CN102887939A - Method for promoting protein crystallization by use of bioglass - Google Patents
Method for promoting protein crystallization by use of bioglass Download PDFInfo
- Publication number
- CN102887939A CN102887939A CN2011102019244A CN201110201924A CN102887939A CN 102887939 A CN102887939 A CN 102887939A CN 2011102019244 A CN2011102019244 A CN 2011102019244A CN 201110201924 A CN201110201924 A CN 201110201924A CN 102887939 A CN102887939 A CN 102887939A
- Authority
- CN
- China
- Prior art keywords
- vitric
- bio
- protein
- crystallization
- storage hole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Crystals, And After-Treatments Of Crystals (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for prompting protein crystallization by the use of bioglass. According to the method, superfine bioglass particles are mixed with a liquid for increasing surface activity of bioglass to form a suspension with long service life and stable properties; and the suspension is mixed with a protein saturated solution so as to form an environment which is beneficial to crystallization. After bioglass is made into the suspension, bioglass can be dripped by the use of a dropper to a place where the bioglass is required. Therefore, an application method of bioglass is greatly improved, and automation can replace manual work. The method is more suitable for large-scale protein crystallization condition screening. As the suspension, bioglass can be fully mixed with a protein solution when in use, and the contact surface with protein is larger, which is beneficial to crystallization under various conditions.
Description
Technical field
The present invention relates to the crystallization field of protein, be specifically related to a kind of method of utilizing bio-vitric to promote crystallization of protein.
Background technology
Along with the development of biotechnology, the functional performance of the uniqueness of protein and other more and more is subject to people's attention, and wherein, the pharmaceutical characteristic of numerous protein constantly is found.As everyone knows, the functional performance of molecule depends primarily on the three-D space structure of its uniqueness, therefore will understand, determine the function of a certain biomacromolecule, just must measure and study its three-dimensional structure; In addition, understand the structure of biomacromolecule for medicinal design, change or decorating molecule structure and make it to have specific application characteristic and all have very important significance.
The method of protein structure determination mainly contains three kinds at present: X ray diffraction technology, nuclear magnetic resonance technique and Electron Microscopy.According to protein structure database (Protein Data Bank, PDB) data that provide, by on April 16th, 2009, existing 52,686 kinds of protein structures are resolved and are submitted in the PDB database, wherein the structure more than 85% (45,620) obtains by X ray diffraction technology.Therefore, X ray diffraction technology is topmost and the most reliable protein structure analytic method of present stage.
Carry out the structure elucidation of protein, just must obtain first to be suitable for the crystal of X-ray diffraction structural analysis, and the crystal that how to obtain high-quality suitable diffraction always is the subject matter of puzzlement structure biology man.Scientist from Imperial College London and Surrey Roehampton (University of Surrey) forms crystal for induced protein, used a kind of material that is called the nuclei of crystallization, it can promote protein molecule to form a kind of lattice, and its theoretical basis is can bottle up protein molecule and impel their crystallizations of the vesicular structure of material; The researchist uses a kind of bio-vitric (BioGlass) of being developed by the material expert of Transactions as bottling up and promoting the scaffolding of protein crystal growth, described BioGlass is a kind of porous material, the hole that has different size on it, the protein of the different sizes of therefore bottling up; The researchist finds with bio-vitric as the nuclei of crystallization, makes the protein can easier crystallization, and BioGlass can induce the protein crystal that utilizes nuclei of crystallization to form that is formed into present maximum.Bio-vitric is hopeful can impel the crystallization of protein of all kinds as a kind of general nuclei of crystallization most.
American documentation literature US7252713 B2(application number US20050534088, open day on July 13rd, 2006) a kind of method (Mesoporous glass as nucleant for macromolecule crystallisation) that mesoporous glass is made to macromolecule crystallization is disclosed, wherein open " adding a mesoporous glass as nucleant to a crystallization sample wherein the mesoporous glass comprises pores having diameters between 4 nm and 100nm and has a surface area of at least 50m
2/ g(adds mesoporous glass as nucleus in crystalline sample, the hole diameter of wherein said mesoporous glass is between 4nm to 100nm, and surface area is 50m at least
2/ g.）”
Technique scheme directly drops into glass and promotes crystallization of protein in the protein soln when using mesoporous glass (also being above-mentioned bio-vitric); During actual use bio-vitric, be to live one with the pincet tweezer, put into protein soln, leave standstill for some time, the observing protein crystalline condition; Because bioglass particles is very little, therefore with tweezers tweezer bio-vitric, experimentation is wanted extreme care, is not very convenient; Bio-vitric is settled down to container bottom after adding solution in addition, and the hole of its surface part has just lost effect; And in order to cooperate the mode of tweezer, the particle of bio-vitric is larger, and the hole of bio-vitric inside has also lost effect like this.
