CN105566443B - Make the method that seed crystal improves crystallization of protein success rate using crosslinked protein crystals - Google Patents
Make the method that seed crystal improves crystallization of protein success rate using crosslinked protein crystals Download PDFInfo
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- 239000013078 crystal Substances 0.000 title claims abstract description 175
- 238000002425 crystallisation Methods 0.000 title claims abstract description 124
- 230000008025 crystallization Effects 0.000 title claims abstract description 124
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 96
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 96
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000012216 screening Methods 0.000 claims abstract description 39
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
- 238000002447 crystallographic data Methods 0.000 claims abstract description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 6
- 239000012498 ultrapure water Substances 0.000 claims abstract description 6
- 235000010335 lysozyme Nutrition 0.000 claims description 34
- 102000016943 Muramidase Human genes 0.000 claims description 33
- 108010014251 Muramidase Proteins 0.000 claims description 33
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 33
- 239000004325 lysozyme Substances 0.000 claims description 33
- 229960000274 lysozyme Drugs 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 31
- 238000004132 cross linking Methods 0.000 claims description 26
- 102000016938 Catalase Human genes 0.000 claims description 15
- 108010053835 Catalase Proteins 0.000 claims description 15
- 108010067770 Endopeptidase K Proteins 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 13
- 229940088598 enzyme Drugs 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 11
- 235000010436 thaumatin Nutrition 0.000 claims description 10
- 239000000892 thaumatin Substances 0.000 claims description 10
- 108010038061 Chymotrypsinogen Proteins 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 239000011549 crystallization solution Substances 0.000 claims description 7
- 239000012460 protein solution Substances 0.000 claims description 5
- 230000007774 longterm Effects 0.000 abstract description 2
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- 238000002474 experimental method Methods 0.000 description 8
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- 238000007710 freezing Methods 0.000 description 5
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000010413 mother solution Substances 0.000 description 3
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 108010062466 Enzyme Precursors Proteins 0.000 description 2
- 102000010911 Enzyme Precursors Human genes 0.000 description 2
- 238000010382 chemical cross-linking Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000000311 Cytosine Deaminase Human genes 0.000 description 1
- 108010080611 Cytosine Deaminase Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101710094902 Legumin Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
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- 239000000872 buffer Substances 0.000 description 1
- 238000002288 cocrystallisation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- NEKNNCABDXGBEN-UHFFFAOYSA-L disodium;4-(4-chloro-2-methylphenoxy)butanoate;4-(2,4-dichlorophenoxy)butanoate Chemical compound [Na+].[Na+].CC1=CC(Cl)=CC=C1OCCCC([O-])=O.[O-]C(=O)CCCOC1=CC=C(Cl)C=C1Cl NEKNNCABDXGBEN-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21001—Chymotrypsin (3.4.21.1)
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Abstract
Make the method that seed crystal improves crystallization of protein success rate using crosslinked protein crystals the present invention provides a kind of, protein crystal is grown first, and protein crystal is chemically crosslinked, obtained crosslinked protein crystals are dissolved in ultrapure water, seed crystal solution is made；Then seed crystal solution is instilled to the sitting drop hole of crystallization plates, naturally dry crystallization plates；Crystallization condition screening finally is carried out to protein using screening reagent box, using the pattern of auto-phot system view protein crystal, and counts the crystallization condition number of protein crystal；Simultaneously diffraction protein crystal is collected, diffraction data is obtained.The present invention chooses crosslinked protein crystals as seed crystal, stable in the air can exist, and is conducive to long-term storage, and can reuse；The present invention can effectively improve the success rate of protein crystallization condition screening.
Description
Technical field
The present invention relates to a kind of methods that raising protein crystallization screening hanks power.
Background technique
Protein is the carrier of vital movement, and protein structure is the important prerequisite for determining its physiological function.Currently, obtaining
The monocrystalline of high quality is the key that the bottleneck using x-ray diffraction technique parsing protein fine three dimensional structure and this technology
Problem.The crystallization condition for how screening to obtain protein is the first step for obtaining high quality protein matter monocrystalline, however, from high-purity egg
White matter to obtain diffraction quality protein crystal success rate less than 20%, therefore, improve protein crystal crystallization success
Rate is extremely urgent.
