CN105062988B - A kind of preparation method of biotin labeling inorganic pyrophosphatase - Google Patents
A kind of preparation method of biotin labeling inorganic pyrophosphatase Download PDFInfo
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- CN105062988B CN105062988B CN201510416726.8A CN201510416726A CN105062988B CN 105062988 B CN105062988 B CN 105062988B CN 201510416726 A CN201510416726 A CN 201510416726A CN 105062988 B CN105062988 B CN 105062988B
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- C12Y306/01001—Inorganic diphosphatase (3.6.1.1)
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Abstract
The invention belongs to biological technical field, be specifically related to the preparation method of a kind of biotin labeling inorganic pyrophosphatase.Step is as follows: dissolves inorganic pyrophosphatase, activated biotin, in conjunction with inorganic pyrophosphatase, dialysis, measures protein content, preserve.The inorganic pyrophosphatase that the present invention prepares, is highly suitable to be applied in Manganic pyrophosphate complex initiation reaction, and the high activity of inorganic pyrophosphatase can ensure that the accuracy of sequencing reaction.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the preparation method of a kind of biotin labeling inorganic pyrophosphatase.
Background technology
Biotinylation (biotinylation) refers to the process being covalently attached to by biotin on protein, nucleic acid or other molecule.Owing to the molecular weight of biotin is little (molecular weight is 244.31), biotinylation reaction is quick, efficient and is difficult to disturbed.Biotinylated molecule by biotin and Streptavidin, Avidin interaction, and can not affected by high heat, pH, proteolysis.Biotin is special and efficient with the combination of Streptavidin, Avidin, and therefore this interaction has a wide range of applications in many fields of biotechnology.It addition, multiple Biotinylated molecules can crosslink forms the albumen that a scientific research personnel is interested, allow simultaneously and multiple Streptavidins, Avidin, neutral affinity prime combine, add the detection sensitivity to this albumen.Application in addition with substantial amounts of Biotinylated molecules has to be developed.
Inorganic pyrophosphatase is the hydrolytic enzyme with pyrophosphoric acid as substrate, and this enzyme is widely present in nature, and participation synthesizes the hydrolysis of the pyrophosphoric acid formed in the multiple metabolic pathways such as saccharide, nucleic acid and protein.Many utilizes the metabolic activity of ATP can produce pyrophosphoric acid, and the pyrophosphoric acid produced in organism metabolic activity can be decomposed by inorganic pyrophosphatase, prevents it from building up.IPPA plays a role the most in vivo, prevents inorganic pyrophosphate from affecting the homergy of organism, and recently as the rise of biocatalyzer, its external function the most gradually displays.
Inorganic pyrophosphatase is that catalysis pyrophosphoric acid is hydrolyzed to ortho-phosphoric class of enzymes.Pyrophosphoric acid is the by-product of the building-up process of some metabolic process such as DNA and RNA polymerization, the synthesis of activation of amino acid aminoacyl tRNA, the synthesis of beta oxidation acyl coenzyme A of fatty acid, the synthesis of cellulosic electrode UDPG and Starch synthesis ADP-glucose.Inorganic pyrophosphatase can make above-mentioned reaction carry out to compound direction by decomposing pyrophosphoric acid and can provide energy for many biosynthesis reactions, thus inorganic pyrophosphatase is necessary to life.
Inorganic pyrophosphatase just can be carried out solid phase affinity interaction with Avidin after biotin modification.Biotin and Avidin are the affinity ligands that a pair specificity is at a relatively high, and have that dissociation rate is slow, be susceptible to the features such as interference.At present, biotinylated means have chemical modification and bio-modification, but the biotinylated protein activity after modifying is the highest.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 being badly in need of finding a kind of workable, that its lytic activity is high preparation method.
Summary of the invention
It is an object of the invention to the preparation method of a kind of biotin labeling inorganic pyrophosphatase, comprise the following steps:
(1) Tris-HCl buffer solution inorganic pyrophosphatase is used；
(2) activated biotin；
(3) balance biotin is to less than 10 DEG C；
(4) biotin of activation is dissolved in dimethylformamide and oxolane mixed solvent, adds inorganic pyrophosphatase, according to biotin with inorganic pyrophosphatase mol ratio (22-25): the ratio of 1 mixes, stirring, obtain mixed solution；
(5) using Tris-HCl buffer to dialyse mixed solution, temperature is 4 DEG C, goes the removal of impurity and free biotin；
(6) the absorbance measurement protein content of 280nm is used；
(7) adding protective agent, 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, making inorganic pyrophosphate enzyme concentration is 1.5-2mg/ml.