Summary of the invention
Technical problem to be solved by this invention provides and a kind ofly implements conveniently, efficient is higher promotes the method for crystallization of protein with bio-vitric.
The technical scheme that realizes the object of the invention is a kind of method of impelling crystallization of protein with bio-vitric, first that bioglass particles is even with the surface-active liquid mixing of increase bio-vitric, carry out sonic oscillation again and obtain the bio-vitric suspension liquid, then with dropper the bio-vitric suspension liquid is transferred to the crystallization of protein environment, thereby the bio-vitric suspension liquid promotes crystallization of protein; The granularity 100nm of described bioglass particles～200nm, mean pore size 90nm～200nm, the concentration of bioglass particles is 1～100/μ L in the suspension liquid.
The concentration of bioglass particles is preferably 2～50/μ L in the above-mentioned bio-vitric suspension liquid.
The surface-active liquid of above-mentioned increase bio-vitric is mixing solutions, and wherein solvent is deionized water, and solute comprises that concentration is the chloroform of 15～25mg/mL, pore-creating agent polyoxyethylene glycol and the 3～8mg/mL promoting agent sulfated oil of 1～3mg/mL.
The surface-active liquid of above-mentioned increase bio-vitric is deionized water.
Above-mentioned crystallization of protein environment is the crystalline environment in microfluidic protein crystallization board; Described microfluidic protein crystallization board is the integrated through injection molding part, the crystalline element that comprises supporting structure section and in supporting structure section, distribute with array format, each crystalline element is comprised of the liquid storage hole section at two ends and the up and down connective slot of connection two ends liquid storage hole section, the intracavity section of described liquid storage hole section is long-pending to be reduced from the top down gradually, and the upper connective slot of connection groove section is up big and down small.
During crystallization of protein, the protein supersaturated solution is splashed in the liquid storage hole of one of them liquid storage hole section of crystalline element of crystallization plates, then in the liquid storage hole of another liquid storage hole section of crystalline element, splash into precipitant solution and bio-vitric suspension liquid, thereby the bio-vitric suspension liquid enters crystalline environment, also namely the material in 2 liquid storage hole sections by lower connective slot so that protein supersaturated solution and the mutual diffusion of bio-vitric suspension liquid phase, and the lower port place of the notch position of time connective slot and liquid storage hole is provided with for the restraining mass that seals; At the interface bio-vitric suspension liquid at both sides solution promotes crystallization of protein at last.
Bio-vitric in the above-mentioned bio-vitric suspension is made by sol-gel method.
The step that is prepared bio-vitric by sol-gel method is as follows: be P according to being converted into oxide mol ratio first
2O
5: CaO:MgO:SiO
2=1:(3～3.5): (1～1.2): the ratio of (4.5～5.5) takes by weighing respectively the P of corresponding amount
2O
5, Ca (NO
3)
2, Mg (NO3)
2, tetraethoxy, each compound is mixed respectively and reflux and being dissolved in the ethanol with dehydrated alcohol, make respectively uniform P
2O
5Ethanolic soln, Ca (NO
3)
2Ethanolic soln, Mg (NO3)
2Ethanolic soln and the ethanolic soln of tetraethoxy, the mol ratio of ethanol and each material is respectively (4～10): 1;
In the ethanolic soln of tetraethoxy, add deionized water, made tetraethoxy prehydrolysis 40～50 minutes;
With P
2O
5Ethanolic soln, Ca (NO
3)
2Ethanolic soln, Mg (NO3)
2Ethanolic soln drop to successively in the ethanolic soln of above-mentioned tetraethoxy, during dropping, front a kind of ethanolic soln dropwise 1～1.5 as a child dripped again after a kind of ethanolic soln, dropwise post-heating and vigorous stirring and obtain treating the mixed solution of solation;
Add deionized water in above-mentioned mixed solution, with nitric acid regulator solution pH=2～2.5, and so that the solution solation, the colloidal sol that makes makes wet gel through ageing, gelation; Be placed on after wet gel and urea mixed in 500 ℃～550 ℃ the stove and light a fire, after burning finished, quenching obtained soft-agglomerated powder, fully grinds in agate mortar, and obtains the super fine biological glass particle, and grain graininess is less than 200nm.