There are many kinds of the methods that power is hanked for improving protein crystallization screening, including cocrystallization, improvement interface, control change
Warm mode, seed-grain method etc..Document 1: " An automated microseed matrix-screening method for
protein crystallization[J].Acta Crystallographica Section D,2007,63(4):550-
554. ", which report seed-grain method, can make the crystallization success rate of protein improve 2.5~65 times；Document 2: " Microseed
matrix screening to improve crystals of yeast cytosine deaminase[J].Acta
Crystallographica Section D, 2004,60 (3): 601-605 " reports the protein crystal using low resolution
As the crystallization condition of seed crystal optimization protein, high-resolution protein crystal can be obtained, and then obtains diffraction data.So
And the seed crystal of this method preparation is dissolved in water and organic solvent, is used only once, it is expensive.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provide it is a kind of utilize crosslinking protein crystal be used for as seed crystal
The method of crystallization of protein is crosslinked using the protein crystal that crosslinking agent prepares conventional method, and obtaining can be steady in air
Fixed existing crosslinked protein crystals.Then using crosslinked protein crystals as seed crystal, protein knot can be effectively improved
The success rate of brilliant conditional filtering.
The technical solution adopted by the present invention to solve the technical problems is: it is molten to prepare crosslinked protein crystals seed crystal first
Then liquid seed crystal solution is added dropwise on crystallization plates, finally carry out the screening of crystallization of protein success rate.Specific step is as follows:
The first step grows protein crystal, and is chemically crosslinked to protein crystal, by obtained cross-linked proteins crystalline substance
Body is dissolved in ultrapure water, and concentration is 5-20mg/ml, and seed crystal solution is made；
Second step instills the seed crystal solution of 0.5-2 μ l, naturally dry crystallization plates in each sitting drop hole of crystallization plates；
Third step utilizes IndexTMScreening reagent box carries out crystallization condition screening to protein, and it is molten to prepare protein first
Liquid；Then the pond liquid of 80 μ l is added into each deep hole of crystallization plates, crystallizing agent and protein solution will be screened according to the matter of 1:1
Amount ratio is mixed to get crystallization solution, and the crystallization drop of 2 μ l is instilled to the sitting drop hole of crystallization plates, seals crystallization plates, and in 277-
It is crystallized 3-7 days under the crystallization temperature of 293K；
4th step, the pattern of view protein crystal count the crystallization condition number of protein crystal；Collect simultaneously diffraction albumen
Matter crystal obtains diffraction data.
The seed crystal solution selects crosslinking lysozyme as seed crystal.
The seed crystal solution selects the crosslinking lysozyme that mass ratio is 1:1 and hydrogen peroxide enzyme crystal as seed crystal.
It is crosslinking lysozyme crystal, catalase, the protein of 1:1:1:1:1 that the seed crystal solution, which selects mass ratio,
Enzyme K, thaumatin matter and chymotrypsinogen A II crystal are as seed crystal.
Crystalline mother solution is all replaced as crosslinker solution during the chemical crosslinking.
The beneficial effects of the present invention are:
1, the present invention chooses crosslinked protein crystals as seed crystal, stable in the air can exist, and is conducive to long-term storage,
And it can reuse；
2, the bigger crosslinked protein crystals of combined use molecular weight and differences between lattice constant of the invention are covered as seed crystal
The molecular weight and lattice constant of most of crystallizable protein have been covered, good inducing action can be played to crystallization of protein,
A possibility that increasing crystallization of protein；
3, the present invention can provide a kind of effective method to improve the success rate of crystallization of protein screening.
Specific embodiment
Below with reference to embodiment, the present invention is further described, and the invention includes, but is not limited to, the following examples.