Described activated biotin uses EDC/NHS and organic solvent, and biotin, the mol ratio of EDC and NHS are 4:5:4, and organic solvent is dimethylformamide, and purity is more than analytical pure.
Described whipping temp is 4 DEG C, continues more than 5 hours, preferably 8 hours.
Described dialysis procedure continues more than 12 hours, preferably 18 hours.
Described dialysis procedure, Tris-HCl buffer volume is more than 10 times of mixed solution, more than Tris-HCl buffer exchange once.
Described protective agent contains Tris-HCl buffer；Possibly together with one or more in glycine, histidine, mannitol, DTT, EDTA, sodium azide and Polyethylene Glycol.
Described protective agent preferably comprises Tris-HCl buffer, glycine, histidine, DTT, EDTA and sodium azide.
After adding protective agent, the mixed liquor of protective agent with enzyme is regulated to pH 8.0.
In the present invention, dimethylformamide and oxolane mixed solvent can make biotin and inorganic pyrophosphatase be dissolved in wherein, the most efficiently, sufficiently combine.The biotinylation inorganic pyrophosphatase prepared, is highly suitable to be applied in Manganic pyrophosphate complex initiation reaction, and the high activity of inorganic pyrophosphatase can ensure that the accuracy of sequencing reaction.In sequencing reaction, enzyme synergism can be vulcanized with pyrophosphatase and ATP, carry out that flux is high, read the sequencing reaction grown up.
Detailed description of the invention
Embodiment
1
(1) using the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concentration is 1.8mg/ml；
(2) activated biotin: by 1mol biotin, 1.25mol EDC, 1mol NHS adds in the analytically pure dimethylformamide of 1L, carries out the activation of biotin；
(3) balance biotin is to 10 DEG C；
(4) being dissolved in by the biotin 0.24mol of activation in the mixed solvent being mixed to get by 90ml dimethylformamide and 100ml oxolane, add inorganic pyrophosphatase 0.01mol, mixing, stir 8 hours, temperature is 4 DEG C, obtains mixed solution；
(5) using 1L Tris-HCl buffer to dialyse 100ml mixed solution, continue 18 hours, temperature is 4 DEG C, goes the removal of impurity and free biotin, and Tris-HCl buffer was changed once at 6 hours；
(6) the absorbance measurement protein content of 280nm is used；
(7) adding the protective agent of enzyme volume 2 times, protective agent contains 10mM Tris-HCl buffer, 20% glycine, 30% histidine, 0.15mM DTT, 0.08mM EDTA and 0.06mM sodium azide, regulates pH to 8.0, preserves to-16 DEG C.
Embodiment
2
(1) using the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concentration is 1.5mg/ml；
(2) activated biotin: by 1mol biotin, 1.25mol EDC, 1mol NHS adds in the analytically pure dimethylformamide of 1L, carries out the activation of biotin；
(3) balance biotin is to 4 DEG C；
(4) being dissolved in by the biotin 0.22mol of activation in the mixed solvent being mixed to get by 90ml dimethylformamide and 100ml oxolane, add inorganic pyrophosphatase 0.01mol, mixing, stir 5 hours, temperature is 4 DEG C, obtains mixed solution；
(5) using 1L Tris-HCl buffer to dialyse 80ml mixed solution, continue 12 hours, temperature is 4 DEG C, goes the removal of impurity and free biotin, and Tris-HCl buffer was changed once at 4 hours；
(6) the absorbance measurement protein content of 280nm is used；
(7) adding the protective agent of enzyme volume 1.5 times, protective agent contains 10mM Tris-HCl buffer and 0.1mM DTT, regulates pH to 8.0, preserves to-20 DEG C.