The present invention has positive effect: (1) the present invention can drop to bio-vitric in the protein soln with dropper after bio-vitric is made suspension liquid, and because the bio-vitric suspension liquid of configuration is in stable condition within a certain period of time.Therefore, the suspension liquid volume that control is drawn then can be controlled the quantity of the bio-vitric that drips in the protein soln; Greatly improve the using method of bio-vitric, and can substitute manually with automation, be more applicable for the screening of large-scale protein matter crystallization condition.
(2) bio-vitric in the suspension liquid is uniformly dispersed, and can fully mix with protein soln and be dispersed in the protein soln during use, and is larger with the protein contact surface, is conducive to the crystallization under the various conditions.
(3) the present invention is the surfactivity that the liquid of the dispersion bio-vitric that configures of prepare suspension has increased bio-vitric, and the surface bonding power of liquid and bio-vitric is stronger, so that bio-vitric is not easy precipitation, the bio-vitric suspension liquid can exist for more time.
(4) the present invention is in order to improve the utilising efficiency of bio-vitric, and the concentration of bioglass particles is 1～100/μ L in the suspension liquid, and preferred 2～50, the suspension liquid that is configured to was longer than 24 hours steady time.
(5) suspension liquid of the present invention can use together in conjunction with micro-fluidic crystallization apparatus, can not only improve crystalline rate and can also increase the range of application of bio-vitric.
Description of drawings
Fig. 1 is the crystal pattern of embodiment 1 zytase protein;
Fig. 2 is the schematic perspective view of the used microfluidic protein crystallization board of embodiment 3;
Fig. 3 is the vertical view of one of them unit of Fig. 2 crystallization plates;
Fig. 4 is that the A of Fig. 3 is to three-dimensional view;
Fig. 5 is that the A of another unit is to three-dimensional view;
Mark in the above-mentioned accompanying drawing is as follows:
Supporting structure section 1, panel 11, support portion 12, upper frame 12-1, connection section 12-2, underframe 12-3;
Lower connective slot 22, U-shaped groove 22-1, V-groove 22-2, upper connective slot 23, retaining wall 24.
Embodiment
(embodiment 1, promotes the method for crystallization of protein with bio-vitric)
The bio-vitric suspension liquid of the present embodiment is that the bio-vitric small-particle is suspended in the deionized water and the mixture that forms, and described bio-vitric small-particle is the super fine biological glass particle, and grain graininess is less than 200nm.In the bio-vitric suspension liquid, the short grained concentration of bio-vitric is 2～50/μ L, and the concentration of the present embodiment is 30/μ L.
The bio-vitric suspension liquid impels the method for crystallization of protein to comprise the steps:
1. the preparation of bio-vitric: be P according to being converted into oxide mol ratio
2O
5: CaO:MgO:SiO
2=1:3:1:5 takes by weighing respectively the P of corresponding amount
2O
5, Ca (NO
3)
2, Mg (NO3)
2, tetraethoxy, then each compound mixes with dehydrated alcohol respectively and reflux and being dissolved in the ethanol, makes respectively uniform P
2O
5Ethanolic soln, Ca (NO
3)
2Ethanolic soln, Mg (NO3)
2Ethanolic soln and the ethanolic soln of tetraethoxy; It is 6:1 that the mol ratio of ethanol and each material is respectively 4～10:1(the present embodiment).
In the ethanolic soln of tetraethoxy, add deionized water, made tetraethoxy prehydrolysis 45 minutes.
With P
2O
5Ethanolic soln, Ca (NO
3)
2Ethanolic soln, Mg (NO3)
2Ethanolic soln drop to successively in the ethanolic soln of above-mentioned tetraethoxy, during dropping before a kind of ethanolic soln dropwise 1 as a child added again after a kind of ethanolic soln, dropwise post-heating and vigorous stirring and obtain treating the mixed solution of solation.
In above-mentioned mixed solution, add deionized water, make catalyzer with nitric acid, regulator solution pH=2, and so that the solution solation.The colloidal sol that makes makes wet gel through ageing, gelation; Be placed on after wet gel and a certain amount of urea (sintering agent) mixed in 500 ℃ the stove and light a fire, after burning finishes, quenching obtains soft-agglomerated powder, in agate mortar, fully grind, and obtain the super fine biological glass particle, grain graininess is less than 200nm, mean pore size 110.2nm, characteristic aperture 96nm.
2. the preparation of bio-vitric suspension liquid: the 2g bioglass particles that 1. step is prepared adds in the 100mL deionized water, form unsettled suspension liquid through fully stirring, the more ultrasonic concussion of unsettled suspension liquid 5 minutes is obtained the bio-vitric suspension liquid of stable and uniform; Bioglass particles concentration is 30/μ L in the suspension liquid that obtains.
