CN105062988A - Preparation method of biotin labeled inorganic pyrophosphatase - Google Patents
Preparation method of biotin labeled inorganic pyrophosphatase Download PDFInfo
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- CN105062988A CN105062988A CN201510416726.8A CN201510416726A CN105062988A CN 105062988 A CN105062988 A CN 105062988A CN 201510416726 A CN201510416726 A CN 201510416726A CN 105062988 A CN105062988 A CN 105062988A
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- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
- C12Y306/01001—Inorganic diphosphatase (3.6.1.1)
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Abstract
The invention belongs to the technical field of biotechnology and particularly relates to a preparation method of biotin labeled inorganic pyrophosphatase. The method includes the steps of dissolving inorganic pyrophosphatase, activating biotin, combining the biotin with the inorganic pyrophosphatase, performing dialyzing, measuring a protein content, and storing. The inorganic pyrophosphatase prepared by the method is very suitable for application in pyrosequencing reactions; high activity of the inorganic pyrophosphatase ensures accurateness of the pyrosequencing reactions.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of biotin labeling inorganic pyrophosphatase.
Background technology
Biotinylation (biotinylation) refers to the process be covalently attached to by vitamin H on protein, nucleic acid or other molecule.Due to the molecular weight little (molecular weight is 244.31) of vitamin H, biotinylation reaction is fast, efficiently and not easily disturbed.Biotinylated molecular energy is done mutually by vitamin H and Streptavidin, avidin, and not by the impact of high heat, pH, proteolysis.Vitamin H and Streptavidin, avidin in conjunction with special and efficient, therefore this does to have a wide range of applications in many fields of biotechnology mutually.In addition, multiple Biotinylated molecules can be cross-linked to form an interested albumen of scientific research personnel, and permission simultaneously and multiple Streptavidin, avidin, neutral affinity prime combine, and add the detection sensitivity to this albumen.In addition the application of a large amount of Biotinylated molecules is also had to have to be developed.
Inorganic pyrophosphatase take tetra-sodium as the lytic enzyme of substrate, and this enzyme is extensively present in nature, participates in the hydrolysis of the tetra-sodium formed in the multiple pathways metabolisms such as synthesis carbohydrate, nucleic acid and protein.The Metabolic activity of many ATP of utilization can produce tetra-sodium, and the tetra-sodium produced in organism Metabolic activity can decompose by inorganic pyrophosphatase, prevents it from excessively accumulating.IPPA not only plays a role in vivo, prevents inorganic pyrophosphate from affecting the eubolism of organism, and in recent years along with the rise of biological catalyst, its external function also displays gradually.
Inorganic pyrophosphatase is that catalysis tetra-sodium is hydrolyzed to ortho-phosphoric class of enzymes.Tetra-sodium is some metabolic process as the by product of the building-up process of the synthesis of β-oxidation aliphatic alcohol of DNA and RNA polymerization, the synthesis of activation of amino acids aminoacyl-tRNA, lipid acid, the synthesis of cellulosic electrode UDPG and Starch synthesis ADP-glucose.Inorganic pyrophosphatase can make above-mentioned reaction carry out and can react for many biosynthesizing providing energy to compound direction by decomposing tetra-sodium, and thus inorganic pyrophosphatase is that life is necessary.
Inorganic pyrophosphatase just can be carried out solid phase affinity interaction with avidin by after biotin modification.Vitamin H and avidin are the affinity ligands that a pair specificity is quite high, and have the features such as dissociation rate slowly, is not easily disturbed.At present, biotinylated means have chemically modified and bio-modification, but biotinylated protein after modifying is active general not high.Biotinylated inorganic pyrophosphatase can be applicable in Manganic pyrophosphate complex initiation reaction, and enzyme work can be directly connected to the accuracy of sequencing reaction, so be badly in need of finding one workable, the preparation method that its lytic activity is high.
Summary of the invention
An object of the present invention is a kind of preparation method of biotin labeling inorganic pyrophosphatase, comprise the following steps:
(1) Tris-HCl buffer solution inorganic pyrophosphatase is used;
(2) activated biotin;
(3) below vitamin H to 10 DEG C is balanced;
(4) vitamin H of activation is dissolved in dimethyl formamide and tetrahydrofuran (THF) mixed solvent, adds inorganic pyrophosphatase, according to vitamin H and inorganic pyrophosphatase mol ratio (22-25): the ratio of 1 mixes, stir, obtain mixing solutions;
(5) use Tris-HCl damping fluid to dialyse to mixing solutions, temperature is 4 DEG C, removes impurity and free biotin;
(6) the absorbance measurement protein content of 280nm is used;
(7) add protective material, stored refrigerated is to-80 DEG C--16 DEG C.
In described step (1)-step (5), Tris-HCl pH of cushioning fluid is 8.5.
