CN102517379A - Formula of 96-condition kit for protein crystallization screening - Google Patents
Formula of 96-condition kit for protein crystallization screening Download PDFInfo
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- CN102517379A CN102517379A CN2011103997545A CN201110399754A CN102517379A CN 102517379 A CN102517379 A CN 102517379A CN 2011103997545 A CN2011103997545 A CN 2011103997545A CN 201110399754 A CN201110399754 A CN 201110399754A CN 102517379 A CN102517379 A CN 102517379A
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Abstract
The invention discloses a formula of a 96-condition kit for protein crystallization screening. The formula contains buffer solutions, salts, precipitants and additives, the types of the precipitants cover all nature compounds, and the formula contains various reagents commonly used for crystallization, so that the success rate of crystallization is enhanced, and the possibility of crystallization is increased; and the anion coverage of the salt matters is comprehensive, the concentration of the precipitants is relatively high, and then, the concentration of a protein sample required in an experiment can be greatly lowered, so that the cost for the experiment is saved, and the formula has a higher value for commercial popularization.
Description
Technical field
The present invention relates to a kind of 96 condition kits that are used for the crystallization of protein screening.
Background technology
At present, be the Index of Hampton Research company for the primary screening reagent box of selecting of the screening of protein crystallization condition
TM96 conditional filtering test kits, concrete composition is seen table 1.
Table 1:Index
TMThe condition component list
Use Index
TM18 kinds of protein are carried out the crystallization condition screening study; Be respectively cellulase, Chymotrypsin, myohaemoglobin, rnase iii, rnase X II, sweet protein, papoid, Proteinase K, opalescin, N,O-Diacetylmuramidase, chymotrypsinogen, canavalin(e), glycase, stomach en-, Lrax, glucophosphatase, oxyphorase, subtilisin; Statistics draws it and grows the crystalline success ratio and be merely 27.78% (5/18), and the result finds Index
TMScreening crystallization condition for most of protein (13/18) is invalid, and it is very necessary therefore to invent a kind of new box of screening reagent efficiently.
Summary of the invention
In order to overcome existing 96 conditional filtering test kits low defective of success ratio in the protein crystallization condition screening; The present invention provides a kind of new precipitation agent to use more comprehensive 96 condition crystallization of protein screening test kit, can effectively improve protein crystallization condition and be filtered into power.
The technical solution adopted for the present invention to solve the technical problems is: a kind of 96 condition kits of high efficiency protein crystallization condition screening, and called after CU-Index, it is specifically formed like table 2.Comprise four parts: damping fluid, salt, precipitation agent and additive.
Table 2:CU-Index 96 condition component lists
The invention has the beneficial effects as follows: 1. the kind of the selected precipitation agent of this test kit covers complete.Its type covers whole nature compounds---inorganics and organism, and comprises all ingredients that crystallization is commonly used, thereby has improved the crystalline success ratio, has increased the crystalline possibility; 2. negatively charged ion covers comprehensively in the salts substances selected for use of this test kit.Inorganic anion and organic anion exist simultaneously, all have related from low valency ion (1) to high valency ion (3); 3. the organism valency selected for use of this test kit covers extensively.(polyoxyethylene glycol 8000) all has and relates to from low-molecular-weight organic matter (ethanol) to high-molecular-weight organic material; 4. the used pH scope from 3.5 to 9.5 of this test kit has covered the crystallizable pH of most of protein interval; 5. the concentration of the selected precipitation agent of this test kit is higher relatively, thereby the concentration of desirable proteins quality sample will significantly reduce the saving experimental cost in therefore testing, and has higher business promotion and is worth.
Embodiment
Preparation CU-Index screening conditions: the condition A2 (0.1M Trisodium Citrate, pH 4.0,0.2M sodiumazide, 10%v/v PEG 8000 and 10%v/v PEG 4000) with preparation 100mL is an example.The first step: compound concentration is 1M, the sodium citrate buffer solution liquid storage of pH4.5; Second step: compound concentration is that the PEG 8000 of 50%w/v is the sodiumazide liquid storage of 2M with PEG 4000 and concentration; The 3rd step: with pipette, extract citrate buffer solution liquid storage 10mL, sodiumazide liquid storage 10mL, PEG 8000 and each 20mL of PEG 4000 liquid storages mixing in the beaker; Be settled to use 0.22 μ m again behind the 100mL membrane filtration in clean test tube, A2 condition preparation finishes.Same other conditions of method preparation.