In the suspension of aforesaid method preparation, bio-vitric is dispersed in the deionized water, in sonic oscillation, deionized water has increased the surfactivity of bio-vitric, the surface bonding power of deionized water and bio-vitric is stronger, and to increase the suspension liquid existence life-span, the suspension liquid life-span was greater than 24 hours.
3. the bio-vitric suspension liquid promotes crystallization of protein: at first configure zytase (xylanase) protein supersaturated solution, 5 μ L zytase protein supersaturated solutions and 4 μ L precipitation agents are fully merged, the solvent of described precipitation agent is deionized water, the concentration of the ammonium sulfate in the precipitation agent is 1mol/L, the concentration of PEG3350 is 1 % w/v, and the concentration of the two dihydroxymethyl aniline of 2,2-is 0.1mol/L, and the pH that the two dihydroxymethyl aniline of 2,2-are regulated precipitation agent is 5.5.PEG3350 also is PEG3350, and CAS number is 25322-68-3.
Drip the bio-vitric suspension liquid 1 μ L that 2. step prepares in the above-mentioned mixed solution, leave standstill after shaking up, after the situation of observation solution internal protein crystallization in 1-3 days, obviously observed the protein crystal (Fig. 1) take bio-vitric as the nuclei of crystallization at 30 hours.
Because bioglass particles adds in the protein soln in the mode of suspension liquid, therefore bio-vitric can fully mix with protein soln and be dispersed in the protein soln, larger with the protein contact surface, improved the crystallization of protein probability, promoted the crystallization of protein.
In addition, promote the method for crystallization of protein to compare with gaseous diffusion process, we have compared catalase, myoglobin, thaumatin, zytase and the ferritin percent crystallization in massecuite with two kinds of diverse ways, and comparative result sees the following form 1:
Table 1
By the increasing degree of table 1 as can be known, add the percent crystallization in massecuite that has greatly improved protein behind the bio-vitric suspension liquid in the protein soln.
(embodiment 2, promote the method for crystallization of protein with bio-vitric)
In the present embodiment, the rest part of the bio-vitric suspension liquid that 2. step obtains is identical with embodiment 1, and difference is: also comprise the chloroform of 20mg/mL, pore-creating agent PEG3350 and the 5mg/mL promoting agent sulfated oil of 2.5mg/mL in the suspension.Also be that the surface-active liquid solvent of the present embodiment increase bio-vitric is deionized water, solute comprises that concentration is the chloroform of 20mg/mL, pore-creating agent PEG3350 and the 5mg/mL promoting agent sulfated oil of 2.5mg/mL.The used sulfated oil of the present embodiment is Viscotrol C and effect of sulfuric acid and the sulphonated oil that makes, and molecular formula is C
18Na
2O
6S, the CAS registration number is 8002-33-3.
The bio-vitric suspension liquid of the present embodiment impels the rest part of method of crystallization of protein identical with embodiment 1, and difference is:
In the suspension of aforesaid method preparation, bio-vitric is dispersed in the liquid, promoting agent sulfated oil in the liquid has further strengthened the surfactivity of bio-vitric, the surface bonding power of liquid and bio-vitric is stronger, to increase the suspension liquid existence life-span, the suspension liquid life-span, the stable existence time was elongated greater than 30 hours.
Step 3. in, when the bio-vitric suspension liquid promotes crystallization of protein, in the mixing solutions of zytase (xylanase) protein supersaturated solution and precipitation agent, add the bio-vitric suspension liquid 1 μ L that 2. step prepares, leave standstill after shaking up, after observe the crystallization of solution internal protein in 1-3 days situation, the protein crystal of obviously observing take bio-vitric as the nuclei of crystallization at 24 hours.
(embodiment 3, promote the method for crystallization of protein with bio-vitric)
The rest part of the present embodiment is identical with embodiment 2, and difference is:
Step 3. in, when promoting crystallization of protein by the bio-vitric suspension liquid, the bio-vitric suspension liquid promotes crystallization of protein in microfluidic protein crystallization board.
See Fig. 2, the used microfluidic protein crystallization board of the present embodiment is the integrated through injection molding part, comprises supporting structure section 1 and is connected to the crystalline element 2 that distributes with array format in the supporting structure section 1.
Supporting structure section 1 is comprised of panel 11 and the support portion 12 that is supported in edge, panel 11 surrounding.The global shape of panel 11 is the rectangle that has lacked an angle, the mark of this vee placement location when using, and the plate body of panel 11 be provided with distribute with array format with each crystalline element 2 upper end cross-sectional shape, special-shaped through hole that size is identical.