Described dissolving inorganic pyrophosphatase, makes inorganic pyrophosphate enzyme concn be 1.5-2mg/ml.
Described activated biotin uses EDC/NHS and organic solvent, and the mol ratio of vitamin H, EDC and NHS is 4:5:4, and organic solvent is dimethyl formamide, and purity is more than analytical pure.
Described whipping temp is 4 DEG C, continues more than 5 hours, is preferably 8 hours.
Described dialysis procedure continues more than 12 hours, is preferably 18 hours.
Described dialysis procedure, Tris-HCl damping fluid volume is more than 10 times of mixing solutions, more than Tris-HCl buffer exchange once.
Described protective material contains Tris-HCl damping fluid; Also containing one or more in glycine, Histidine, N.F,USP MANNITOL, DTT, EDTA, sodium azide and polyoxyethylene glycol.
Described protective material is preferably containing Tris-HCl damping fluid, glycine, Histidine, DTT, EDTA and sodium azide.
After adding protective material, the mixed solution of protective material and enzyme is adjusted to pH8.0.
In the present invention, dimethyl formamide and tetrahydrofuran (THF) mixed solvent can make vitamin H and inorganic pyrophosphatase be dissolved in wherein, efficiently, fully combine wherein.The biotinylation inorganic pyrophosphatase prepared, be applicable to very much being applied in Manganic pyrophosphate complex initiation reaction, the high reactivity of inorganic pyrophosphatase can guarantee the accuracy of sequencing reaction.In sequencing reaction, can act synergistically with Pyrophosphate phosphohydrolase and ATP sulfuration enzyme, carry out flux high, read the sequencing reaction of growing up.
Embodiment
embodiment 1
(1) use the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concn is 1.8mg/ml;
(2) activated biotin: by 1mol vitamin H, 1.25molEDC, 1molNHS add in the analytically pure dimethyl formamide of 1L, carry out the activation of vitamin H;
(3) vitamin H to 10 DEG C is balanced;
(4) be dissolved in by the vitamin H 0.24mol of activation in the mixed solvent be mixed to get by 90ml dimethyl formamide and 100ml tetrahydrofuran (THF), add inorganic pyrophosphatase 0.01mol, mixing, stir 8 hours, temperature is 4 DEG C, obtains mixing solutions;
(5) use 1LTris-HCl damping fluid to dialyse to 100ml mixing solutions, continue 18 hours, temperature is 4 DEG C, removes impurity and free biotin, and Tris-HCl damping fluid was changed once at 6 hours;
(6) the absorbance measurement protein content of 280nm is used;
(7) add the protective material of enzyme volume 2 times, protective material contains 10mMTris-HCl damping fluid, 20% glycine, 30% Histidine, 0.15mMDTT, 0.08mMEDTA and 0.06mM sodium azide, regulates pH to 8.0, is saved to-16 DEG C.
embodiment 2
(1) use the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concn is 1.5mg/ml;
(2) activated biotin: by 1mol vitamin H, 1.25molEDC, 1molNHS add in the analytically pure dimethyl formamide of 1L, carry out the activation of vitamin H;
(3) vitamin H to 4 DEG C is balanced;
(4) be dissolved in by the vitamin H 0.22mol of activation in the mixed solvent be mixed to get by 90ml dimethyl formamide and 100ml tetrahydrofuran (THF), add inorganic pyrophosphatase 0.01mol, mixing, stir 5 hours, temperature is 4 DEG C, obtains mixing solutions;
(5) use 1LTris-HCl damping fluid to dialyse to 80ml mixing solutions, continue 12 hours, temperature is 4 DEG C, removes impurity and free biotin, and Tris-HCl damping fluid was changed once at 4 hours;
(6) the absorbance measurement protein content of 280nm is used;
(7) add the protective material of enzyme volume 1.5 times, protective material contains 10mMTris-HCl damping fluid and 0.1mMDTT, regulates pH to 8.0, is saved to-20 DEG C.
embodiment 3
(1) use the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concn is 1.6mg/ml;
(2) activated biotin: by 1mol vitamin H, 1.25molEDC, 1molNHS add in analytically pure dimethyl formamide, carry out the activation of vitamin H;
(3) vitamin H to 4 DEG C is balanced;
(4) be dissolved in dimethyl formamide and tetrahydrofuran (THF) mixed solvent by the vitamin H 0.25mol of activation, add inorganic pyrophosphatase 0.01mol, mixing, stir 6 hours, temperature is 4 DEG C, obtains mixing solutions;
(5) use 1LTris-HCl damping fluid to dialyse to 90ml mixing solutions, continue 15 hours, temperature is 4 DEG C, removes impurity and free biotin, and Tris-HCl damping fluid is changed 4 hours and 8 hours;
(6) the absorbance measurement protein content of 280nm is used;
(7) add the protective material of enzyme volume 2 times, protective material contains 10mMTris-HCl damping fluid, 20% glycine, 0.2mM N.F,USP MANNITOL, 0.15mMDTT and 0.08mMEDTA, regulates pH to 8.0, is saved to-70 DEG C.
