CN104946611B - A kind of purification process of ATP sulfurylase - Google Patents
A kind of purification process of ATP sulfurylase Download PDFInfo
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- CN104946611B CN104946611B CN201510414393.5A CN201510414393A CN104946611B CN 104946611 B CN104946611 B CN 104946611B CN 201510414393 A CN201510414393 A CN 201510414393A CN 104946611 B CN104946611 B CN 104946611B
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Abstract
The invention belongs to biological technical field, be specifically related to a kind of purification process of ATP sulfurylase. Comprise the following steps: adjust pH value; Add slaine; Add stabilizing agent; Organic solvent deposit; Aseptic filtration; Ni-sepharose purification; Do you adopt superdex? 200 carry out molecular exclusion; Carry out freeze drying. The product purity obtaining with the inventive method is high, and the suitable pyrophosphoric acid that is applied to checks order.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of purification process of ATP sulfurylase.
Background technology
ATP sulfurylase (ATPsulfurylase, ATPS) is extensively present in plant, animal and microorganism, but its biological action in the middle of various organisms is slightly different. In plant and part microorganism, it is the key enzyme of the catalysis sulfate anabolic reaction first step. Under the catalysis of ATPS, sulfate ion reacts with ATP, produces adenine-5 '-phosphoric acid sulfuric acid ATS and pyrophosphate PPi. And in some chemoautotrophic bacteriums and chemolithotrophic bacteria, ATPS is the enzyme that catalysis Oxidation of Hydrogen Sulfide generates mineral sulfates final step, its effect is that catalysis ATS and PPi reaction generate ATP and sulfate ion, the i.e. back reaction of above-mentioned reaction. ATPS from the organisms such as penicillium chrysogenum, mouse liver, cabbage, has extracted and purifying obtains, and the gene of coding ATPS is also cloned and out and in suitable host expressed from prokaryotes, eucaryote, animal and plant. The ATPS structure difference of separate sources, the ATPS in Escherichia coli sources is heterodimer and needs GTP to activate, the ATPS of yeast, fungi and plant origin is monomer or homology oligomer. Aspect molecular biology, study at present the relation of its 26S Proteasome Structure and Function by the means such as amino acid modified, deepen the understanding to ATPS mechanism of action. Coupled ATP sulfurylase can be applied to a lot of aspects, as: PPi quantitatively, RNA mensuration, DNA sequencing, archaeal dna polymerase enzyme activity determination etc.
Gene order-checking is one of forward position core technology of contemporary Life Sci-Tech, and pyrophosphoric acid PCR sequencing PCR has wherein adopted ATP sulfurylase to detect, and reads to grow up, and accuracy is high. In existing ATP sulfurylase purification technique, the product purity obtaining is lower, easily affects the precision of sequencing reaction, can not meet the needs of gene order-checking.
Summary of the invention
The purification process that an object of the present invention is a kind of ATP sulfurylase, comprises the following steps:
(1) adjust pH value: regulate the pH value of crude enzyme liquid to 7.5-8.0;
(2) add slaine: add magnesium salts and calcium salt;
(3) add stabilizing agent;
(4) organic solvent deposit;
(5) aseptic filtration;
(6) carry out ni-sepharose purification;
(7) carry out the dialysis of protein liquid;
(8) adopt superdex200 to carry out molecular exclusion;
(9) carry out freeze drying.
Described magnesium salts is one or more in magnesium sulfate, magnesium chloride and magnesium nitrate, and described calcium salt is one or more in calcium sulfate, calcium chloride and potassium nitrate.
Described step adds magnesium salts and calcium salt in (2), and the final concentration that makes magnesium ion is 20-30mmol/L, and the final concentration of calcium ion is 10-20mmol/L.
Described stabilizing agent is one or more in glycerine, ethylene glycol, soybean polyoses and DTT. The final concentration of stabilizing agent is 5-15g/L.
The organic solvent that described organic solvent deposit uses is one or more in methyl alcohol, acetonitrile and acetone.
Described aseptic filtration is used 0.22 μ m filter membrane.
In the step of described purification process, keeping the environment temperature of ATP sulfurylase is below 16 DEG C.
Described freeze drying adopts-20 ~-16 DEG C.
Described ATP sulfurylase is with His-tag.
When described ni-sepharose purification, use the imidazoles of 20-500mM to carry out gradient elution.
Described dialysis is used Tris-HCl buffer solution.
Described superdex200 is GE molecular exclusion prepacked column.
The magnesium salts adding in the present invention and calcium salt can increase the stability of ATP sulfurylase, keep its high activity in purge process simultaneously. The activity of further having guaranteed ATP sulfurylase that adds of stabilizing agent is not suffered a loss in purge process. DEAESepharoseCL-6B ion exchange column, superdex200 molecular exclusion and organic solvent deposit combine, and can effectively remove foreign protein and other impurity molecules in enzyme liquid. The firefly ATP sulfurylase vigor that uses this method purifying to obtain is large, and purity is high, and catalytic efficiency is high, can guarantee to react precision, meets the needs of gene order-checking, suitable being widely used in gene order-checking.