Support portion 12 comprises upper frame 12-1, underframe 12-3 and the connection section 12-2 that is connected upper and lower framework.The shape of the upper surface of upper frame 12-1, big or small corresponding with panel 11, support portion 12 is connected on the panel 11 from the below along the edge, lower surface by its upper frame 12-1, and the angle between upper frame 12-1 and the panel 11 is 80 °; The length and width of underframe 12-3 is all greater than upper frame 12-1, and underframe 12-3 is fixedly linked by connection section 12-2 and upper frame 12-1 and has formed the bottom outside flange, and formed flange is stacking for stablizing between the different crystallization plates.
See Fig. 3, each crystalline element 2 all is connected on the panel 11 of supporting structure section 1 from the lower end along a corresponding edge as the special-shaped through hole of connecting portion, and each crystalline element 2 comprises the liquid storage hole section 21 at two ends and is communicated with the connection groove section of two ends liquid storage hole section 21.The between centers of the liquid storage hole section 21 at two ends is 9mm, 12mm, 15mm or 18mm.
See Fig. 4, the liquid storage hole of described liquid storage hole section 21 is the through hole that arranges along the vertical direction, and the intracavity section of liquid storage hole section 21 is long-pending to be reduced from the top down gradually, is divided into successively upper hole section 21-1, lower hole section 21-2 and base apertures section 21-3 according to from top to bottom order; The upper hole section 21-1 of liquid storage hole is the up big and down small truncated cone-shaped with lateral notch, described lateral notch is that the cross-sectional shape that communicates with top connective slot opening upwards is up big and down small trapezoidal breach 21-4, and the lower end of the lower end of this breach 21-4 and upper hole section 21-1 is in the sustained height position; The lower hole section 21-2 of liquid storage hole is the semisphere of opening upwards, and the axis of hole section is identical up and down; The base apertures section 21-3 of liquid storage hole is the strip rectangular opening along the length direction setting of connection groove section.
The connective slot of connection groove section comprises the upper connective slot 23 of opening upwards of mutual isolation and the lower connective slot 22 that Open Side Down, upper connective slot 23 and lower connective slot 22 are symmetrical arranged with respect to a vertical guide along its length direction setting, and described vertical guide is through the axis of 2 liquid storage holes of liquid storage hole section 21.
The upper connective slot 23 of connective slot is that cross-sectional shape is up big and down small trapezoidal holding tank, upper connective slot 23 be positioned on the length direction two ends each be connected with the lateral notch 21-4 of the upper hole section 21-1 of the liquid storage hole of a corresponding liquid storage hole section 21.Upper connective slot 23 narrows down from the top down gradually, and the upper end of the lower end of upper connective slot 23 and lower hole section 21-2 is in the sustained height position.The liquid storage hole of the liquid storage hole section 21 of upper connective slot 23 and crystalline element 2 all communicates with the space outerpace of top.
The lower connective slot 22 of connective slot is groove, and lower connective slot 22 is arranged on the bottom of crystalline element 2, and the liquid storage hole of the liquid storage hole section of lower connective slot 22 and crystalline element all communicates with the space outerpace of below.The cross-sectional shape of lower connective slot 22 is rectangle, and as U-shaped groove 22-1, the width W of described U-shaped groove 22-1 is 0.2mm or 0.3mm, and U-shaped groove height D is 0.2mm or 0.3mm, and the crystalline element 2 that comprises U-shaped groove 22-1 is U-shaped unit.The width of the width of U-shaped groove 22-1 and the base apertures section 21-3 of liquid storage hole section 21 and highly identical, the height of U-shaped groove 22-1 be liquid storage hole section 21 lower hole section 21-2 height 1/10 to 1/20; The end that is positioned on the length direction of U-shaped groove 22-1 is connected with the base apertures section 21-3 of the liquid storage hole of a liquid storage hole section 21, the other end that is positioned on the length direction of U-shaped groove 22-1 is connected with the base apertures section 21-3 of the liquid storage hole of another liquid storage hole section 21, and the base apertures section 21-3 of U-shaped groove 22-1 and the liquid storage hole that links to each other with its two ends has formed the downward rectangular slot that has that links up in the bottom of crystalline element 2 thus.
See Fig. 5, the cross-sectional shape of lower connective slot 22 can also be V-type, and as V-groove 22-2, the crystalline element 2 that comprises V-groove 22-2 is the V-type unit.The width W bottom of V-groove 22-2 is 0.2mm or 0.3mm, and the height D of V-groove 22-2 is 0.2mm or 0.3mm.