Verify the effect of this test kit below with the crystallization experiment of some protein, with the Index of Hampton Research company
TMThe screening reagent box is as contrast.
Embodiment 1:
CU-Index 96 conditions with preparation are carried out conditional filtering to cellulase (cellulase).Concrete prescription is seen table 3.
The CU-Index 96 condition component lists that use among table 3: the embodiment 1
The first step: with the article No. of U.S. Sigma company is the cellulase of C0615; Being dissolved in pH is 7.0; Concentration is in N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid sodium damping fluid of 0.025mol/L, is mixed with the cellulase solution that concentration is 10mg/mL, with the filter membrane elimination impurity of 0.22 μ m.
Second step: present embodiment uses CU-Index 96 conditions of preparation that cellulase is carried out the crystallization condition screening.
The 3rd step: use to sit and drip plate, add the pond liquid of 90 μ L in every deep hole, use automatic liquor-transferring system that 1 μ L screening crystallizing agent and 1 μ L protein soln are mixed into crystallization drop, dropping in sits for a while drips in the hole, the sealing crystallization plates.
The 4th goes on foot: above-mentioned crystallization solution system is placed 6 days in 20 ℃ of temperature control boxs after, utilize automatic photomicrograph systematic observation and statistics cellulase crystalline condition number to occur.
Through statistics, it is following to obtain the result: the crystallization condition number of cellulase is 6, compares its success ratio with Index screening reagent box (the crystallization number is 0) and has improved no several times, is improved largely.
Embodiment 2:
CU-Index 96 conditions with preparation are carried out conditional filtering to Chymotrypsin (a-chymotrypsin).Concrete prescription is seen table 4.
The CU-Index 96 condition component lists that use among table 4: the embodiment 2
The first step: with the article No. of U.S. Sigma company is the Chymotrypsin of C4129; Being dissolved in pH is 7.0; Concentration is in N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid sodium damping fluid of 0.025mol/L, and being mixed with concentration is the Chymotrypsin solution of 10mg/mL, with the filter membrane elimination impurity of 0.22 μ m.
Second step: present embodiment uses CU-Index 96 conditions of preparation that Chymotrypsin is carried out the crystallization condition screening.
The 3rd step: use to sit and drip plate, add the pond liquid of 90 μ L in every deep hole, use automatic liquor-transferring system that 1 μ L screening crystallizing agent and 1 μ L protein soln are mixed into crystallization drop, dropping in sits for a while drips in the hole, the sealing crystallization plates.
The 4th goes on foot: above-mentioned crystallization solution system is placed 6 days in 20 ℃ of temperature control boxs after, utilize automatic photomicrograph systematic observation and statistics to grow Chymotrypsin crystalline condition number.
It is following to obtain the result through statistics: the crystallization condition number of Chymotrypsin is 4, compares (the crystallization number is 0) its success ratio with the contrast agents box and has improved no several times, is improved largely.
Embodiment 3:
CU-Index 96 conditions with preparation are carried out conditional filtering to myohaemoglobin (myoglobin).Concrete prescription is seen table 5.
The CU-Index 96 condition component lists that use among table 5: the embodiment 3
The first step: with the article No. of U.S. Sigma company is the myohaemoglobin of M0603; Being dissolved in pH is 7.0; Concentration is in N-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid sodium damping fluid of 0.025mol/L, and being mixed with concentration is the myohaemoglobin solution of 10mg/mL, with the filter membrane elimination impurity of 0.22 μ m.
Second step: present embodiment uses CU-Index 96 conditions of preparation that myohaemoglobin is carried out the crystallization condition screening.
The 3rd step: use to sit and drip plate, add the pond liquid of 90 μ L in every deep hole, use automatic liquor-transferring system that 1 μ L screening crystallizing agent and 1 μ L protein soln are mixed into crystallization drop, dropping in sits for a while drips in the hole, the sealing crystallization plates.
The 4th goes on foot: above-mentioned crystallization solution system is placed 6 days in 20 ℃ of temperature control boxs after, utilize automatic photomicrograph systematic observation and statistics to grow myohaemoglobin crystalline condition number.
It is following to obtain the result through statistics: the crystallization condition number of myohaemoglobin is 2, compares (the crystallization number is 0) its success ratio with the contrast agents box and has improved no several times, is improved largely.
Embodiment 4:
CU-Index 96 conditions with preparation are carried out conditional filtering to Proteinase K (proteinase K).Concrete prescription is seen table 6.