Detailed description of the invention
Embodiment 1
(1) adjust pH value: the pH value to 7.6 that regulates crude enzyme liquid;
(2) get 1L luciferase crude enzyme liquid, add magnesium chloride 10mmol, magnesium sulfate 15mmol and calcium chloride 16mmol;
(3) add soybean polyoses 6g and DTT 3g;
(4) organic solvent deposit: slowly add 15ml acetone and 20ml acetonitrile, stir, leave standstill 10 minutes, 13000rpm is centrifugal, and supernatant discarded adds 10mlTris-HCl buffer solution, stirs, and 13000rpm is centrifugal, collects supernatant;
(5) use 0.22 μ m filter membrane to carry out aseptic filtration;
(6) carry out ni-sepharose purification, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, when wash-out, use the imidazoles of 20-500mM to carry out gradient elution;
(7) use Tris-HCl buffer solution to carry out the dialysis of protein liquid;
(8) adopt GE molecular exclusion prepacked column superdex200 to carry out molecular exclusion, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, obtain enzyme liquid 100ml;
(9) freeze drying, temperature is-16 DEG C.
Above step is all carried out under 16 DEG C of environment.
Embodiment 2
(1) adjust pH value: the pH value to 8.0 that regulates crude enzyme liquid;
(2) get 1L luciferase crude enzyme liquid, add magnesium nitrate 20mmol, calcium sulfate 12mmol, calcium chloride 8mmol;
(3) add glycerine 5g;
(4) organic solvent deposit: slowly add 20ml methyl alcohol and 12ml acetonitrile, stir, leave standstill 10 minutes, 13000rpm is centrifugal, and supernatant discarded adds 10mlTris-HCl buffer solution, stirs, and 13000rpm is centrifugal, collects supernatant;
(5) use 0.22 μ m filter membrane to carry out aseptic filtration;
(6) carry out ni-sepharose purification, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, when wash-out, use 20-500mM imidazoles to carry out gradient elution;
(7) use Tris-HCl buffer solution to carry out the dialysis of protein liquid;
(8) adopt GE molecular exclusion prepacked column superdex200 to carry out molecular exclusion, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, obtain enzyme liquid 100ml;
(9) freeze drying, temperature is-20 DEG C.
Carry out at 4 DEG C above step (6) and (8), and other steps are all carried out under 16 DEG C of environment.
Embodiment 3
(1) adjust pH value: the pH value to 7.5 that regulates crude enzyme liquid;
(2) get 1L luciferase crude enzyme liquid, add magnesium chloride 30mmol, calcium sulfate 2mmol, calcium chloride 4mmol and calcium nitrate 4mmol;
(3) add glycerine 8g, ethylene glycol 2g and soybean polyoses 5g;
(4) organic solvent deposit: slowly add 30ml acetonitrile, stir, leave standstill 10 minutes, 13000rpm is centrifugal, and supernatant discarded adds 10mlTris-HCl buffer solution, stirs, and 13000rpm is centrifugal, collects supernatant;
(5) use 0.22 μ m filter membrane to carry out aseptic filtration;
(6) carry out ni-sepharose purification, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, when wash-out, use 20-500mM imidazoles to carry out gradient elution;
(7) use Tris-HCl buffer solution to carry out the dialysis of protein liquid;
(8) adopt GE molecular exclusion prepacked column superdex200 to carry out molecular exclusion, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, obtain enzyme liquid 100ml;
(9) freeze drying, temperature is-18 DEG C.
Carry out at 4 DEG C above step (6) and (8), and other steps are all carried out under 16 DEG C of environment.
Embodiment 4
(1) adjust pH value: the pH value to 7.8 that regulates crude enzyme liquid;
(2) get 1L luciferase crude enzyme liquid, add ammonium chloride 2mmol, potassium sulfate 3mmol, magnesium chloride 15mmol, magnesium sulfate 6mmol, calcium nitrate 5mmol and calcium sulfate 12mmol;
(3) add ethylene glycol 5g and DTT 8g;
(4) organic solvent deposit: slowly add 16ml methyl alcohol and 20ml acetone, stir, leave standstill 10 minutes, 13000rpm is centrifugal, and supernatant discarded adds 10mlTris-HCl buffer solution, stirs, and 13000rpm is centrifugal, collects supernatant;
(5) use 0.22 μ m filter membrane to carry out aseptic filtration;
(6) carry out ni-sepharose purification, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, when wash-out, use 20-500mM imidazoles to carry out gradient elution;
(7) use Tris-HCl buffer solution to carry out the dialysis of protein liquid;
(8) adopt GE molecular exclusion prepacked column superdex200 to carry out molecular exclusion, use the Tris-HCl buffer solution of pH8.0 to carry out column equilibration and wash-out, obtain enzyme liquid 100ml;
(9) freeze drying, temperature is-20 DEG C.