The width W bottom of V-groove 22-2 and height D respectively with the width of the base apertures section 21-3 of liquid storage hole section 21 and highly identical, the height D of V-groove 22-2 be liquid storage hole section 21 lower hole section 21-2 height 1/10 to 1/20, V-groove 22-2 be positioned on the length direction two ends each be communicated with the base apertures section 21-3 of a corresponding liquid storage hole section 21.
Be used to form crystalline element 2 upper connective slot 23 about two retaining walls 24 interval S topmost be that the retaining wall interval S is 4mm, 5mm or 7mm.The degree of depth that the retaining wall depth H namely goes up connective slot 23 also is that the vertical range of the lower end of panel 11 supreme hole section 21-1 is 2.5mm, 3mm, 3.5mm or 4mm.
The liquid storage hole section 21 of above-mentioned crystalline element 2 adopts the form of reverse frustoconic inner chamber and semisphere combination of lumens, so that when operation solution can splash into easily, and using at present, the upper end of connective slot 22 is capped, only the base apertures section 21-3 of two ends liquid storage hole section 21 upper end is uncovered, therefore only needs a small amount of oil that covers can seal the solution that splashes into fully.
The long 127.76mm of described crystallization plates, wide 85.48mm has 5 * 8=40 crystalline element 2.The crystalline element 2 that distributes take array format is for distributing with box formation, and between each neighbouring crystalline element 2, the between centers of neighbouring liquid storage hole section 21 is equal; Between the adjacent crystalline element 2 in each left and right sides, the between centers of the liquid storage hole section 21 that the left and right sides is adjacent all equates, thereby can realize crystallization and the screening of protein on protein crystallization board of different sorts, different concns.
Crystallization plates adopts the PC resin injection molded as raw material, and the crystallization plates of making transparent and difficult generation cut and damage also have enough stabilitys simultaneously; The material of crystallization plates also can be selected the plastics of other similar quality such as PE etc.Described crystallization plates has adopted corona method to carry out surface treatment, and its surface has hydrophilic properties.
Before microfluidic protein crystallization board (hereinafter to be referred as crystallization plates) uses, first cover thin film fully in the bottom of crystallization plates, the restraining mass that seals as the base apertures section 21-3 to lower connective slot 22 and liquid storage hole, thus the seal channel that 2 liquid storage holes are communicated with formed; Then crystallization plates is faced up and be placed on the worktable.
When the bio-vitric suspension liquid promotes crystallization of protein, 1.5 μ L zytase protein supersaturated solutions are splashed in the liquid storage hole of one of them liquid storage hole section 21 of crystalline element 2 of crystallization plates, then splash into the mixing solutions of precipitant solution and bio-vitric suspension in the liquid storage hole of another liquid storage hole section 21 of crystalline element 2, by the lower connective slot 22 of the connection groove section between the liquid storage hole of 2 liquid storage hole sections 21, so that the material phase mutual diffusion in two liquid storage hole sections 21.Then in the at the interface observation crystallization situation of both sides solution, the protein crystal of obviously observing take bio-vitric as the nuclei of crystallization at 24 hours.
Described mixing solutions is mixed and is obtained by the precipitation agent of 1 μ L and the bio-vitric suspension liquid of 0.5 μ L.The solvent of described precipitation agent is deionized water, and the concentration of ammonium sulfate is 1mol/L in the precipitation agent, and the concentration of PEG3350 is 1 % w/v, 2, the concentration of the two dihydroxymethyl aniline of 2-is 0.1 mol/L, and the pH that the two dihydroxymethyl aniline of 2,2-are regulated precipitation agent is 5.5.
Above each embodiment and application examples are the explanations to the specific embodiment of the present invention; but not limitation of the present invention; person skilled in the relevant technique is not in the situation that break away from the spirit and scope of the present invention; can also make various conversion and obtain the corresponding technical scheme that is equal to variation, so all technical schemes that are equal to all should be included into scope of patent protection of the present invention.
Claims (7)
1. method of impelling crystallization of protein with bio-vitric, it is characterized in that: first that bioglass particles is even with the surface-active liquid mixing of increase bio-vitric, carry out sonic oscillation again and obtain the bio-vitric suspension liquid, then with dropper the bio-vitric suspension liquid is transferred to the crystallization of protein environment, thereby the bio-vitric suspension liquid promotes crystallization of protein;
The granularity 100nm of described bioglass particles～200nm, mean pore size 90nm～200nm, the concentration of bioglass particles is 1～100/μ L in the suspension liquid.
2. the method for impelling crystallization of protein with bio-vitric according to claim 1, it is characterized in that: the concentration of bioglass particles is 2～50/μ L in the bio-vitric suspension liquid.