The present invention proposes a kind of using crosslinked protein crystals as seed crystal, hanks power for improving protein crystallization screening
Method.Specific technical solution mainly includes two aspects: 1, preparing crosslinked protein crystals；2, crosslinked protein crystals are utilized
Crystallization success rate as seed crystal screening protein crystal.
1. preparing seed crystal
The preparation of crosslinked protein crystals mainly includes two steps: (1) crystallization of protein；(2) chemistry of crystal is handed over
Connection.Step 1: the crystallization of protein.It is added in each hole of 40 orifice plates from the pond liquid for drawing 80 μ l in same precipitating reagent.By egg
White matter solution and precipitant solution are mixed with to obtain crystallization solution by 1:1, and the solution for then drawing 2 μ l is added drop-wise to coverslip
On, it, will be on coverslip overturning lid to 40 orifice plates as crystallization of protein hanging drop.40 orifice plates are put into temperature-controlled box at 293k
It is incubated for 2 days, protein crystal is prepared.Step 2: the chemical crosslinking of crystal.Protein crystal is collected into the EP pipe of 5ml
In, crosslinking agent is added, crystal is chemically crosslinked.After 1 day, cross-linking agent solution is siphoned away, and with ultrapure water crystal wash clean.
Crosslinked protein crystals prepared by the present invention include: crosslinking lysozyme, catalase, protease K, thaumatin
Matter, chymotrypsinogen A II crystal.Seed crystal used in the present invention mainly includes three classes: 1, selecting crosslinking lysozyme as seed crystal；
2, select crosslinking lysozyme and hydrogen peroxide enzyme crystal as seed crystal, mass ratio 1:1；3, select crosslinking lysozyme crystal,
Catalase, protease K, thaumatin matter, chymotrypsinogen A II crystal are as seed crystal, mass ratio 1:1:
1:1:1.In experiment, seed crystal is dissolved in ultrapure water, is configured to the seed crystal solution that concentration is 5-20mg/ml.
2. using crosslinked protein crystals as the crystallization success rate of seed crystal screening protein crystal
Firstly, the sitting drop hole that the seed crystal solution that 0.5-2 μ l step 1 obtains instills crystallization plates is drawn, naturally dry crystallization plates.
Then, the protein solution and precipitant solution of 2 μ l is added into each sitting drop hole of crystallization plates using automatic liquor-transferring system, it will
Crystallization plates are put into temperature-controlled box, crystallization temperature 277-293K, and crystallization time is 3-7 days.The present invention selects Hampton
Research company article No. is the Index of No.HR2-134TMScreening reagent box, crystallization experiment use sitting-drop methods.With 20 kinds of albumen
Matter carries out crystallization screening experiment, and observation counts its selection result, and verifies the crystallization success rate of 20 kinds of protein；By to crystal
Collection, freezing, diffraction analysis, obtain the diffraction data of protein crystal.
Specific step is as follows:
Step 1: preparing crosslinked protein crystals, including grows protein crystal and crystal is chemically crosslinked.?
It needs crystalline mother solution to be all replaced as crosslinker solution in cross-linking reaction, avoids unreacted protein and crosslinking in crystalline mother solution
Agent reaction generates floccule precipitating；Then crosslinked protein crystals are dissolved in ultrapure water, concentration is 5-20mg/ml, system
Obtain seed crystal solution；
Step 2: the seed crystal solution for drawing 0.5-2 μ l instills the sitting drop hole of crystallization plates, naturally dry crystallization plates；
Step 3: utilizing IndexTMScreening reagent box carries out crystallization condition screening to protein.It is molten that protein is prepared first
Liquid；Then the pond liquid of 80 μ l is added into each deep hole of crystallization plates, (is sieved the crystallization drop of 2 μ l using automatic liquor-transferring system
Crystallizing agent and protein solution 1:1 is selected to mix) drop is in the sitting drop hole of experimental group, with crystallization rubber belt sealing crystallization plates.Then
Crystallization plates are all placed in temperature control box, crystallization temperature 277-293K, and crystallization time is 3-7 days；
Step 4: using the pattern of auto-phot system view protein crystal, and count the crystallization item of protein crystal
Number of packages；Simultaneously diffraction protein crystal is collected, diffraction data is obtained.
