CN102517379B - 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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- CN102517379B CN102517379B CN201110399754.5A CN201110399754A CN102517379B CN 102517379 B CN102517379 B CN 102517379B CN 201110399754 A CN201110399754 A CN 201110399754A CN 102517379 B CN102517379 B CN 102517379B
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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 for crystallization of protein screening.
Background technology
At present, the primary screening reagent box of selecting of screening for protein crystallization condition is the Index of Hampton Research company
tM96 conditional filtering test kits, concrete composition is in Table 1.
Table 1:Index
tMcondition component list
Use Index
tM18 kinds of protein are carried out to 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), amylase, stomach en-, cytochrome C, glucophosphatase, oxyphorase, subtilisin, statistics show that its success ratio that grows crystal is only 27.78% (5/18), found that Index
tMscreening crystallization condition for most of protein (13/18) is invalid, therefore invents a kind of new efficient screening reagent box very necessary.
Summary of the invention
In order to overcome existing 96 conditional filtering test kits low defect of success ratio in protein crystallization condition screening, the invention provides a kind of new precipitation agent and use more comprehensive 96 condition crystallization of protein screening test kits, 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 specifically forms as 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 conventional, thereby has improved the success ratio of crystallization, has increased the possibility of crystallization; 2. in the selected salts substances of this test kit, negatively charged ion covers comprehensively.Inorganic anion and organic anion exist simultaneously, from low valency ion (1), to high valency ion (3), all have related; 3. the selected organism valency of this test kit covers extensively.From low-molecular-weight organic matter (ethanol), to high-molecular-weight organic material (PEG 8000), all have and relate to; 4. this test kit pH scope from 3.5 to 9.5 used, has covered the crystallizable pH of most of protein interval; 5. the relative concentration of the selected precipitation agent of this test kit is higher, thereby in therefore testing, the concentration of desirable proteins quality sample will significantly reduce saving experimental cost, has higher business promotion and is worth.
Embodiment
Preparation CU-Index screening conditions: be example to prepare the condition A2 (0.1M Trisodium Citrate, pH 4.0,0.2M sodiumazide, 10%v/v PEG 8000 and 10%v/v PEG 4000) of 100mL.The first step: compound concentration is 1M, the sodium citrate buffer solution liquid storage of pH4.5; Second step: the sodiumazide liquid storage that the PEG 8000 that compound concentration is 50%w/v and PEG 4000 and concentration are 2M; The 3rd step: mix in beaker with pipette, extract citrate buffer solution liquid storage 10mL, sodiumazide liquid storage 10mL, PEG 8000 and each 20mL of PEG 4000 liquid storages, after being settled to 100mL, use the membrane filtration of 0.22 μ m in clean test tube, the preparation of A2 condition is complete again.Same method is prepared other conditions.
With the crystallization experiment of some protein, verify the effect of this test kit below, with the Index of Hampton Research company
tMscreening reagent box in contrast.
Embodiment 1:
By CU-Index 96 conditions of preparation, cellulase (cellulase) is carried out to conditional filtering.Specifically fill a prescription in Table 3.
The CU-Index 96 condition component lists that use in table 3: embodiment 1
The first step: the cellulase that is C0615 by the article No. of U.S. Sigma company, being dissolved in pH is 7.0, concentration is in the HEPES 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: the present embodiment is used CU-Index 96 conditions of preparation to carry out crystallization condition screening to cellulase.
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 hole, sealing crystallization plates.
The 4th step: above-mentioned crystallization solution system is placed after 6 days in 20 ℃ of temperature control boxs, utilize automatic photomicrograph systematic observation statistics to occur the condition number of cellulase crystal.
Through statistics, obtain result as follows: the crystallization condition number of cellulase is 6, compare its success ratio with Index screening reagent box (crystallization number is 0) and improved without several times, be improved largely.
Embodiment 2:
By CU-Index 96 conditions of preparation, Chymotrypsin (a-chymotrypsin) is carried out to conditional filtering.Specifically fill a prescription in Table 4.
The CU-Index 96 condition component lists that use in table 4: embodiment 2
The first step: the Chymotrypsin that is C4129 by the article No. of U.S. Sigma company, being dissolved in pH is 7.0, concentration is in the HEPES 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: the present embodiment is used CU-Index 96 conditions of preparation to carry out crystallization condition screening to Chymotrypsin.
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 hole, sealing crystallization plates.
The 4th step: above-mentioned crystallization solution system is placed after 6 days in 20 ℃ of temperature control boxs, utilize automatic photomicrograph systematic observation statistics to grow the condition number of Chymotrypsin crystal.
Through statistics, obtain result as follows: the crystallization condition number of Chymotrypsin is 4, compares (crystallization number is 0) its success ratio improved without several times, be improved largely with contrast agents box.
Embodiment 3:
By CU-Index 96 conditions of preparation, myohaemoglobin (myoglobin) is carried out to conditional filtering.Specifically fill a prescription in Table 5.
The CU-Index 96 condition component lists that use in table 5: embodiment 3
The first step: the myohaemoglobin that is M0603 by the article No. of U.S. Sigma company, being dissolved in pH is 7.0, concentration is in the HEPES 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: the present embodiment is used CU-Index 96 conditions of preparation to carry out crystallization condition screening to myohaemoglobin.
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 hole, sealing crystallization plates.
The 4th step: above-mentioned crystallization solution system is placed after 6 days in 20 ℃ of temperature control boxs, utilize automatic photomicrograph systematic observation statistics to grow the condition number of myohaemoglobin crystal.
Through statistics, obtain result as follows: the crystallization condition number of myohaemoglobin is 2, compares (crystallization number is 0) its success ratio improved without several times, be improved largely with contrast agents box.
Embodiment 4:
By CU-Index 96 conditions of preparation, Proteinase K (proteinase K) is carried out to conditional filtering.Specifically fill a prescription in Table 6.
The CU-Index 96 condition component lists that use in table 6: embodiment 4
The first step: the Proteinase K that is P6556 by the article No. of U.S. Sigma company, 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: the present embodiment is used CU-Index 96 conditions of preparation to carry out crystallization condition screening to Proteinase K.
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 hole, 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 the condition number of Proteinase K crystal.
Through statistics, obtain result as follows: the crystallization condition number of Proteinase K is 13, compares (crystallization number is 2) its success ratio improved 650% with contrast agents box, is improved largely.
Except the above embodiments, the inventive method is 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), amylase, stomach en-, cytochrome C, glucophosphatase, oxyphorase, subtilisin, it is 66.67% (12/18) that the crystallization of protein obtaining is filtered into power, with Index
tMcomparing success ratio significantly improves.
Claims (1)
1. 96 condition crystallization of protein screening test kit formulas, is characterized in that: comprise damping fluid, salt, precipitation agent and additive, shown in concrete table composed as follows
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| CN102517379B true CN102517379B (en) | 2014-02-05 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103333219A (en) * | 2013-04-17 | 2013-10-02 | 西北工业大学 | Method for selecting buffer solution for protein crystallization |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| 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 |
Non-Patent Citations (4)
| Title |
|---|
| 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》.2011,全文. |
| 尹大川等.筛选蛋白质结晶条件的试剂盒研发进展.《生物化学和生物物理进展》.2001,第38卷(第3期),全文. |
| 筛选蛋白质结晶条件的试剂盒研发进展;尹大川等;《生物化学和生物物理进展》;20011231;第38卷(第3期);全文 * |
| 蛋白质结晶过程的模拟优化和实验研究;刘晶晶等;《中国优秀硕博论文数据库》;20110418;全文 * |
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