embodiment 4
(1) use the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concn is 2mg/ml;
(2) activated biotin: by 1mol vitamin H, 1.25molEDC, 1molNHS add in analytically pure dimethyl formamide, carry out the activation of vitamin H;
(3) vitamin H to 0 DEG C is balanced;
(4) be dissolved in dimethyl formamide and tetrahydrofuran (THF) mixed solvent by the vitamin H 0.23mol of activation, add inorganic pyrophosphatase 0.01mol, mixing, stir 7 hours, temperature is 4 DEG C, obtains mixing solutions;
(5) use 1LTris-HCl damping fluid to dialyse to 85ml mixing solutions, continue 13 hours, temperature is 4 DEG C, removes impurity and free biotin, and Tris-HCl damping fluid is changed 2 hours and 9 hours;
(6) the absorbance measurement protein content of 280nm is used;
(7) add with enzyme isopyknic protective material, protective material contains 10mMTris-HCl damping fluid, 25% glycine, 0.15mMDTT and 0.2mM polyoxyethylene glycol, regulates pH to 8.0, is saved to-80 DEG C.
Claims (8)
1. a preparation method for biotin labeling inorganic pyrophosphatase, is characterized in that, comprises the following steps:
(1) Tris-HCl buffer solution inorganic pyrophosphatase is used;
(2) activated biotin;
(3) below vitamin H to 10 DEG C is balanced;
(4) vitamin H of activation is dissolved in dimethyl formamide and tetrahydrofuran (THF) mixed solvent, adds inorganic pyrophosphatase, according to vitamin H and inorganic pyrophosphatase mol ratio (22-25): the ratio of 1 mixes, stir, obtain mixing solutions;
(5) use Tris-HCl damping fluid to dialyse to mixing solutions, temperature is 4 DEG C, removes impurity and free biotin;
(6) the absorbance measurement protein content of 280nm is used;
(7) add protective material, stored refrigerated is to-80 DEG C--16 DEG C.
2. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterized in that, in described step (1)-step (5), Tris-HCl pH of cushioning fluid is 8.5.
3. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, is characterized in that, described dissolving inorganic pyrophosphatase, makes inorganic pyrophosphate enzyme concn be 1.5-2mg/ml.
4. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterized in that, described activated biotin uses EDC/NHS and organic solvent, and the mol ratio of vitamin H, EDC and NHS is 4:5:4, organic solvent is dimethyl formamide, and purity is more than analytical pure.
5. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterized in that, described whipping temp is 4 DEG C, continues more than 5 hours, is preferably 8 hours.
6. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, is characterized in that, described dialysis procedure continues more than 12 hours, is preferably 18 hours.
7. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterized in that, described dialysis procedure, Tris-HCl damping fluid volume is more than 10 times of mixing solutions, more than Tris-HCl buffer exchange once.
8. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterized in that, described protective material contains Tris-HCl damping fluid; Also containing one or more in glycine, Histidine, N.F,USP MANNITOL, DTT, EDTA, sodium azide and polyoxyethylene glycol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400749A (en) * | 2015-12-30 | 2016-03-16 | 北京中科紫鑫科技有限责任公司 | Biotinylation inorganic pyrophosphatase expression method |
| CN105483096A (en) * | 2015-12-31 | 2016-04-13 | 北京中科紫鑫科技有限责任公司 | Storage reagent for biotinylation inorganic pyrophosphatase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102858965A (en) * | 2010-04-30 | 2013-01-02 | 霍夫曼-拉罗奇有限公司 | System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102858965A (en) * | 2010-04-30 | 2013-01-02 | 霍夫曼-拉罗奇有限公司 | System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus |
Non-Patent Citations (2)
| Title |
|---|
| JEAN-LUC GUESDON等: "The use of avidin-biotin interaction in immunoenzymatic techniques.", 《THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY》 * |
| THERMO SCIENTIFIC公司: "《Avidin-biotin technical handbook》", 31 December 2009 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105400749A (en) * | 2015-12-30 | 2016-03-16 | 北京中科紫鑫科技有限责任公司 | Biotinylation inorganic pyrophosphatase expression method |
| CN105483096A (en) * | 2015-12-31 | 2016-04-13 | 北京中科紫鑫科技有限责任公司 | Storage reagent for biotinylation inorganic pyrophosphatase |
| CN105483096B (en) * | 2015-12-31 | 2017-04-05 | 北京中科紫鑫科技有限责任公司 | A kind of storage reagent of biotinylation inorganic pyrophosphatase |
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| CN105062988B (en) | 2016-08-24 |
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Granted publication date: 20160824 Termination date: 20190716 |