Use the crystallization experiment of some protein below to verify crosslinking lysozyme crystal as the effect of seed crystal, seed is not added
Crystalline substance is as a control group.
Embodiment 1: the screening of crystallization condition is carried out to lysozyme as seed crystal using crosslinking lysozyme crystal, control group is not
Do any processing.
Step 1: drawing the sitting drop that the crosslinking lysozyme crystal seed crystal solution that 0.5 μ l concentration is 5mg/ml instills crystallization plates
Hole, as experimental group, naturally dry crystallization plates.The crystallization plates of control group are without any processing.
Step 2: being dissolved in pH by six recrystallization lysozymes of the NO.100940 purchased from Seikagaku company, Japan and being
4.6, concentration be 0.I M sodium-acetate buffer in, initial concentration be 20mg/ml to get arrive antalzyme crystallization solution.
Step 3: utilizing IndexTMScreening reagent box carries out crystallization condition screening to lysozyme.This experiment uses sitting-drop methods,
The pond liquid of 80 μ l is added into each deep hole of crystallization plates, the crystallization drop of 2 μ l (is screened into crystallizing agent using automatic liquor-transferring system
Being mixed with lysozyme soln 1:1) drop is in the sitting drop hole of experimental group, with crystallization rubber belt sealing crystallization plates.In kind knot
Brilliant drop is added in the sitting drop hole of control group.
Step 4: the crystallization plates of experimental group and control group are all placed in 277K temperature control box, crystallization time is 3 days, benefit
The pattern of lysozyme crystal is observed with automatic photomicrograph system, and counts the crystallization condition number of lysozyme.
Step 5: collecting and freezing protein crystal, the diffraction of crystal is carried out using household x-ray diffractometer, is collected simultaneously
Analyze diffraction data.By statistics and analysis, it is as follows to obtain result: being molten by the above-mentioned crystal of X-ray diffraction experimental verification
Bacterium zymoprotein crystal, macroscopically can produce X-ray diffraction phenomenon, and the resolution ratio of diffraction isWherein experimental group is molten
The crystallization condition number of bacterium enzyme is 80, compared with control group (crystallization number is 40), to be crosslinked lysozyme crystal as the sieve of seed crystal
Power is hanked to be significantly increased.
Embodiment 2: crystallization condition sieve is carried out to Proteinase K as seed crystal using crosslinking lysozyme and hydrogen peroxide enzyme crystal
Choosing, control group select the lysozyme crystal of crosslinking to make seed crystal.
Step 1: drawing the crosslinking lysozyme and catalase crystal seeds solution instillation knot that 1 μ l concentration is 10mg/ml
The crosslinking lysozyme crystal that 1 μ l concentration is 10mg/ml, naturally dry is added as experimental group in the sitting drop hole of brilliant plate in control group
Crystallization plates.
Step 2: being dissolved in pH is 4.60 by the No.P6556 Proteinase K of Sigma Co., USA, concentration is 0.08M acetic acid
Magnesium, in the buffer of 0.05M natrium cacodylicum, initial concentration is 20mg/ml to get protease K crystallization solution is arrived.
Step 3: utilizing IndexTMScreening reagent box carries out crystallization condition screening to Proteinase K.This experiment uses sitting drop
The pond liquid of 80 μ l is added into each deep hole of crystallization plates for method, and using automatic liquor-transferring system, by the crystallization drop of 2 μ l, (screening is tied
Brilliant agent and Proteinase K Solution 1:1 mixing) drop is in the sitting drop hole of experimental group, with crystallization rubber belt sealing crystallization plates.In kind
Crystallization drop is added in the sitting drop hole of control group.