Above step is all carried out under 16 DEG C of environment.
Comparative example 1
(1) get 1L luciferase crude enzyme liquid, add magnesium sulfate 3mmol and calcium chloride 5mmol;
(2) add glycerine 1g;
(3) organic solvent deposit: slowly add 6ml methyl alcohol, stir, 13000rpm is centrifugal, and supernatant discarded adds 10mlTris-HCl buffer solution, stirs, and 13000rpm is centrifugal, collects supernatant;
(4) use 0.22 μ m filter membrane to carry out aseptic filtration;
(5) carry out ni-sepharose purification, use the Tris-HCl buffer solution of pH7.1 to carry out column equilibration and wash-out, when wash-out, use 20-500mM imidazoles to carry out gradient elution, dry;
Above step is all carried out under 26 DEG C of environment.
The mensuration of embodiment 1, embodiment 2 and comparative example 1 being carried out to product enzyme purity, result is as follows:
Table 1
As shown in Table 1, the product purity obtaining in the present invention is significantly higher than comparative example 1, reached 98.81%, and comparative example 1 product purity being obtained by different condition is only 95.02%. Highly purified ATP sulfurylase can be guaranteed carrying out smoothly of sequencing reaction.
Above-mentioned detailed description is for the illustrating of one of them possible embodiments of the present invention, and this embodiment is not in order to limit the scope of the claims of the present invention, and the equivalence that all the present invention of disengaging do is implemented or changed, and all should be contained in the scope of technical solution of the present invention.
Claims (9)
1. a purification process for ATP sulfurylase, is characterized in that, comprises the following steps:
(1) adjust pH value: regulate the pH value of crude enzyme liquid to 7.5-8.0;
(2) add slaine: add magnesium salts and calcium salt;
(3) add stabilizing agent;
(4) organic solvent deposit;
(5) aseptic filtration;
(6) carry out ni-sepharose purification;
(7) carry out the dialysis of protein liquid;
(8) adopt superdex200 to carry out molecular exclusion;
(9) carry out freeze drying.
2. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, described magnesium salts is one or more in magnesium sulfate, magnesium chloride and magnesium nitrate, and described calcium salt is one or more in calcium sulfate, calcium chloride and calcium nitrate.
3. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, described step adds magnesium salts and calcium salt in (2), and the final concentration that makes magnesium ion is 20-30mmol/L, and the final concentration of calcium ion is 10-20mmol/L.
4. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, described stabilizing agent is one or more in glycerine, ethylene glycol, soybean polyoses and DTT.
5. the purification process of ATP sulfurylase as claimed in claim 4, is characterized in that, described stabilizing agent is soybean polyoses and DTT.
6. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, the organic solvent that described organic solvent deposit uses is one or more in methyl alcohol, acetonitrile and acetone.
7. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, described aseptic filtration is used 0.22 μ m filter membrane.
8. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, in the step of described purification process, keeping the environment temperature of ATP sulfurylase is below 16 DEG C.
9. the purification process of ATP sulfurylase as claimed in claim 1, is characterized in that, described freeze drying adopts-20 ~-16 DEG C.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006089660A2 (en) * | 2005-02-24 | 2006-08-31 | Lohmann & Rauscher Gmbh & Co. Kg | Method for cleaning marine collagen and the treatment thereof to form porous sponges |
| CN103966175A (en) * | 2014-05-06 | 2014-08-06 | 国家海洋局第三海洋研究所 | Extraction separation technology of marine organism source SOD (superoxide dismutase) |
| CN104593339A (en) * | 2014-12-31 | 2015-05-06 | 唯美度科技（北京）有限公司 | Bio-active enzyme and extraction process thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006089660A2 (en) * | 2005-02-24 | 2006-08-31 | Lohmann & Rauscher Gmbh & Co. Kg | Method for cleaning marine collagen and the treatment thereof to form porous sponges |
| CN103966175A (en) * | 2014-05-06 | 2014-08-06 | 国家海洋局第三海洋研究所 | Extraction separation technology of marine organism source SOD (superoxide dismutase) |
| CN104593339A (en) * | 2014-12-31 | 2015-05-06 | 唯美度科技（北京）有限公司 | Bio-active enzyme and extraction process thereof |
Non-Patent Citations (1)
| Title |
|---|
| 萤火虫荧光素酶及酿酒酵母ATP硫酸化酶在大肠杆菌中的表达，纯化及其应用;蒋庆庆;《南京农业大学硕士学位论文》;20120630;第1-71页全文 * |
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