3. the method for impelling crystallization of protein with bio-vitric according to claim 1, it is characterized in that: the surface-active liquid of described increase bio-vitric is mixing solutions, wherein solvent is deionized water, and solute comprises that concentration is the chloroform of 15～25mg/mL, pore-creating agent polyoxyethylene glycol and the 3～8mg/mL promoting agent sulfated oil of 1～3mg/mL.
4. the method for impelling crystallization of protein with bio-vitric according to claim 1, it is characterized in that: the surface-active liquid of described increase bio-vitric is deionized water.
5. the method for impelling crystallization of protein with bio-vitric according to claim 1 is characterized in that: described crystallization of protein environment is the crystalline environment in microfluidic protein crystallization board;
Described microfluidic protein crystallization board is the integrated through injection molding part, comprise supporting structure section (1) and the crystalline element (2) that upward distributes with array format in supporting structure section (1), each crystalline element (2) is comprised of the liquid storage hole section (21) at two ends and the up and down connective slot (23,22) of connection two ends liquid storage hole sections (21), the intracavity section of described liquid storage hole section (21) is long-pending to be reduced from the top down gradually, and the upper connective slot (23) of connection groove section is up big and down small;
During crystallization of protein, the protein supersaturated solution is splashed in the liquid storage hole of one of them liquid storage hole section (21) of crystalline element (2) of crystallization plates, then in the liquid storage hole of another liquid storage hole section (21) of crystalline element (2), splash into precipitant solution and bio-vitric suspension liquid, thereby the bio-vitric suspension liquid enters crystalline environment, also namely the material in 2 liquid storage hole sections (21) by lower connective slot (22) so that protein supersaturated solution and the mutual diffusion of bio-vitric suspension liquid phase, and the lower port place of the notch position of time connective slot (22) and liquid storage hole is provided with for the restraining mass that seals; At the interface bio-vitric suspension liquid at both sides solution promotes crystallization of protein at last.
6. the method that makes crystallization of protein with bio-vitric according to claim 1, it is characterized in that: the bio-vitric in the bio-vitric suspension is made by sol-gel method.
7. the method that makes crystallization of protein with bio-vitric according to claim 6, it is characterized in that: the step that is prepared bio-vitric by sol-gel method is as follows: be P according to being converted into oxide mol ratio first
2O
5: CaO:MgO:SiO
2=1:(3～3.5): (1～1.2): the ratio of (4.5～5.5) takes by weighing respectively the P of corresponding amount
2O
5, Ca (NO
3)
2, Mg (NO3)
2, tetraethoxy, each compound is mixed respectively and reflux and being dissolved in the ethanol with dehydrated alcohol, make respectively uniform P
2O
5Ethanolic soln, Ca (NO
3)
2Ethanolic soln, Mg (NO3)
2Ethanolic soln and the ethanolic soln of tetraethoxy, the mol ratio of ethanol and each material is respectively (4～10): 1;
In the ethanolic soln of tetraethoxy, add deionized water, made tetraethoxy prehydrolysis 40～50 minutes;
With P
2O
5Ethanolic soln, Ca (NO
3)
2Ethanolic soln, Mg (NO3)
2Ethanolic soln drop to successively in the ethanolic soln of above-mentioned tetraethoxy, during dropping, front a kind of ethanolic soln dropwise 1～1.5 as a child dripped again after a kind of ethanolic soln, dropwise post-heating and vigorous stirring and obtain treating the mixed solution of solation;
Add deionized water in above-mentioned mixed solution, with nitric acid regulator solution pH=2～2.5, and so that the solution solation, the colloidal sol that makes makes wet gel through ageing, gelation; Be placed on after wet gel and urea mixed in 500 ℃～550 ℃ the stove and light a fire, after burning finished, quenching obtained soft-agglomerated powder, fully grinds in agate mortar, and obtains the super fine biological glass particle, and grain graininess is less than 200nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110201924.4A CN102887939B (en) | 2011-07-19 | 2011-07-19 | The method of crystallization of protein is impelled with bio-vitric |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110201924.4A CN102887939B (en) | 2011-07-19 | 2011-07-19 | The method of crystallization of protein is impelled with bio-vitric |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102887939A true CN102887939A (en) | 2013-01-23 |
| CN102887939B CN102887939B (en) | 2015-08-19 |
Family
ID=47531633