Step 4: the crystallization plates of experimental group and control group are all placed in temperature control box, crystallization temperature 293K, when crystallization
Between 7 days, using the pattern of automatic photomicrograph system observation Proteinase K crystal, and count the conditions number of crystal.
Step 5: collecting and freezing protein crystal, the diffraction of crystal is carried out using household x-ray diffractometer, is collected simultaneously
Analyze diffraction data.By observation and statistics, it is as follows to obtain result: being egg by the above-mentioned crystal of X-ray diffraction experimental verification
White enzyme K crystal, macroscopically can produce X-ray diffraction phenomenon, and the resolution ratio of diffraction isWherein test histone
The crystallization condition number of matter is 54, compared with control group (crystallization number is 26), is made with being crosslinked lysozyme and hydrogen peroxide enzyme crystal
It is far longer than only for the screening success rate of seed crystal to be crosslinked lysozyme as the success rate of seed crystal.
Embodiment 3: crosslinking lysozyme crystal, catalase, protease K, thaumatin matter, rotten albumen are utilized
Proenzyme A II crystal carries out crystallization condition screening to catalase as seed crystal, and control group selects crosslinking lysozyme and peroxidating
Hydrogen enzyme crystal is as seed crystal.
Step 1: draw 2 μ l concentration be the crosslinking lysozyme crystal of 15mg/ml, it is catalase, protease K, unusual
Fruit thaumatin T matter, chymotrypsinogen A II crystal seeds solution instill the sitting drop hole of crystallization plates, as experimental group, the knot of control group
It is the crosslinking lysozyme and hydrogen peroxide enzyme crystal of 15mg/ml as seed crystal, naturally dry crystallization that 2 μ l concentration are added in brilliant plate
Plate.
Step 2: the No.C40 catalase that Sigma Co., USA is produced, being dissolved in pH is 7.00, concentration 0.IM
Fourth acetate buffer in, initial concentration 12mg/ml is filtered to remove impurity, as peroxide with 0.22 μm of sterilised membrane filter
Change hydrogen enzyme crystallization solution.
Step 3: utilizing IndexTMScreening reagent box carries out crystallization condition screening to catalase.This experiment is using seat
The pond liquid of 80 μ l is added into each deep hole for drop method, using automatic liquor-transferring system by the crystallization drop of 2 μ l (screening crystallizing agent and
Catalase solution 1:1 mixing) drop is in the sitting drop hole of experimental group, with crystallization rubber belt sealing crystallization plates.In kind
Crystallization drop is added in the sitting drop hole of control group.
Step 4: the crystallization plates of experimental group and control group are all placed in temperature control box, crystallization temperature 283K, when crystallization
Between 6 days, using the pattern of automatic photomicrograph system observation catalase crystal, and count the conditions number of crystal.
Step 5: collecting and freezing protein crystal, the diffraction of crystal is carried out using household x-ray diffractometer, is collected simultaneously
Analyze diffraction data.By observation and statistics, it is as follows to obtain result: being by the above-mentioned crystal of X-ray diffraction experimental verification
Hydrogen oxide enzyme crystal, macroscopically can produce X-ray diffraction phenomenon, and the resolution ratio of diffraction isWherein experimental group egg
The crystallization condition number of white matter is 46, compared with control group (crystallization number be 30), be crosslinked lysozyme crystal, catalase,
Protease K, thaumatin matter, chymotrypsinogen A II crystal are greatly improved as the screening success rate of seed crystal.
Embodiment 4: crosslinking lysozyme crystal, catalase, protease K, thaumatin matter, rotten albumen are utilized
Proenzyme A II crystal seeds as seed crystal to canavaline carry out crystallization condition screening, naturally dry crystallization plates, control group
Crystallization plates are without any processing.
Step 1: draw 1 μ l concentration be the crosslinking lysozyme crystal of 20mg/ml, it is catalase, protease K, unusual
Fruit thaumatin T matter, chymotrypsinogen A II crystal seeds solution instill the sitting drop hole of crystallization plates, as experimental group, the knot of control group
Brilliant plate is without any processing.