The CU-Index 96 condition component lists that use among table 6: the embodiment 4
The first step: with the article No. of U.S. Sigma company is the Proteinase K of P6556; Being dissolved in pH is 7.00; Concentration is in 0.025mol/LN-2-hydroxyethyl piperazine-N '-2-ethanesulfonic acid sodium damping fluid, and being mixed with concentration is the Proteinase K solution of 10mg/mL, with the filter membrane elimination impurity of 0.22 μ m.
Second step: present embodiment uses CU-Index 96 conditions of preparation that Proteinase K is carried out the crystallization condition screening.
The 3rd step: use to sit and drip plate, add the pond liquid of 90 μ L in every deep hole, use automatic liquor-transferring system that 1 μ L screening crystallizing agent and 1 μ L protein soln are mixed into crystallization drop, dropping in sits for a while drips in the hole, the sealing crystallization plates.
The 4th step: above-mentioned crystallization solution system is placed after 6 days in 20 ℃ of temperature control boxs, utilizes automatic photomicrograph systematic observation and counts Proteinase K crystalline condition number.
It is following to obtain the result through statistics: the crystallization condition number of Proteinase K is 13, compares (the crystallization number is 2) its success ratio with the contrast agents box and has improved 650%, is improved largely.
Except that the above embodiments; The inventive method is used for the screening of 14 kinds of other albumen crystallization conditions; Be respectively rnase iii, rnase X II, sweet protein, papoid, opalescin, N,O-Diacetylmuramidase, chymotrypsinogen, canavalin(e), glycase, stomach en-, Lrax, glucophosphatase, oxyphorase, subtilisin; It is 66.67% (12/18) that the crystallization of protein that obtains is filtered into power, with Index
TMComparing success ratio significantly improves.
Claims (1)
1. a condition crystallization of protein screening test kit prescription is characterized in that: comprise damping fluid, salt, precipitation agent and additive, specifically form as shown in the table.
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| CN201110399754.5A CN102517379B (en) | 2011-12-05 | 2011-12-05 | Formula of 96-condition kit for protein crystallization screening |
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| CN201110399754.5A CN102517379B (en) | 2011-12-05 | 2011-12-05 | Formula of 96-condition kit for protein crystallization screening |
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| CN102517379A true CN102517379A (en) | 2012-06-27 |
| CN102517379B CN102517379B (en) | 2014-02-05 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333219A (en) * | 2013-04-17 | 2013-10-02 | 西北工业大学 | Method for selecting buffer solution for protein crystallization |
| CN103436591A (en) * | 2013-08-30 | 2013-12-11 | 西北工业大学 | Kit for screening protein crystals by taking salt as precipitator |
| CN109870472A (en) * | 2019-01-10 | 2019-06-11 | 西北工业大学 | Utilize the method for kit formulation screening protein self assembly condition |
| CN111073870A (en) * | 2018-10-19 | 2020-04-28 | 南京理工大学 | Crystallization method of Roc mutant protein |
| CN114437165A (en) * | 2021-12-22 | 2022-05-06 | 厦门医学院 | 96-condition crystallization screening reagent composition suitable for small-molecule natural product and application thereof |
Citations (1)
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| CN101833009A (en) * | 2010-04-29 | 2010-09-15 | 浙江康特生物科技有限公司 | Double antibody complex retinol-binding protein assay kit |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101833009A (en) * | 2010-04-29 | 2010-09-15 | 浙江康特生物科技有限公司 | Double antibody complex retinol-binding protein assay kit |
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| 刘晶晶等: "蛋白质结晶过程的模拟优化和实验研究", 《中国优秀硕博论文数据库》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333219A (en) * | 2013-04-17 | 2013-10-02 | 西北工业大学 | Method for selecting buffer solution for protein crystallization |
| CN103436591A (en) * | 2013-08-30 | 2013-12-11 | 西北工业大学 | Kit for screening protein crystals by taking salt as precipitator |
| CN103436591B (en) * | 2013-08-30 | 2015-06-10 | 西北工业大学 | Kit for screening protein crystals by taking salt as precipitator |
| CN111073870A (en) * | 2018-10-19 | 2020-04-28 | 南京理工大学 | Crystallization method of Roc mutant protein |
| CN109870472A (en) * | 2019-01-10 | 2019-06-11 | 西北工业大学 | Utilize the method for kit formulation screening protein self assembly condition |
| CN114437165A (en) * | 2021-12-22 | 2022-05-06 | 厦门医学院 | 96-condition crystallization screening reagent composition suitable for small-molecule natural product and application thereof |
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| CN102517379B (en) | 2014-02-05 |
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