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110201924.4A Expired - Fee Related CN102887939B (en) | 2011-07-19 | 2011-07-19 | The method of crystallization of protein is impelled with bio-vitric |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102887939B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108137647A (en) * | 2015-10-09 | 2018-06-08 | 东丽株式会社 | Fibre-bearing crystal, the manufacturing method of fibre-bearing crystal, the manufacturing device of fibre-bearing crystal and chemical reagent infuser device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004041847A1 (en) * | 2002-11-07 | 2004-05-21 | Imperial College Innovations Limited | Mesoporous glass as nucleant for macromolecule crystallisation |
| CN101161871A (en) * | 2006-10-11 | 2008-04-16 | 博亚捷晶科技（北京）有限公司 | Protein crystal board and crystallization method |
-
2011
- 2011-07-19 CN CN201110201924.4A patent/CN102887939B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004041847A1 (en) * | 2002-11-07 | 2004-05-21 | Imperial College Innovations Limited | Mesoporous glass as nucleant for macromolecule crystallisation |
| CN101161871A (en) * | 2006-10-11 | 2008-04-16 | 博亚捷晶科技（北京）有限公司 | Protein crystal board and crystallization method |
Non-Patent Citations (2)
| Title |
|---|
| N.E.CHAYEN ET AL.: "Porous Silicon: an Effective Nucleation-inducing Material for Protein Crystallization", 《JMB》, vol. 312, 31 December 2001 (2001-12-31), pages 591 - 595, XP004451631, DOI: doi:10.1006/jmbi.2001.4995 * |
| NAOMI E. CHAYEN ET AL.,: "Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium", 《PNAS》, vol. 103, no. 3, 17 January 2006 (2006-01-17), pages 597 - 601, XP002668874, DOI: doi:10.1073/pnas.0504860102 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108137647A (en) * | 2015-10-09 | 2018-06-08 | 东丽株式会社 | Fibre-bearing crystal, the manufacturing method of fibre-bearing crystal, the manufacturing device of fibre-bearing crystal and chemical reagent infuser device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102887939B (en) | 2015-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Valtchev et al. | Porous nanosized particles: preparation, properties, and applications | |
| Ogura et al. | Aluminosilicate species in the hydrogel phase formed during the aging process for the crystallization of FAU zeolite | |
| JP4650489B2 (en) | Zeolite membrane production equipment | |
| CN105271299B (en) | A kind of preparation method of the zeolites of mesoporous ZSM 5 | |
| Shao et al. | Influences of seeds on the properties of zeolite NaA membranes on alumina hollow fibers | |
| Fu et al. | Uniformly Oriented Zeolite ZSM‐5 Membranes with Tunable Wettability on a Porous Ceramic | |
| JP6106589B2 (en) | A film formed by secondary growth of seed crystals in which all three crystal axes are uniformly aligned on the substrate | |
| CN102826564A (en) | Preparation method for Beta zeolite molecular sieve with hierarchical porous structure | |
| CN104437602A (en) | Multistage meso-porous ZSM-5 catalyst as well as preparation method and application method thereof | |
| CN102674387A (en) | Synthesis method of multi-stage nanometer crystal accumulation structure melt flow index (MFI) type molecular sieves | |
| KR20170046712A (en) | Zeolitic materials having a distinctive monocrystal macroporosity, and method for the production thereof | |
| CN103318910A (en) | Method for preparing large-size analcite by utilizing hydrothermal crystallization of geopolymer | |
| CN102887939A (en) | Method for promoting protein crystallization by use of bioglass | |
| CN102286067B (en) | Method for simultaneously improving protein crystallization success rate and crystal quality | |
| Jain et al. | In situ imaging of faujasite surface growth reveals unique pathways of zeolite crystallization | |
| CN105073640A (en) | Method for producing ddr-type zeolite crystals and method for producing ddr-type zeolite film | |
| US11434140B2 (en) | Hierarchical zeolites and preparation method therefor | |
| CN107758688B (en) | Nano-aggregated disk-shaped mordenite with different compactness | |
| CN100360405C (en) | LTA and FAU molecular screen nanocrystalline preparation method | |
| CN105905919A (en) | Meso-porous EU-1 zeolite molecular sieve and preparation method thereof | |
| CN105174284B (en) | Efficient two-silicon-source amine-free synthesis method for morphology-controllable large-size mordenite | |
| KR20110000297A (en) | Hollow mesoporous silica capsule and method for preparing the same | |
| CN102502685B (en) | Preparation method of mesoporous LTA zeolite | |
| CN107265479A (en) | A kind of preparation method of the molecular sieves of nanometer MCM 41 of high dispersive Al containing skeleton | |
| CN113086989B (en) | Preparation method of hierarchical pore NaY molecular sieve |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150819 Termination date: 20180719 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |