district of microarray.Therefore, there are the crystallization condition of separating out and concentration and the inconsistent possibility of kind of distinguishing the crystallizing agent that contains in the interior gelling material in advance.
In addition, use such device to supply with proteinaceous sample to the crystallizing agent maintaining part by handwork, operation becomes miscellaneous.And supply with the automatic gear valency height of proteinaceous sample automatically.
The invention that the present invention makes in order to solve above-mentioned problem just.The object of the present invention is to provide and the gel gonorrhoeaization takes place do not make protein crystallizing agent that gelation reaction carries out and preparation method thereof, and can be rapidly and proteinic crystallization experiment or crystallization condition screening are implemented in high reliability ground economically protein crystallization apparatus.
Summary of the invention
The invention provides following content.
A kind of protein crystallization apparatus is characterized in that: have the used for protein crystalling microarray, it has 2 or its above crystallizing agent maintaining part that keeps protein crystallizing agent; With with the stacked plate of above-mentioned used for protein crystalling microarray, above-mentioned plate has the crystallizing field that contains protein example corresponding to filling of above-mentioned crystallizing agent maintaining part, and is arranged on the recess between this crystallizing field.
A kind of gel used for protein crystalling, it maintains sodium-chlor in gelling material, and described gelling material comprises at least a monomer that is selected from acrylamide, 2-acrylamide-2-methyl propane sulfonic acid, the methacryloyl dimethyl aminoethyl Methochloride salt.
A kind of gel used for protein crystalling, it maintains MPD in gelling material, and described gelling material comprises DMAA.
A kind of protein crystallizing agent, it maintains phosphoric acid Na/K in gelling material, and described gelling material comprises 2-acrylamide-2-methyl propane sulfonic acid.
A kind of gel used for protein crystalling, it maintains ammonium sulfate in gelling material, and described gelling material comprises methacryloyl dimethyl aminoethyl Methochloride salt.
A kind of gel used for protein crystalling, it maintains sodium malonate in gelling material, and described gelling material comprises acrylamide.
A kind of gel used for protein crystalling, it maintains PEG6k in gelling material, and described gelling material comprises the polyoxyethylene mono acrylic ester.
Description of drawings
Fig. 1 is the mode chart of the user mode of expression protein crystallization apparatus of the present invention.
Fig. 2 represents the example of the microarray of the used for protein crystalling that uses among the present invention.
Fig. 3 is the mode chart of an example of expression protein crystallization apparatus of the present invention.
Fig. 4 is the sectional view of the part of the plate that uses among the present invention.
Fig. 5 is the orthographic plan of the plate that uses among the present invention.
Fig. 6 is plate and the sample orthographic plan of filling the user mode of auxiliary means of expression among the present invention.
Fig. 7 is the orthographic plan of the support that uses among the present invention.
Fig. 8 is the orthographic plan of the user mode of expression protein crystallization apparatus of the present invention.
Nomenclature
12 tubular fibres; 16 crystallizing agent maintaining parts; 18 used for protein crystalling microarraies; 20 supports; 24 plates; 32 crystallizing fields; 34 recesses
Embodiment
Followingly embodiments of the present invention are described with reference to accompanying drawing.
Fig. 1 is the mode chart of an example of expression protein crystallization apparatus of the present invention.
This example protein crystallization apparatus is represented just as Fig. 1, has liner 22, used for protein crystalling microarray 18, the plate 24 of support 20, silicon rubber system.
Fig. 3 is the sectional view that the central part of plate 24 is exaggerated.In Fig. 4, provided a part of sectional view of the assembled state of support 20, used for protein crystalling microarray 18, plate 24.
This plate 24 is laminated in by above the used for protein crystalling microarray 18 of support 20 supports.That is, as shown in Figure 4, crystallizing field 32 is by 16 sealings of crystallizing agent maintaining part.And then each recess 34 is in by the crystallizing field 32 of crystallizing field support section 31 supports and equally by the position between the crystallizing field 32 of crystallizing field support section 31 supports.
Below, will support the face of used for protein crystalling microarray 18 to be called microarray seating surface 100 in the support 20, the face that will contact with used for protein crystalling microarray 18 in plate 24 is called reaction surface 102, and the face that face is opposite therewith is called outer side 104.
In addition, in plate 24, the outward flange of arranging the zone of crystallizing field 32 becomes recess.Therefore in plate 24, formed the zone that becomes certain area of concavity.Below, the zone that will arrange the recess of crystallizing field 32 in the reaction surface of plate 24 is called sealing 30.
In this embodiment, used for protein crystalling microarray 18 is set in the microarray support section 26 of support 20.As long as and used for protein crystalling microarray 18 is set to crystallizing agent maintaining part 16 corresponding crystallizing fields 32, these crystallizing field 32 sealings are got final product like that.Be that used for protein crystalling microarray 18 can be bonded on the support 20, also can make microarray support section 26 form concavities, be not bonded in the bottom of this microarray support section and be layered on this bottom.
But the material of support 20 and shape so long as the fixing protein crystallization with the material and the shape of microarray 18, just be not particularly limited.As the material of support 20, by using translucent material, crystallization apparatus itself just can be confirmed the crystallization of generation, observe the crystalline process of growth at any time with microscope rapidly and easily, and is therefore preferred.As translucent material, for example can enumerate acrylic resin, polycarbonate resin, polystyrene resin, polydimethylsiloxane (PDMS), glass etc.
Implement to be used to determine the mark of the fixed position of used for protein crystalling microarray 18 for support 20.Marking method is so long as can then be not particularly limited used for protein crystalling microarray 18 correct fixed methods.For example can enumerate, have and the same shape of used for protein crystalling microarray 18 and the impression of area, on support 20 corresponding to the position spraying point of institute's allocation of used for protein crystalling microarray 18 etc.
In this example, just as shown in Figure 7, further be provided with plate pilot hole 44 on support 20, this hole makes plate 24 and support 20 carry out making crystallizing field 32 corresponding with crystallizing agent maintaining part 16 when stacked.
Used for protein crystalling microarray 18 has 2 or its above crystallizing agent maintaining part 16 that keeps protein crystallizing agent.
As crystallizing agent maintaining part 16, can use the maintaining part that in gel etc. can keep the porous plastid (hereinafter referred to as keeping phase) of protein crystallizing agent, has kept protein crystallizing agent.Keep just as shown in Figure 4, so long as when the proteinaceous sample that is filled into crystallizing field 32 contacts with crystallizing agent maintaining part 16, protein crystallizing agent can by this keep that crystallizing field 32 in opposite directions moves just passable.
As above-mentioned maintenance phase, preferably use gel.By using gel, protein crystallizing agent can be controlled in appropriateness speed slowly by crystallizing agent maintaining part 16 to the proteinaceous movement of sample that is filled into crystallizing field 32.Therefore can realize stable crystallization.
The material of gel is not particularly limited, but can use for example acrylamide, N,N-DMAA, N-N-isopropylacrylamide, N-acrylamido ethoxy ethanol, N-acrylamido propyl alcohol, N hydroxymethyl acrylamide, N-vinyl pyrrolidone, methacrylic acid hydroxyethyl ester, (methyl) vinylformic acid, allyl group dextrin etc. monomeric a kind or 2 kinds or its above with multi-functional monomers such as two (methyl) acrylamides of methene, polyoxyethylene glycol two (methyl) the acrylate gel that copolymerization obtains in aqueous medium for example.Also can use the gel of agarose, Lalgine, dextran, polyvinyl alcohol, polyoxyethylene glycol etc. as gel in addition or with the gel of their crosslinked acquisitions.
For gel as described above is remained in the used for protein crystalling microarray 18, for example the solution that contains as the monomers such as acrylamide of gel constituent, multi-functional monomer and initiator can be injected this container, their polymerizations, gelation are got final product.As the method that makes its gelation,, also can be the method for after not having to make their copolymerizations in the presence of the multi-functional monomer, using linking agent except making in the presence of the multi-functional monomer the method for their copolymerizations.In addition, when using agarose, can carry out gelation by reducing temperature as gelatinous material.
When making protein crystallizing agent remain in the gel, this method is had no particular limits.For example, can be in advance with protein crystallizing agent with import to earlier in the proper container after above-mentioned polymerizable monomer mixes, carry out polyreaction then and make it form gel.Protein crystallizing agent is remained in the gel.Here, protein crystallizing agent is contained be immersed in porousness particle etc., this particle is included in the gel.
As the material of used for protein crystalling microarray 18, shape etc., so long as reaction with the most permutations configurations of material, does not hinder and observes crystallization and separate out, just there is no particular limitation.