Embodiment
3
(1) using the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concentration is 1.6mg/ml；
(2) activated biotin: by 1mol biotin, 1.25mol EDC, 1mol NHS adds in analytically pure dimethylformamide, carries out the activation of biotin；
(3) balance biotin is to 4 DEG C；
(4) being dissolved in dimethylformamide and oxolane mixed solvent by the biotin 0.25mol of activation, add inorganic pyrophosphatase 0.01mol, mixing, stir 6 hours, temperature is 4 DEG C, obtains mixed solution；
(5) using 1L Tris-HCl buffer to dialyse 90ml mixed solution, continue 15 hours, temperature is 4 DEG C, goes the removal of impurity and free biotin, and Tris-HCl buffer was changed at 4 hours and 8 hours；
(6) the absorbance measurement protein content of 280nm is used；
(7) adding the protective agent of enzyme volume 2 times, protective agent contains 10mM Tris-HCl buffer, 20% glycine, 0.2mM mannitol, 0.15mM DTT and 0.08mM EDTA, regulates pH to 8.0, preserves to-70 DEG C.
Embodiment
4
(1) using the Tris-HCl buffer solution inorganic pyrophosphatase of pH8.5, inorganic pyrophosphate enzyme concentration is 2mg/ml；
(2) activated biotin: by 1mol biotin, 1.25mol EDC, 1mol NHS adds in analytically pure dimethylformamide, carries out the activation of biotin；
(3) balance biotin is to 0 DEG C；
(4) being dissolved in dimethylformamide and oxolane mixed solvent by the biotin 0.23mol of activation, add inorganic pyrophosphatase 0.01mol, mixing, stir 7 hours, temperature is 4 DEG C, obtains mixed solution；
(5) using 1L Tris-HCl buffer to dialyse 85ml mixed solution, continue 13 hours, temperature is 4 DEG C, goes the removal of impurity and free biotin, and Tris-HCl buffer was changed at 2 hours and 9 hours；
(6) the absorbance measurement protein content of 280nm is used；
(7) adding and the isopyknic protective agent of enzyme, protective agent contains 10mM Tris-HCl buffer, 25% glycine, 0.15mM DTT and 0.2mM Polyethylene Glycol, regulates pH to 8.0, preserves to-80 DEG C.
Claims (8)
1. the preparation method of a biotin labeling inorganic pyrophosphatase, it is characterised in that comprise the following steps:
(1) using Tris-HCl buffer solution inorganic pyrophosphatase, making inorganic pyrophosphate concentration is 1.5-1.8mg/mL；
(2) using EDC and NHS activated biotin, be dissolved in organic solvent dimethylformamide, the purity of organic solvent is more than analytical pure, and wherein biotin, the mol ratio of EDC and NHS are 4:5:4；
(3) balance biotin is to less than 10 DEG C；
(4) biotin of activation is dissolved in dimethylformamide and oxolane mixed solvent, adds inorganic pyrophosphatase, according to biotin with inorganic pyrophosphatase mol ratio (22-25): the ratio of 1 mixes, stirring, obtain mixed solution；
(5) using Tris-HCl buffer to dialyse mixed solution, temperature is 4 DEG C, goes the removal of impurity and free biotin；
(6) the absorbance measurement protein content of 280nm is used；
(7) adding protective agent, 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 characterised in that in described step (1) and 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, it is characterised in that described whipping temp is 4 DEG C, continues more than 5 hours
4. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 3, it is characterised in that described whipping temp is 4 DEG C, continues 8 hours.
5. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterised in that described dialysis procedure continues more than 12 hours.
6. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 5, it is characterised in that described dialysis procedure continues 18 hours.
7. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterised in that described dialysis procedure, Tris-HCl buffer volume is more than 10 times of mixed solution, more than Tris-HCl buffer exchange once.
8. the preparation method of biotin labeling inorganic pyrophosphatase as claimed in claim 1, it is characterised in that described protective agent contains Tris-HCl buffer；Possibly together with one or more in glycine, histidine, mannitol, DTT, EDTA, sodium azide and Polyethylene Glycol.
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| CN105400749B (en) * | 2015-12-30 | 2017-05-31 | 北京中科紫鑫科技有限责任公司 | A kind of expression of 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 |
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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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 (1)
| Title |
|---|
| The use of avidin-biotin interaction in immunoenzymatic techniques.;Jean-Luc Guesdon等;《The Journal of Histochemistry and Cytochemistry》;19790930;第27卷(第8期);参见第1132页右栏第1段"Biotin-labeling"，表1， 第1133页右栏第2段 * |
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