Step 2: the L7647 canavaline that Sigma Co., USA is produced, is dissolved in that pH is 6.50, concentration is 0.1M's
In Bis-tris buffer, initial concentration be 80mg/ml to get arrive canavaline crystallization solution
Step 3: utilizing IndexTMScreening reagent box carries out crystallization condition screening to canavaline.This experiment is using seat
The pond liquid of 80 μ l is added into each deep hole for drop method, using automatic liquor-transferring system by the crystallization drop of 2 μ l (screening crystallizing agent and
Canavaline solution 1:1 mixing) drop is in the sitting drop hole of experimental group, with crystallization rubber belt sealing crystallization plates.In kind
Crystallization drop is added in the sitting drop hole of control group.
Step 4: the crystallization plates of experimental group and control group are all placed in temperature control box, crystallization temperature 288K, when crystallization
Between 5 days, using the pattern of automatic photomicrograph system observation canavaline crystal, and count the conditions number of crystal.
Step 5: collecting and freezing protein crystal, the diffraction of crystal is carried out using household x-ray diffractometer, is collected simultaneously
Analyze diffraction data.By observation and statistics, it is as follows to obtain result: being knife by the above-mentioned crystal of X-ray diffraction experimental verification
Legumin albumin crystal, macroscopically can produce X-ray diffraction phenomenon, and the resolution ratio of diffraction is Wherein test
The crystallization condition number of group canavaline is 24, compared with control group (crystallization number is 12), to be crosslinked lysozyme crystal, peroxide
Change hydrogen enzyme, protease K, thaumatin matter, the screening success rate that chymotrypsinogen A II crystal is seed crystal, which have, substantially to be mentioned
It is high.
Claims (4)
1. a kind of make the method that seed crystal improves crystallization of protein success rate using crosslinked protein crystals, it is characterised in that including under
State step:
The first step grows protein crystal, and is chemically crosslinked to protein crystal, and obtained crosslinked protein crystals are molten
In ultrapure water, concentration is 5-20mg/ml, and seed crystal solution is made；
Second step instills the seed crystal solution of 0.5-2 μ l, naturally dry crystallization plates in each sitting drop hole of crystallization plates；
Third step carries out crystallization condition screening to protein using screening reagent box, prepares protein solution first；Then to knot
The pond liquid of 80 μ l is added in each deep hole of brilliant plate, screening crystallizing agent and protein solution are mixed according to the mass ratio of 1:1
To crystallization solution, the crystallization drop of 2 μ l is instilled to the sitting drop hole of crystallization plates, seals crystallization plates, and in the crystallization temperature of 277-293K
Degree lower crystallization 3-7 days；
4th step, the pattern of view protein crystal count the crystallization condition number of protein crystal；It collects and diffraction protein is brilliant
Body obtains diffraction data.
2. a kind of side for making seed crystal raising crystallization of protein success rate using crosslinked protein crystals according to claim 1
Method, it is characterised in that: the seed crystal solution selects crosslinking lysozyme as seed crystal.
3. a kind of side for making seed crystal raising crystallization of protein success rate using crosslinked protein crystals according to claim 1
Method, it is characterised in that: the seed crystal solution selects the crosslinking lysozyme that mass ratio is 1:1 and hydrogen peroxide enzyme crystal as seed
It is brilliant.
4. a kind of side for making seed crystal raising crystallization of protein success rate using crosslinked protein crystals according to claim 1
Method, it is characterised in that: the seed crystal solution select the crosslinking lysozyme crystal that mass ratio is 1:1:1:1:1, catalase,
Protease K, thaumatin matter and chymotrypsinogen AII crystal are as seed crystal.
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Non-Patent Citations (4)
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| A method to produce microseed stock for use in the crystallization of biological macromolecules;Joseph R. Luft,等。;《Acta Crystallographica》;19990202;第988-993页 |
| An automated microseed matrix-screening method for protein crystallization;Allan D’Arcy，等;《Acta Crystallographica》;20070214;第550-551、553页 |
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