As the material of used for protein crystalling microarray 18, can enumerate for example glass, resin, metal etc.Also can use the latter made complex body of these combinations of materials in addition.But separate out in order to confirm that easily crystallization of protein has or not, preferably use the high material of light transmission.
As the shape of used for protein crystalling microarray 18, can enumerate for example shapes such as circle, square, rectangle.In addition, its thickness considers that the raising of crystalline rate and facilitation and rapidization that observation is separated out in crystallization can select arbitrarily.For example can make 0.1～5mm, preferred 0.2～2mm.
Fig. 2 has showed the microarray that a plurality of hollow tubular bodies arrangements constitute as the example that is fit to of the used for protein crystalling microarray that uses in protein crystallization apparatus of the present invention.Represented just as Fig. 2, as hollow tubular body, at least 2 tubular fibre 12 is arranged by the district to be fixed on the substrate 10.These tubular fibres 12 have hollow bulb 14.Fill the gel that maintains protein crystallizing agent in the empty in these portion 14, constitute crystallizing agent maintaining part 16.That is, at least 2 crystallizing agent maintaining parts 16 are arranged formation used for protein crystalling microarray 18 by the district on substrate 10.
The inner wall surface of tubular fibre 12 can be used under the state of non-processor.Also can implement Cement Composite Treated by Plasma or gamma-rays, the processing of electron rays isoradial as required.And then tubular fibre 12 also can import reactive functional groups as required.
The external diameter of tubular fibre 12, many for the number of the crystallizing agent maintaining part 16 of per unit area being done, preferably at 2mm or below it.More preferably 0.7mm or below it.And the internal diameter of tubular fibre can suitably be selected in the scope of above-mentioned external diameter.
As on substrate 10 to 2 or tubular fibre 12 more than it carry out by the district arrange, the fixed method, can use following method.That is, tubular fibre spaced and parallel is according to the rules arranged.With these tubular fibres 12 bring together the back bonding, can form fibre array body (three-dimensional arrangement body).The device that uses makings section such as ultramicrotome with the three-dimensional arrangement body that obtains according to the direction of reporting to the leadship after accomplishing a task with fibre axis, preferably with respect to the vertical direction cut-out of fibre axis.Thus, can obtain the microarray that constitutes by the thin slice with tubular fibre arrange body section (Fig. 2).The thickness of thin slice can be made 100～5000 μ m usually and use.Preferred 200～2000 μ m.
At this moment, bonding with resin adhesive etc. by with the regular arrangement of tubular fibre, for example can obtain at the tubular fibre arrange body of direction tubular fibre 12 whole right regular arrangements in length and breadth.The shape of tubular fibre arrange body is not particularly limited.Usually by with the regular arrangement of fiber, can form square, rectangle, circle etc.
So-called " regular " refers to the certain ordered arrangement like that of radical of the fiber that contains in the framework that can make a certain size.For example after making the fiber bunchy of diameter 1mm, be arranged as section Cheng Zong and be 10mn, horizontal square for 10mm like that the time, (1cm in this foursquare frame
2) the fiber number that contains of 1 limit be 10.Then these 10 fibers are tied into 1 row, made 1 synusia.Then this sheet is become 10 layers such overlapping.The result can be arranged in vertical 10, horizontal 10, adds up to 100 fiber.But, make the gimmick of the regular arrangement of fiber be not limited to the above-mentioned method that sheet is stacked like that.
As used for protein crystalling microarray 18, resemble by use and a plurality of hollow tubular bodies to be arranged the microarray that constitutes above-mentioned, the kind of thickness that can fine control used for protein crystalling microarray 18, the volume of crystallizing agent maintaining part 16, maintained protein crystallizing agent and concentration etc. can be made, and protein crystallization apparatus can be made expeditiously.
In addition, used for protein crystalling microarray 18 is preferably by with the container that can prevent the good leak tightness that contact with outside air or pass through airtight preservation such as sealing.As the container of good leak tightness, can enumerate for example gas or little macromolecular material or glass, the metal etc. of water transmitance.And such microarray is preferably preserved at low temperatures, also can freezingly preserve when prolonged preservation especially.
In addition, each crystallizing agent maintaining part 16 preferably is made of the gel that is manufactured to different protein crystallization conditions.So, when carrying out the screening of crystallization condition, on one piece of microarray, can screen rapidly.
The kind that the so-called protein crystallization condition here refers to protein crystallizing agent and concentration, the maintenance that protein crystallizing agent is kept mutually, for example make the temperature, time, cooling distribution plan etc. of composition, degree of crosslinking, crystallizing agent maintaining part 16 and crystallizing field 32 of pH, the gel of the gel after protein crystallizing agent keeps, solidifies.
As the kind of protein crystallizing agent, can enumerate precipitation agent for example, pH buffer reagent, their arbitrary combination etc.
As precipitation agent, can enumerate for example sodium-chlor, polyoxyethylene glycol, 2-methyl-2,4-pentanediol, ammonium sulfate etc.As the pH buffer reagent, can enumerate for example sodium-acetate trihydrate, potassiumphosphate, imidazoles, Trisodium Citrate, sodium dimethylarsonate etc.These reagent can use separately, also can use 2 kinds or its above combination.In addition, as protein crystallizing agent, can use " WIZARD II " that commercially available product EmeraldBioStructures company produces, " Crystal screen " that HamptonResearch company produces, " Grid Screen " etc.
As the concentration of protein crystallizing agent,, for example, under the situation of the precipitation agent that comprises polyoxyethylene glycol 5～50 volume %, preferred 10～35 volume % because of the kind of the protein crystallizing agent that uses is different.On the other hand, be 0.05～0.5mol/L, preferred 0.1～0.2mol/L under the situation of pH buffer reagent.
As above-mentioned,, can carry out the screening of the proteinic crystallization condition of object rapidly by each crystallizing agent maintaining part 16 is prepared into each different protein crystallization condition.Here, preference is set at the multistage as the concentration with protein crystallizing agent.Specifically, when use comprises the precipitation agent of sodium-chlor, in the scope of 0.5～4.0mol/L, make 5 stages, 10 stages, such dilution series of 20 stages.The protein crystallizing agent of each concentration can be filled in the crystallizing agent maintaining part 16 then.
In order to carry out crystallization at more next piece of crystallization condition, the preferred protein crystallization has 10～1000 crystallizing agent maintaining parts 16 with microarray 18.For example, in common protein crystallization condition screening, need study about 800 crystallization condition.Therefore, the number of crystallizing agent maintaining part is preferably at 10 or more than it.On the other hand, if surpass 1000 crystallizing agent maintaining part 16 if used for protein crystalling microarray 18 has, then the interval between the crystallizing agent maintaining part 16 becomes very narrow, and the efficient of processing is poor.
Used for protein crystalling microarray 18 preferably is made of the high material of light transmission in order to carry out the affirmation that protein is separated out easily.
In this example, liner 22 is a silastic product.The material of liner is so long as the material that other integrant (for example, used for protein crystalling microarray 18, plate 24, support 20) in suitable and the protein crystallization apparatus is used in combination is just passable.
In plate 24, as filling the location division that the auxiliary means position coincide with sample described later, preference as, form sample and fill auxiliary means pilot hole 50.
In this embodiment, further be provided with plate align member 44 on support 20, this plate align member 44 is carrying out making crystallizing field 32 corresponding with crystallizing agent maintaining part 16 when stacked to plate 24 and support 20.Be formed with on the plate 24 and the plate align member 44 corresponding plate pilot holes 46 that are arranged on the support 20.
In this embodiment, shown in Fig. 1,5, the outer side 104 that further is equipped with slave plate 24 penetrates into the sealing agent inlet 48 of reaction surface 102.Just as shown in Figure 5, in this embodiment, two sealing agent inlets 48 run through two relative both sides by means of sealing 30.Thus, when from a side sealing agent inlet 48 when sealing 30 injects sealing agents, because the air that comes into existence in sealing 30 is discharged by the opposing party's sealing agent inlet 48, so the crystallizing field between recess 34 32 is more positively sealed in sealing 30.
The material of plate 24 and shape are so long as can be superimposed with used for protein crystalling microarray 18, and just there is no particular limitation, consider the shape of used for protein crystalling microarray 18, can use material and shape arbitrarily.As the material of plate, for example enumerate glass, resin, metal etc.Can from these materials, select material, a kind of separately arbitrarily, or will use after two kinds of combinations.In addition in order to observe the proteinic crystalline state in the crystallizing field 32, preferably when superimposed, partly has light transmission with each crystallizing agent maintaining part 16 corresponding eclipsed with used for protein crystalling microarray 18.Therefore, as the material of plate 24, preferred translucent material, for example transparent resin, glass etc.
As such crystallization recovering mechanism, can enumerate for example following mechanism etc., wherein sealing 30 by with plate 24 independently member constitute, be connected through the hinge with plate 24, sealing 30 outside sides 104 can be opened as required.
The shape of crystallizing field 32 be so long as can fill proteinaceous sample, sealed shape when contacting with crystallizing agent maintaining part 16, and just there is no particular limitation.
The capacity of crystallizing field 32 when being purpose with the crystallization condition of screening carry out to(for) object protein, reduces preferred less than 0.5 μ l for crystallization condition being screened needed protein mass.On the other hand, if continue to resolve for x-ray structure with the crystallization that will separate out, or be used to make for the crystalline kind of structure elucidation brilliant during for purpose here, the capacity of crystallizing field 32 is preferably at 0.5 μ l or more than it.At this moment, as carrying out the 0.1mm of x-ray structure parsing or the big crystallization more than it in order to obtain, as long as the relation of the area of crystallizing agent maintaining part 16 and crystallizing field 32 satisfies following condition, the area that is the face that contacts with crystallizing field 32 of crystallizing agent maintaining part 16 can be so that remaining on the crystallizing agent of crystallizing agent maintaining part 16 moves to crystallizing field 32, and can with the proteinaceous example reaction that is filled in this crystallizing field 32.Compare with crystallizing agent maintaining part 16, crystallizing field 32 can be bigger.But crystallizing field 32 does not preferably contact with the substrate 12 that constitutes used for protein crystalling microarray 18.
In this embodiment, as shown in Figure 4, all be filled with sealing agent 35 at all recesses 34 that are arranged between the crystallizing field 32.
As sealing agent 35, so long as with the dissolving mutually of proteinaceous sample, do not corrode plate 24, liner 22, constitute used for protein crystalling microarray 18 members such as substrate 12 can.For example can use paraffin oil, silicone oil etc.
Even do not use sealing agent 35,, can prevent that also proteinaceous sample is to the intrusion of the district of adjacency owing to have recess 34.If yet use sealing agent 35, can more positively prevent between the proteinaceous sample mixing and when the crystallizing field 32 of adjacency is invaded, can also prevent the evaporation of proteinaceous sample.
In this embodiment, also shown the mechanism that used for protein crystalling microarray 18 that support 20 is supported and plate 24 carry out crimping.Specifically as shown in Figure 1, be provided with first threaded hole 40 that connects support 20 and corresponding to second threaded hole 42 of the first threaded hole run-through board 24.Just as shown in Figure 1, screw 41 is screwed into first threaded hole 40, second threaded hole 42.By using such mechanism, can make crystallizing field 32 more stably remain on air-tight state.
In this example protein crystallization apparatus, the feeler mechanism of the proteinic crystallization in the monitoring crystallizing field 32 can also be set.
As above-mentioned feeler mechanism, for example can enumerate, comprise the microscope of the outer side 104 that is arranged on plate 24 and the feeler mechanism of carrying the CCD photographic camera on microscope.In such feeler mechanism, can by the image document of record is handled, can judge rapidly whether successfully crystallization to the movement that crystallization the is separated out record of photographing by the CCD photographic camera that carries on microscope.Therefore can determine protein crystallization condition rapidly.
Here, enumerate the suitable using method of this example protein crystallization apparatus.
[filling of proteinaceous sample]
In this embodiment, use sample to fill auxiliary means, can in crystallizing field 32, fill proteinaceous sample.
(crystallizing field)
When using the present invention with proteinic crystallization during, preferably the capacity of crystallizing field 32 is made 0.5 μ l or more than it as purpose.When more promptly screening protein crystallization condition and be purpose, preferably the capacity of crystallizing field 32 is made less than 0.5 μ l.
(proteinaceous sample)
In the present invention, so-called proteinaceous sample is to contain to carry out crystalline, or contain will be to the sample of crystallization condition particular proteins (hereinafter referred to as object protein).As protein, can enumerate natural or synthetic peptide, polypeptide, protein and protein complex.After these materials preferably extract, separate or generate by genetically engineered gimmick or chemosynthesis gimmick etc. from natural or synthetic materials, by the common method of purification of combination utilization, for example solvent extraction, column chromatography, liquid chromatography (LC) etc. are carried out the material that purifying obtains in advance to object protein.
Protein concn in the proteinaceous sample and purity also are essential factors of protein crystallization condition.Therefore, can prepare the proteinaceous sample of the concentration and the purity in a plurality of stages, itself and protein crystallizing agent are reacted.For example, preferred protein concentration changes a plurality of stages in 1～50mg/ml scope.In addition, with the amount of the proteinaceous sample of protein crystallizing agent reaction, can be according to suitably changes such as the capacity of the crystallizing field 32 that uses and numbers.In addition, the viscosity of proteinaceous sample, so long as will maintain contain this proteinic sample crystallizing field 32 down and during holding plate 24, proteinaceous sample does not spill from crystallizing field 32, just there is no particular limitation.
Proteinaceous sample except object protein, but also can contain stabilization agents such as helping protein dissolved albumen solvation, reductive agent etc.But as the protein solvation, for example can enumerate, can make membranin dissolved tensio-active agent etc.If the use tensio-active agent in proteinaceous sample, for example can make water-soluble low protein such as membranin disperse well.Therefore, use this example protein crystallization apparatus, can more effectively carry out proteinic crystallization.
At first, as shown in Figure 1, plate 24 resembled reaction surface 102 is left standstill up.Fill in auxiliary means pilot hole 50 and the plate pilot hole 46 at the sample that passes plate 24, each inserts a columniform steady brace 52 with this sample filling auxiliary means pilot hole 50 and plate pilot hole 46 same diameters.
Next,, as shown in Figure 6, lamellar sample is set fills auxiliary means 54 above the reaction surface 102 at this.This auxiliary means has the perforation 56 of arranging corresponding to the crystallizing field on the plate 24 32, and as making 2 the position mating holes 58 of this perforation 56 corresponding to the position matching mechanism of crystallizing field 32 on the plate 24.At this moment, make the position mating holes 58 that passes sample filling auxiliary means 54 aim at the back and insert the steady brace 52 that the sample that is individually fixed in plate 24 is filled auxiliary means pilot hole 50 and plate pilot hole 46.Then, sample being filled auxiliary means 54 is arranged on the plate 24.By such operation, can make the crystallizing field 32 of plate 24 and the perforation 56 of sample filling auxiliary means 54 that sample filling auxiliary means 54 is set accordingly.
Then, sample on being arranged on plate 24 is filled above the auxiliary means 54, loads proteinaceous sample, and its component is filled and disposed 56 the whole zone of boring a hole in the auxiliary means 54 for spreading all over sample.
And then proteinaceous sample is filled on the surface of using the sample filling auxiliary meanss 54 that nuzzle up such as scraper plate in corresponding to the crystallizing field 32 of perforation 56.
Then, with reaction surface 102 side direction outer sides 104 extrusions of steady brace slave plate 24, sample filled on the auxiliary means slave plate 24 unload.So, all filled proteinaceous sample at whole crystallizing field 32.
Just as above explanation, being arranged on the reaction surface 102 of plate 24 by sample being filled auxiliary means 54, face adds proteinaceous sample from it, proteinaceous sample correctly can be filled in the crystallizing field 32.
In addition, sample is filled auxiliary means 54, from viewpoints such as the easy degree that is shaped, intensity, salt tolerances, preferably makes with stainless steel.In addition, preferably fill and the position of operating by tweezers is set on the auxiliary means 54 unloads so that easily sample is filled auxiliary means 54 after proteinaceous sample is filled into crystallizing field 32 at sample.For example, a preferred end is designed to the bending part 59 to the upside bending.
Steady brace 52 is separately fixed at sample herein, and fills auxiliary means pilot hole 50 and plate pilot hole 46.But in plate 24, sample is filled auxiliary means 54 pilot holes 50 and also can be independent of with 46 formation of plate pilot hole a plurality of.
As the method that proteinaceous sample is filled into crystallizing field 32,, prevent that the method that the constitutive requirements beyond crystallizing field 32 are adhered to can so long as can proteinaceous sample be filled in all crystallizing fields 32 according to same component.For example, can not use sample to fill auxiliary means 54, use pipette etc. is filled proteinaceous sample in each crystallizing field 32.Also can use sample to add in addition and use robot.
But,, preferably use above-mentioned sample to fill auxiliary means 54 for rapidly and carry out the filling of proteinaceous sample economically.
In addition, when using pipette in the filling that is containing protein example, the mechanism that does not preferably use the proteinaceous sample that is attracted to pipette and keeps to spue, but make near the micro-drop contact crystallizing field 32 attached to the front end of pipette.
[assembling of protein crystallization apparatus]
Just as shown in Figure 1, be equipped with liner 22 with mark 28 on the support 20.Then used for protein crystalling microarray 18 is arranged on the support 20 like that according to the hollow bulb 23 that is collected in this liner 22.
At this moment, make the plate align member 44 that is arranged on the support 20 be set to connect the plate pilot hole 46 that passes plate 24.
In addition, in this embodiment, plate 24 reaction surfaces 102 be layered in down be supported used for protein crystalling microarray 18 that body 20 supports above.But also plate 24 reaction surfaces 102 of having filled proteinaceous sample at crystallizing field 32 can be left standstill up, make the support 20 of having supported used for protein crystalling microarray 18 according to the microarray seating surface be layered in down this plate 24 above.If the outer side 104 of plate 24 is provided with up and since carry out easily self-sealing substance inlet 48 sealing agent injection or observe the crystallization of separating out at crystallizing field 32, thereby preferred.
Use microscope to confirm the occupied state of proteinaceous sample then.
After confirming that proteinaceous sample is filled into each crystallizing field 32, shown in Fig. 5,8, from the sealing agent inlet 48 that passes plate 24 sealing agent is filled into sealing 30 and recess 34 with pipette.So, escaping to the proteinaceous sample of the position beyond the crystallizing agent maintaining part in the used for protein crystalling microarray 18 16 can sealed dose of displacement.And can further prevent really because proteinaceous sample moves pollution and the condition variation that causes between the crystallizing field 32 of adjacency.Can also prevent proteinaceous sample evaporation.In addition, the injection rate of sealing agent is preferably the recess 34 that sealing agent spreads all over the whole zone of sealing 30, and just as shown in Fig. 5,8, when a side sealing agent inlet 48 injects sealing agents, does not spill the maximum of sealing agent from the opposing party.
[screening of protein crystallization condition]
Use the protein crystallization apparatus of assembling, carry out the screening of protein crystallization condition, can obtain the suitableeest crystallization condition in the target protein matter.Use the crystallization of separating out also can make the crystal that is suitable for structure elucidation in addition.
In protein crystallization apparatus, make protein crystallizing agent and proteinaceous example reaction after, protein crystallization apparatus is statically placed in air-tight state or the atmosphere in protein is separated out the needed sufficient time, under a certain temperature condition.
So-called protein is separated out the sufficient time that needs, and different because of specified protein, concentration, crystallization condition etc., about 1 hour～10 days, in situation about approximately not separating out through 30 days or its above crystallization, it was improper to regard this crystallization condition as.In addition, because temperature condition is an essential factor of protein crystallization condition, therefore also can the changing temperature condition carry out crystallization.Temperature condition is preferably set a plurality of stages like that according to for example 4 ℃, 15 ℃, 18 ℃, 22 ℃ etc.
Separate out through after the sufficient time at protein, situation is separated out in proteinic crystallization observed.This moment is the outer side 104 of slave plate 24 preferably, for example observes by opticmicroscope.
Through operation can decision objects the suitableeest proteinic crystallization condition like this.
[the crystalline making that structure elucidation is used]
With proteinic crystallization as purpose, the capacity of crystallizing field 32 is made 0.5 μ l or its when above, separating out of all crystallizing fields 32 continue to make crystalline growth in the crystalline crystallizing field 32 or reclaim the crystallization of separating out from crystallizing field 32 as kind brilliant, by well-known method of protein crystallization, under the crystallization condition same, can make the crystallization that structure elucidation is used with reclaiming kind of brilliant crystallizing field 32.As the method for well-known crystallization of protein, for example can enumerating, sessile drop method, seat drip a method, dialysis method, batch method etc.
When being purpose with rapider screening protein crystallization condition, when the capacity of crystallizing field 32 is made less than 0.5 μ l, under the suitableeest crystallization condition that requires, carry out the crystallization of target protein matter, can obtain the crystallization that structure elucidation is used.At this moment, as obtaining the crystalline method that structure elucidation is used, can use well-known method, for example vapor diffusion method, sessile drop method etc.
Reclaim by the crystallization that the structure elucidation that obtains is used, with well-known method x-ray structure is carried out in the crystallization of reclaiming and resolve, can the proteinic space structure of decision objects.
In this example protein crystallization apparatus, be filled into proteinaceous sample in the crystallizing field 32 after, stacked by plate 24 and used for protein crystalling microarray 18 are carried out, crystallizing agent maintaining part 16 and filled crystallizing field 32 corresponding coincidences of proteinaceous sample.So, the crystallizing agent that remains on crystallizing agent maintaining part 16 moves in crystallizing field 32, with proteins react.When each crystallizing agent maintaining part 16 is set to different crystallization conditions, separating out for object protein being carried out crystallizing field 32 intercrystallines that crystallization is set at suitable crystallization condition.
Just as above explanation, in this example protein crystallization apparatus, proteinaceous sample is maintained in the crystallizing field 32, and is airtight by crystallizing agent maintaining part 16.Each crystallizing field 32 is isolated by recess 34 in addition.Therefore, can prevent that proteinaceous sample is moving and/or the mixing of protein crystallizing agent between crystallizing agent maintaining part 16 between the crystallizing field 32.And then, can prevent evaporation from the solution of proteinaceous sample, also can stablize the proteinaceous sample of the trace that keeps the nl level.Therefore, can carry out to high reliability proteinic crystallization.Can reliability more judge that whether suitable crystallization condition is in the highland in addition.
In addition, if sealing agent is filled into recess 34, can more effectively prevent to contain between the protein example mixing and to the intrusion of the crystallizing field 32 of adjacency.Also can prevent to contain the evaporation of protein example in addition.Therefore, even contain the protein example trace, also can fine crystallization control condition.
In addition, if will support the support 20 of used for protein crystalling microarray 18 and plate 24 to carry out crimping, can stablize and airtight crystallizing field 32.Therefore more high reliability carry out crystallization.
By the capacity in suitable selective freezing district 32, can more rapidly and correctly carry out the screening of protein crystallization condition and make the crystallization that structure elucidation is used.
And then, fill auxiliary means if re-use sample, can be easy and promptly carry out the screening of protein crystallization condition.
Protein crystallization apparatus of the present invention is well suited for the screening of research, the protein crystallization condition in the exploitation in for example field of medicaments etc. and protein structure and resolves and use in making of crystalline.
The feature of gel used for protein crystalling of the present invention is by the solution that contains protein crystallizing agent, unsaturated monomer is carried out gelation, to make this protein crystallizing agent be disperseed, be dissolved in the gel by homogeneous.
As protein crystallizing agent, so long as the reagent that the protein solubility of protein soln is descended, just there is no particular limitation.For example, can enumerate ammonium sulfate, sodium-chlor, sodium phosphate, potassiumphosphate, lithium chloride, sodium malonate, Trisodium Citrate, sal epsom, Lithium Sulphate, SODIUMNITRATE, Cadmium Sulphate, sodium sulfate etc. as salt.Can enumerate 2-methyl-2 as organic solvent, 4-pentanediol (hereinafter referred to as MPD), ethanol, Virahol, two alkane, methyl alcohol, the trimethyl carbinol, n-propyl alcohol etc.As water-soluble high-molecular compound, can enumerate polyoxyethylene glycol (hereinafter referred to as PEG), polyalkylene glycol monoalkyl ether, polymine etc.
These precipitation agents are two kinds or its above use alone or in combination.Wherein ammonium sulfate, sodium-chlor, potassiumphosphate/sodium, lithium chloride, sodium malonate, MPD, PEG are suitable especially.
As commercially available product, can use " WIZARDII " that Emerald BioStructures company produces, " Crystal screen " that Hampton Research company produces, " Grid Screen " etc.The concentration that precipitation agent uses, if salt, preferred 0.1～5.0mol/L is if organic solvent, preferred 1～80 volume % is if water-soluble high-molecular compound, preferred 1～50 weight %.
Proteinic crystallization is preferably carried out in particular pH range, can use damping fluid in order to keep pH.The damping fluid that uses is not particularly limited, can enumerate and for example contain citric acid, 2-(N-morpholino) ethyl sulfonic acid, N-2-hydroxyethyl piperazine-N-ethyl sulfonic acid, three (methylol) aminomethane, N, damping fluids such as N-two (hydroxyethyl) glycine, can with separately or two kinds or its above mixture neutralize with acid or alkali etc. as required, adjust to the pH of regulation.The scope of pH preferred 3.0～10.0, more preferably 4.0～9.0 scope.
In the present invention, can use unsaturated monomer as gelating agent.Unsaturated monomer is so long as can form gel by carry out polymerization in aqueous medium, and just there is no particular limitation, preferred (methyl) acrylamide monomer, (methyl) acrylic monomer.
As (methyl) acrylamide monomer, be fit to use (methyl) acrylamide, N,N-DMAA, N, N such as N-diethyl acrylamide, N-dialkyl amido (methyl) acrylamide; (methyl) acrylamide methylsulfonic acid, (methyl) acrylamide ethyl sulfonic acid, 2-(methyl) acrylamide-(methyl) acrylamide alkyl sulfonic acids such as 2-methyl propane sulfonic acid; Dialkyl aminoalkyl (methyl) acrylamides such as dimethylaminopropyl (methyl) acrylamide, diethylamino propyl group (methyl) acrylamide, dimethyl aminoethyl (methyl) acrylamide; Dialkyl amido (methyl) acrylamide quaternary ammonium salts such as dimethylaminopropyl (methyl) acrylamide Methochloride salt, diethylamino propyl group (methyl) acrylamido methylethyl chloride salt, dimethyl aminoethyl (methyl) acrylamide Methochloride salt.
In addition, as (methyl) acrylic monomer, be fit to use (methyl) vinylformic acid 2-hydroxyethyl ester, (methyl) vinylformic acid 4-hydroxyl butyl ester, (methyl) vinylformic acid 4-hydroxyl polyhexamethylene, diglycol monotertiary (methyl) acrylate, triglycol list (methyl) acrylate, polyethyleneglycol (methyl) acrylate etc. to contain (methyl) acrylate of hydroxyl; The quaternary ammonium salt of (methyl) propenoic acid dialkyl aminoalkyl esters such as diethylaluminum monochloride salt of (methyl) propenoic acid dialkyl aminoalkyl esters such as (methyl) vinylformic acid dimethyl aminoethyl ester, (methyl) vinylformic acid diethylamino ethyl ester, (methyl) vinylformic acid dimethyl aminoethyl METH chloride salt, (methyl) vinylformic acid diethylamino ethyl ester.
Monomer concentration is with respect to crystallization of protein agent solution 100 quality %, preferred 1～50 quality %, more preferably 1～10 quality %.
In addition, in the present invention as use as required can with the cross-linkable monomer of above-mentioned monomer copolymerizable, if have 2 functional groups or its above free-radical polymerised monomer, just there is no particular limitation, can enumerate for example N, two (methyl) acrylamides of N '-methene, ethylene glycol bisthioglycolate (methyl) acrylate, glycol ether two (methyl) acrylate, triglycol two (methyl) acrylate, polyoxyethylene glycol two (methyl) acrylate, trimethylolpropane tris (methyl) acrylate, TriMethylolPropane(TMP) oxyethane modification three (methyl) acrylate etc., but N, two (methyl) acrylamides of N '-methene, ethylene glycol bisthioglycolate (methyl) acrylate, polyoxyethylene glycol two (methyl) acrylate is suitable especially.The monomer that the addition of cross-linkable monomer is organized or (B) organized for (A) is 0.01～10 mass parts, is preferably 0.1～5 mass parts.
Protein crystallizing agent of the present invention by with specific precipitation agent and specific unsaturated monomer combination, can obtain clear gel.If gel is transparent, the crystallization of protein of observing generation becomes and is easy to.Therefore utilize the automatization of optical detection system also to become easy.
Combination as unsaturated monomer that can obtain clear gel and protein crystallizing agent, at least a kind of monomer that has that (1) elect from acrylamide, 2-acrylamide-2-methyl propane sulfonic acid, methacryloyl dimethyl aminoethyl Methochloride salt and sodium-chlor, (2) DMAA and MPD, (3) 2-acrylamide-2-methyl propane sulfonic acid and phosphoric acid Na/K, (4) methacryloyl dimethyl aminoethyl Methochloride salt and ammonium sulfate, (5) acrylamide and sodium malonate, (6) polyethylene oxide mono acrylic ester and PEG6k.
Protein crystallizing agent of the present invention is prepared the aqueous solution thermopolymerization that contains precipitation agent, damping fluid, unsaturated monomer formation at least.Perhaps can be by in the presence of heat and/or optical free radical polymerization starter, making its polymerization, gelation, precipitation agent is remained in the gel equably and prepare.It is suitable carrying out polymerization in the presence of radical polymerization initiator.As the radical polymerization initiator that in the aqueous solution, is fit to use, can enumerate for example superoxide such as tertbutyl peroxide, hydrogen peroxide, ammonium persulphate, Potassium Persulphate; 2, two (2-amidine propane) 2 hydrochlorides, 2 of 2-azo, two (2-amidino groups butane) 2 hydrochlorides, 2 of 2-azo, azos such as two [2-(2-tetrahydroglyoxaline-2-yl) propane] 2 hydrochlorides of 2-azo are polymerization starter.These radical polymerization initiators can be separately or are used as two kinds or mixture more than it.Also can use the reducto oxydative system polymerization starter that in above-mentioned superoxide, has made up reductive agents such as tertiary amine, sulphite, ferrous salt in addition, and then also can use reducto oxydative system polymerization starter and azo is the polymerization starter of polymerization starter combination and usefulness.
Providing under the light source of specific wavelength in addition, using the optical free radical polymerization starter, also can carry out polymerization.At this moment, so long as employed optical free radical polymerization starter decomposes by the rayed of particular range of wavelengths, produce the initiator of free radical, just there is no particular limitation.As the initiator that is fit to utilize; can enumerate the initiator that uses in for example normal light polymerizations such as acylphosphine oxide, bitter almond oil camphor, benzoin alkylether, benzil, benzophenone, anthraquinone; other is as 2; two (the 2-methyl-prop amidine) hydrochlorides, 4 of 2 '-azo; 4 '-azo two (the lucky oxalic acid of cyano group) sodium salt, 2, the two azos such as [2-methyl-N-(2-hydroxyethyl) propionic acid amides] of 2-azo are polymerization starter.Can from these initiators, select a kind of or initiator use arbitrarily more than it according to the wavelength that utilizes.In addition, above-mentioned so-called specific wavelength is if this 2 point of photon energy that photoabsorption, free radical that the monomer of considering to contain in the reaction solution itself causes utilize in generating wishes to use the light in wavelength 200～650nm zone.As the typical example of the light-struck light source that can be used for wavelength 200～650nm zone, can enumerate high pressure mercury vapour lamp, Cooper-Hewitt lamp, metal halide lamp, fluorescence chemical lamp, fluorescent blue colored lights etc.
Embodiment
(making of tubular fibre arrange body)
As the constitutive requirements of supporting the crystallizing agent maintaining part, make the tubular fibre arrange body.
2 blocks of porous plates are overlapping, and described porous plate has been arranged has the hole that diameter is 0.32mm, and the width between centers in hole is 0.42mm, and each 10 row adds up to 100 holes in length and breadth, and thickness is 0.1mm.With polyethylene system tubular fibre (Li Yang company of Mitsubishi produce, the about 500 μ m of external diameter, the about 300 μ m of internal diameter are about 100 μ m) 100 pieces each holes by these porous plates.In that tubular fibre is fixed in the position of the 10cm that begins in the end from tubular fibre one side of state of tension and two places, position of 40cm, 30mm is made at the interval of 2 blocks of porous plates then.
Then, the plate object of spatial 3 directions between 2 blocks of porous plates with silicon system fenced up.From an open side, as resin raw material, the resin that will constitute by the urethane resin tackiness agent and be that the sooty mixture of 2.5 quality % flows into 2 blocks of porous plates with respect to the total mass of resin raw material.Next under room temperature, left standstill for 1 week, make hardening of resin.Remove the plate object that is arranged between porous plate and the porous plate then, obtain the tubular fibre arrange body.
(high-molecular gel is to the importing immobilization of tubular fibre arrange body)
Preparation is by the following mixing solutions that constitutes of forming.
Acrylamide: 3.7 mass parts
Methylene diacrylamide: 0.3 mass parts
2, two (2-amidine propane) dihydrochlorides of 2 '-azo: 0.1 mass parts
Above-mentioned mixing solutions and the above-mentioned tubular fibre arrange body that obtains are put into moisture eliminator, a long side's end is impregnated under the state in this mixed solution in the free end of each tubular fibre, by moisture eliminator is made decompression state, in the hollow bulb of tubular fibre, import above-mentioned mixed solution.Then this tubular fibre arrange body is moved on to inside by in the steam-laden airtight Glass Containers, under 80 ℃, carry out 4 hours polyreactions.Obtain the tubular fibre arrange body that acrylamide gel is fixed on the hollow bulb of tubular fibre thus.
(making of gel filled tubular fibre arrange body thin slice)
The above-mentioned tubular fibre arrange body that contains acrylamide gel that obtains is being cut into the thickness of about 2mm with the vertical vertical direction of tubular fibre with the thin slice slicing machine, and the hollow bulb that obtains tubular fibre is filled with each 10 in length and breadth of gel, amount to that 100 tubular fibre is regular to be arranged as dimetric arrange body thin slice.Fig. 2 represents the tubular fibre arrange body thin slice that contains gel that obtains by above-mentioned operation.Just as shown in Figure 2, in the hollow bulb 14 of tubular fibre 12, be filled with the acrylamide gel of above-mentioned making.
(making of used for protein crystalling microarray)
In the tubular fibre arrange body thin slice that contains gel of above-mentioned making, in the acrylamide gel of the hollow bulb 14 that is filled into tubular fibre 12, add to respectively in the gel that remains on hollow bulb by the protein crystallizing agent 1 μ l that the aqueous solution by following A, B, C, D, E, F, G, H, I, J is constituted, protein crystallizing agent is remained in the acrylamide gel, make used for protein crystalling microarray 18.
The A:2.0mol/L sodium chloride aqueous solution
The B:0.5mol/L sodium chloride aqueous solution
C:10 volume % polyoxyethylene glycol (molecular weight 400) aqueous solution
D:20 volume % polyoxyethylene glycol (molecular weight 400) aqueous solution
E:10 volume % polyoxyethylene glycol (molecular weight 6000) aqueous solution
F:20 volume % polyoxyethylene glycol (molecular weight 6000) aqueous solution
G:20 volume %2-methyl-2, the 4-pentanediol aqueous solution
H:20%2-methyl-2, the 4-pentanediol aqueous solution
The I:0.5mol/L ammonium sulfate solution
The J:1.5mol/L ammonium sulfate solution
Below, will keep the crystallizing agent maintaining part 16 of the protein crystallizing agent of above-mentioned A～J to be referred to as an A～J respectively.
The screening of protein crystallization condition
Use protein crystallization apparatus shown in Figure 1, carry out the screening of the crystallization condition of N,O-Diacetylmuramidase (production of Sigma Aldrich company).Use the used for protein crystalling microarray 18 of above-mentioned making as the used for protein crystalling microarray, use acrylic resin as the material of support 20, plate 24.
(interpolation of proteinaceous sample)
Use sample shown in Figure 6 to fill auxiliary means 54, fill proteinaceous sample to crystallizing field 32.Sample is filled auxiliary means 54 and is used the auxiliary means with the perforation 56 of arranging corresponding to the crystallizing agent maintaining part 16 of the above-mentioned used for protein crystalling microarray 18 that obtains.
As proteinaceous sample, use the aqueous solution (80mg/ml) of lysozyme.
At first sample is filled auxiliary means 54 and be arranged on like that on the plate 24 according to Fig. 6, the automatic pipette of usefulness 20 μ l is added above proteinaceous sample 1.5～2 μ l are spreaded all over perforation 56 whole according to sample like that on it.Fill the top of auxiliary means 54 by sample then and pass through proteinaceous sample is pressed on plate 24, proteinaceous sample is filled in the crystallizing field all on the plate 24 32 with scraper plate.
(assembling of protein crystallization apparatus, the screening of protein crystallization condition)
Then, protein crystallization apparatus was left standstill under 20 ℃ 3 hours.Next by in the observation by light microscope crystallizing field 32, the result has kept the some B of the polyoxyethylene glycol aqueous solution not see the crystallization of N,O-Diacetylmuramidase making as protein crystallizing agent, and sees at the some A that has kept the 2.0mol/L sodium chloride aqueous solution and to be suitable for the column crystallization that x-ray structure is resolved most.
(structure elucidation is made of crystalline)
And then, according to above-mentioned result, use the 2.0mol/L sodium chloride aqueous solution as protein crystallizing agent, make antalzyme crystallization from the sample solution of lysozyme by sessile drop method.The size of the antalzyme crystallization that obtains at this moment, is 0.3 * 0.3 * 0.5mm.
By above result the kind and the concentration of protein crystallizing agent in the antalzyme crystallization condition are studied, the result shows that the 2.0mol/L sodium chloride aqueous solution is the suitableeest crystallization condition, and uses the suitableeest crystallization condition that filters out can obtain being suitable for the crystallization that x-ray structure is resolved.
That is proteinaceous sample that, can be by trace rapidly and also economy, high reliability ground implement the screening of protein crystallization condition.
Damping fluid in the gel used for protein crystalling of the present invention uses following damping fluid, is prepared by ordinary method.
0.1M-citrate buffer solution (pH4.0), 0.1M-citrate buffer solution (pH5.0), 0.1M-MES (pH6.0), 0.1M-HEPES (pH7.0), 0.1M-Tris (pH8.0), 0.1M-Bicine (pH9.0).
＜embodiment 1〉weighing is put in the 10ml volumetric flask as the sodium-chlor 0.48g of precipitation agent, with 0.1M-citrate buffer solution (pH4.0) constant volume, the preparation 1.2M-NaCl aqueous solution (this solution is decided to be a liquid).In addition, in 50% acrylamide solution (production of Li Yang company of Mitsubishi) 90g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 5g, deionized water 5g dissolves, and prepares 50% monomer solution (this solution is decided to be b liquid).And then preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
The N,O-Diacetylmuramidase aqueous solution 1ml that adds 40mg/ml in the sample hose of the hydrogel that forms according to above-mentioned steps in 20 ℃ of following incubations 24 hours, separates out antalzyme crystallization in the N,O-Diacetylmuramidase aqueous solution.And the crystallization generation all can be confirmed by naked eyes and stereomicroscope.
＜embodiment 2〉except the concentration with a liquid among the embodiment 1 changed to 2.4M, other all prepared the crystallizing agent aqueous solution (2.0M-NaCl, pH4.0, monomer concentration 8%) according to same step, obtained transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 3〉except the concentration with a liquid among the embodiment 1 changed to 3.6M, other all prepared the crystallizing agent aqueous solution (3.0M-NaCl, pH4.0, monomer concentration 8%) according to same step, obtained transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 4〉except the concentration with a liquid among the embodiment 1 changed to 4.8M, other all prepared the crystallizing agent aqueous solution (4.0M-NaCl, pH4.0, monomer concentration 8%) according to same step, obtained transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 5～8〉except damping fluid among the embodiment 1～4 being changed to 0.1M-citrate buffer solution (pH5.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 9～12〉except damping fluid among the embodiment 1～4 being changed to 0.1M-MES damping fluid (pH6.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 13～16〉except damping fluid among the embodiment 1～4 being changed to 0.1M-HEPES damping fluid (pH7.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 17～20〉except damping fluid among the embodiment 1～4 being changed to 0.1M-Tris damping fluid (pH8.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 21～24〉except damping fluid among the embodiment 1～4 being changed to 0.1M-Bicine damping fluid (pH9.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 25〉weighing as the polyoxyethylene glycol of precipitation agent (with the pure pharmaceutical worker of light manufacturing already, PEG6000, weight-average molecular weight: 6000) 0.55g is put in the 10ml volumetric flask, with 0.1M-citrate buffer solution (pH4.0) constant volume, the preparation 5.5%-PEG aqueous solution (this solution is decided to be d liquid).In addition, the monomer solution (this solution is decided to be e liquid) of polyethyleneglycol diacrylate (Xin Zhong village chemistry system NKESTERA-200) has been dissolved in preparation in polyoxyethylene glycol mono acrylic ester (Japanese grease system Block レ Application マ one AE90) 95g.And then preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 920 μ l, b liquid 80 μ l, the preparation protein crystallizing agent aqueous solution (5%-PEG, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.Below confirm the generation of antalzyme crystallization with method similarly to Example 1.The result is shown in table-1.
＜embodiment 26〉except the concentration with d liquid among the embodiment 25 changes to 11%, other all prepares the crystallizing agent aqueous solution (10%-PEG, pH4.0, monomer concentration 8%) according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 27〉except will the concentration of d liquid changing to 22% in embodiment 25, other all prepares the crystallizing agent aqueous solution (20%-PEG, pH4.0, monomer concentration 8%) according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 28〉except the concentration with d liquid among the embodiment 25 changes to 33%, other all prepares the crystallizing agent aqueous solution (30%-PEG, pH4.0, monomer concentration 8%) according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 29～32〉except damping fluid among the embodiment 25～28 being changed to 0.1M-citrate buffer solution (pH5.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 33～36〉except damping fluid among the embodiment 25～28 being changed to 0.1M-MES damping fluid (pH6.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 37～40〉except damping fluid among the embodiment 25～28 being changed to 0.1M-HEPES damping fluid (pH7.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 41～44〉except damping fluid among the embodiment 25～28 being changed to 0.1M-Tris damping fluid (pH8.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
＜embodiment 45～48〉except damping fluid among the embodiment 25～28 being changed to 0.1M-Bicine damping fluid (pH9.0), other all prepares the crystallizing agent aqueous solution according to same step, obtains transparent hydrogel according to same step again.Use the same method in addition and confirm the generation of antalzyme crystallization.The result is shown in table-1.
Table-1
| Precipitation agent | Monomer | PH | Gel character | The crystallization of protein state | |
| Embodiment 1 | ?1.0M-NaCl | Acrylamide | 4.0 | Clear gel | Crystallization |
| Embodiment 2 | ?2.0M-NaCl | Acrylamide | 4.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 3 | ?3.0M-NaCl | Acrylamide | 4.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 4 | ?4.0M-NaCl | Acrylamide | 4.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 5 | ?1.0M-NaCl | Acrylamide | 5.0 | Clear gel | Crystallization |
| Embodiment 6 | ?2.0M-NaCl | Acrylamide | 5.0 | Clear gel | Crystallization |
| Embodiment 7 | ?3.0M-NaCl | Acrylamide | 5.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 8 | ?4.0M-NaCl | Acrylamide | 5.0 | Clear gel | Precipitation |
| Embodiment 9 | ?1.0M-NaCl | Acrylamide | 6.0 | Clear gel | Crystallization |
| Embodiment 10 | ?2.0M-NaCl | Acrylamide | 6.0 | Clear gel | Crystallization |
| Embodiment 11 | ?3.0M-NaCl | Acrylamide | 6.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 12 | ?4.0M-NaCl | Acrylamide | 6.0 | Clear gel | Precipitation |
| Embodiment 13 | ?1.0M-NaCl | Acrylamide | 7.0 | Clear gel | Crystallization |
| Embodiment 14 | ?2.0M-NaCl | Acrylamide | 7.0 | Clear gel | Crystallization |
| Embodiment 15 | ?3.0M-NaCl | Acrylamide | 7.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 16 | ?4.0M-NaCl | Acrylamide | 7.0 | Clear gel | Precipitation |
| Embodiment 17 | ?1.0M-NaCl | Acrylamide | 8.0 | Clear gel | Crystallization |
| Embodiment 18 | ?2.0M-NaCl | Acrylamide | 8.0 | Clear gel | Crystallization |
| Embodiment 19 | ?3.0M-NaCl | Acrylamide | 8.0 | Clear gel | Crystallization |
| Embodiment 20 | ?4.0M-NaCl | Acrylamide | 8.0 | Clear gel | Crystallization |
| Embodiment 21 | ?1.0M-NaCl | Acrylamide | 9.0 | Clear gel | Crystallization |
| Embodiment 22 | ?2.0M-NaCl | Acrylamide | 9.0 | Clear gel | Crystallization |
| Embodiment 23 | ?3.0M-NaCl | Acrylamide | 9.0 | Clear gel | Precipitation |
| Embodiment 24 | ?4.0M-NaCl | Acrylamide | 9.0 | Clear gel | Precipitation |
| Embodiment 25 | ?5％-PEG | The polyoxyethylene glycol mono acrylic ester | 4.0 | Clear gel | No resultant |
| Embodiment 26 | ?10％-PEG | The polyoxyethylene glycol mono acrylic ester | 4.0 | Clear gel | No resultant |
| Embodiment 27 | ?20％-PEG | The polyoxyethylene glycol mono acrylic ester | 4.0 | Clear gel | Crystallization |
| Embodiment 28 | ?30％-PEG | The polyoxyethylene glycol mono acrylic ester | 4.0 | Clear gel | Crystallization |
| Embodiment 29 | ?5％-PEG | The polyoxyethylene glycol mono acrylic ester | 5.0 | Clear gel | Crystallization |
| Embodiment 30 | ?10％-PEG | The polyoxyethylene glycol mono acrylic ester | 5.0 | Clear gel | Crystallization |
| Embodiment 31 | ?20％-PEG | The polyoxyethylene glycol mono acrylic ester | 5.0 | Clear gel | Crystallization |
| Embodiment 32 | ?30％-PEG | The polyoxyethylene glycol mono acrylic ester | 5.0 | Clear gel | Crystallization, precipitation mixture |
| Embodiment 33 | ?5％-PEG | The polyoxyethylene glycol mono acrylic ester | 6.0 | Clear gel | No resultant |
| Embodiment 34 | ?10％-PEG | The polyoxyethylene glycol mono acrylic ester | 6.0 | Clear gel | No resultant |
| Embodiment 35 | ?20％-PEG | The polyoxyethylene glycol mono acrylic ester | 6.0 | Clear gel | No resultant |
| Embodiment 36 | ?30％-PEG | The polyoxyethylene glycol mono acrylic ester | 6.0 | Clear gel | No resultant |
| Embodiment 37 | ?5％-PEG | The polyoxyethylene glycol mono acrylic ester | 7.0 | Clear gel | No resultant |
| Embodiment 38 | ?10％-PEG | The polyoxyethylene glycol mono acrylic ester | 7.0 | Clear gel | No resultant |
| Embodiment 39 | ?20％-PEG | The polyoxyethylene glycol mono acrylic ester | 7.0 | Clear gel | No resultant |
| Embodiment 40 | ?30％-PEG | The polyoxyethylene glycol mono acrylic ester | 7.0 | Clear gel | No resultant |
| Embodiment 41 | ?5％-PEG | The polyoxyethylene glycol mono acrylic ester | 8.0 | Clear gel | No resultant |
| Embodiment 42 | ?10％-PEG | The polyoxyethylene glycol mono acrylic ester | 8.0 | Clear gel | No resultant |
| Embodiment 43 | ?20％-PEG | The polyoxyethylene glycol mono acrylic ester | 8.0 | Clear gel | No resultant |
| Embodiment 44 | ?30％-PEG | The polyoxyethylene glycol mono acrylic ester | 8.0 | Clear gel | No resultant |
| Embodiment 45 | ?5％-PEG | The polyoxyethylene glycol mono acrylic ester | 9.0 | Clear gel | No resultant |
| Embodiment 46 | ?10％-PEG | The polyoxyethylene glycol mono acrylic ester | 9.0 | Clear gel | No resultant |
| Embodiment 47 | ?20％-PEG | The polyoxyethylene glycol mono acrylic ester | 9.0 | Clear gel | No resultant |
| Embodiment 48 | ?30％-PEG | The polyoxyethylene glycol mono acrylic ester | 9.0 | Clear gel | No resultant |
＜embodiment 49〉weighing is put in the 10ml volumetric flask as the sodium-chlor 0.48g of precipitation agent, with 0.1M-citrate buffer solution (pH4.0) constant volume, the preparation 1.2M-NaCl aqueous solution (this solution is decided to be a liquid).
In addition, in 2-acrylamide-2-methyl propane sulfonic acid 47.5g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 2.5g, deionized water 50g dissolves, and prepares 50% monomer solution (this solution is decided to be b liquid).And then preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
＜embodiment 50〉weighing is put in the 10ml volumetric flask as the sodium-chlor 0.48g of precipitation agent, with 0.1M-citrate buffer solution (pH4.0) constant volume, the preparation 1.2M-NaCl aqueous solution (this solution is decided to be a liquid).
In addition, in 80% methacryloyl dimethyl aminoethyl Methochloride salt brine solution 62.5g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 2.5g, deionized water 35g, dissolve, prepare 50% monomer solution (this solution is decided to be b liquid).In addition, preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
＜embodiment 51〉weighing is as the 2-methyl-2 of precipitation agent, and 4-pentanediol 2g is put in the 10ml volumetric flask, with 0.1M-citrate buffer solution (pH4.0) constant volume, preparation 20%2-methyl-2, the 4-pentanediol aqueous solution (this solution is decided to be a liquid).
In addition, in DMAA 47.5g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 2.5g, deionized water 50g dissolves, and prepares 50% monomer solution (this solution is decided to be b liquid).In addition, preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
＜embodiment 52〉weighing is put in the 10ml volumetric flask as sodium phosphate salt 1.176g, the potassium phosphate salt 0.035g of precipitation agent, with 0.1M-citrate buffer solution (pH4.0) constant volume, preparation 1.0M-sodium phosphate/aqueous solutions of potassium (this solution is decided to be a liquid).
In addition, in 2-acrylamide-2-methyl propane sulfonic acid 47.5g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 2.5g, deionized water 50g dissolves, and prepares 50% monomer solution (this solution is decided to be b liquid).Refabrication is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
＜embodiment 53〉weighing is put in the 10ml volumetric flask as the ammonium sulfate 3.17g of precipitation agent, with 0.1M-citrate buffer solution (pH4.0) constant volume, preparation 2.4M-ammonium sulfate solution (this solution is decided to be a liquid).
In addition, in 80% methacryloyl dimethyl aminoethyl Methochloride salt brine solution 62.5g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 2.5g, deionized water 35g, dissolve, prepare 50% monomer solution (this solution is decided to be b liquid).In addition, preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
＜embodiment 54〉weighing is put in the 10ml volumetric flask as the propanedioic acid 1.04g of precipitation agent, after adding 20%-aqueous sodium hydroxide solution 4g and neutralizing, with 0.1M-citrate buffer solution (pH4.0) constant volume, the preparation 1.0M-sodium malonate aqueous solution (this solution is decided to be a liquid).
In addition, in 50% acrylamide solution (the beautiful positive corporate system of Mitsubishi) 90g, add N, N '-methylene diacrylamide (below be abbreviated as MBAAm) 5g, deionized water 5g dissolves, and prepares 50% monomer solution (this solution is decided to be b liquid).In addition, preparation is as 2 of water-soluble polymerization initiator, 10% aqueous solution (this solution is decided to be c liquid) of two [2-(2-tetrahydroglyoxaline-2-yl) propane] dihydrochlorides of 2 '-azo (already make with the pure pharmaceutical worker of light, VA-044).Mix after in the sample hose of 2ml, injecting a liquid 840 μ l, b liquid 160 μ l, the preparation protein crystallizing agent aqueous solution (1.0M-NaCl, pH4.0, monomer concentration 8%), add thorough mixing behind the c liquid 10 μ l again, under 55 ℃ of temperature are bathed, carry out polymerization in 3 hours then, obtain transparent hydrogel.
Usability on the industry
By protein crystallization apparatus of the present invention, can be rapidly and also economy, high reliability ground implement the crystallization experiment of protein or the screening of crystallization condition. In crystallization of protein, can finish gel reaction and form the gelling material that does not produce gonorrhoea by gel used for protein crystalling of the present invention in addition, the transparent gelling material that has kept protein crystallizing agent can be provided.
Claims (13)
1. a protein crystallization apparatus is characterized in that: have
Used for protein crystalling microarray, this microarray have the crystallizing agent maintaining part more than 2 or 2 that keeps protein crystallizing agent; With
With the stacked plate of above-mentioned used for protein crystalling microarray, above-mentioned plate has corresponding to the crystallizing field of filling proteinaceous sample of above-mentioned crystallizing agent maintaining part and is arranged on recess between this crystallizing field.
2. the described protein crystallization apparatus of claim 1, it is characterized in that: above-mentioned crystallizing agent maintaining part is made of the gel that is prepared into various different proteins crystallization conditions.
3. the described protein crystallization apparatus of claim 1 is characterized in that: above-mentioned used for protein crystalling microarray is that a plurality of hollow tubular bodies are arranged the microarray that constitutes.
4. the described protein crystallization apparatus of claim 1 is characterized in that: also have the mechanism that above-mentioned used for protein crystalling microarray and above-mentioned plate are carried out crimping.
5. the described protein crystallization apparatus of claim 1 is characterized in that: be filled with sealing agent at above-mentioned recess.
6. the described protein crystallization apparatus of claim 1 is characterized in that: the off-capacity 0.5 μ l of above-mentioned crystallizing field.
7. the described protein crystallization apparatus of claim 1 is characterized in that: the capacity of above-mentioned crystallizing field 0.5 μ l or more than.
8. the described protein crystallization apparatus of claim 1, it is characterized in that: above-mentioned used for protein crystalling microarray has 10～1000 crystallizing agent maintaining parts.
9. the described protein crystallization apparatus of claim 1 is characterized in that: above-mentioned plate also has the crystallization recovering mechanism that crystallization that above-mentioned crystallizing field is separated out is reclaimed.
10. the described protein crystallization apparatus of claim 1 is characterized in that: also have the feeler mechanism that the proteinic crystallization in the above-mentioned crystallizing field is monitored.
11. a sample is filled auxiliary means, be that the sample that is used for filling to the above-mentioned crystallizing field of the protein crystallization apparatus of claim 1 to 10 described in each proteinaceous sample is filled auxiliary means, it is characterized in that: have the perforation of arranging and make this perforation contraposition mechanism corresponding on above-mentioned plate with above-mentioned crystallizing field corresponding to above-mentioned crystallizing field.
12. each described protein crystallization apparatus of claim 1 to 10 is characterized in that: on above-mentioned plate, be formed with the location division that makes this plate and the contraposition of the described sample filling of claim 11 auxiliary means.
13. the sieve method of a protein crystallization condition, be to use the described sample of described protein crystallization apparatus of claim 12 and claim 11 to fill the protein crystallization condition sieve method of auxiliary means, it is characterized in that: the above-mentioned perforation that the described sample filling of claim 11 auxiliary means is filled auxiliary means according to this sample is arranged on the above-mentioned plate like that corresponding to above-mentioned crystallizing field, above this sample filling auxiliary means, to above-mentioned perforation, add proteinaceous sample, unload above-mentioned sample after being filled in the above-mentioned crystallizing field and fill auxiliary means, according to making above-mentioned crystallizing field and above-mentioned crystallizing agent maintaining part correspondence, contact like that above-mentioned plate and above-mentioned used for protein crystalling microarray is stacked.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP291308/2003 | 2003-08-11 | ||
| JP2003291308A JP4323258B2 (en) | 2003-08-11 | 2003-08-11 | Protein crystallizing agent and method for preparing the same |
| JP291433/2003 | 2003-08-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200710092292 Division CN101029078A (en) | 2003-08-11 | 2004-08-10 | Gel used for protein crystalling |
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| CN1863946A CN1863946A (en) | 2006-11-15 |
| CN100355949C true CN100355949C (en) | 2007-12-19 |
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| CN 200710092292 Pending CN101029078A (en) | 2003-08-11 | 2004-08-10 | Gel used for protein crystalling |
| CNB2004800295182A Expired - Fee Related CN100355949C (en) | 2003-08-11 | 2004-08-10 | Protein crystallization apparatus, method of protein crystallization, protein crystallizing agent and process for preparing the same |
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| CN 200710092292 Pending CN101029078A (en) | 2003-08-11 | 2004-08-10 | Gel used for protein crystalling |
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| JP (1) | JP4323258B2 (en) |
| CN (2) | CN101029078A (en) |
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| CN102675412B (en) * | 2011-03-16 | 2014-09-24 | 中国科学院化学研究所 | Method for preparing protein crystal |
| JP6848173B2 (en) * | 2015-10-09 | 2021-03-24 | 東レ株式会社 | Fiber-containing crystals, method for producing fiber-containing crystals, equipment for producing fiber-containing crystals, and chemical soaking equipment |
| CN105810072A (en) * | 2016-03-25 | 2016-07-27 | 淮海工学院 | Protein crystallization teaching kit and method thereof |
| CN107099846B (en) * | 2017-05-25 | 2019-05-10 | 西北工业大学 | A kind of pre- cloth is micro-, nanoscale seed crystal protein crystallization board preparation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07500806A (en) * | 1991-10-09 | 1995-01-26 | シェリング・コーポレーション | Crystal forming equipment and automated crystallization equipment |
| JP2001136972A (en) * | 1999-11-15 | 2001-05-22 | Mitsubishi Rayon Co Ltd | Nucleic acid-fixed polymer gel and production method |
| WO2003053998A1 (en) * | 2001-12-11 | 2003-07-03 | Mitsubishi Rayon Co., Ltd. | Array for crystallizing protein, device for crystallizing protein and method of screening protein crystallization using the same |
-
2003
- 2003-08-11 JP JP2003291308A patent/JP4323258B2/en not_active Expired - Fee Related
-
2004
- 2004-08-10 CN CN 200710092292 patent/CN101029078A/en active Pending
- 2004-08-10 CN CNB2004800295182A patent/CN100355949C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07500806A (en) * | 1991-10-09 | 1995-01-26 | シェリング・コーポレーション | Crystal forming equipment and automated crystallization equipment |
| JP2001136972A (en) * | 1999-11-15 | 2001-05-22 | Mitsubishi Rayon Co Ltd | Nucleic acid-fixed polymer gel and production method |
| WO2003053998A1 (en) * | 2001-12-11 | 2003-07-03 | Mitsubishi Rayon Co., Ltd. | Array for crystallizing protein, device for crystallizing protein and method of screening protein crystallization using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1863946A (en) | 2006-11-15 |
| JP4323258B2 (en) | 2009-09-02 |
| JP2005060284A (en) | 2005-03-10 |
| CN101029078A (en) | 2007-09-05 |
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