CN103172727A - Recombinant human proinsulin renaturation and purification method - Google Patents
Recombinant human proinsulin renaturation and purification method Download PDFInfo
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- CN103172727A CN103172727A CN2013100658153A CN201310065815A CN103172727A CN 103172727 A CN103172727 A CN 103172727A CN 2013100658153 A CN2013100658153 A CN 2013100658153A CN 201310065815 A CN201310065815 A CN 201310065815A CN 103172727 A CN103172727 A CN 103172727A
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Abstract
The invention discloses a method for performing high performance size exclusion chromatography (HPSEC) and purifying recombinant human proinsulin renaturation. The method comprises the following steps: (1) performing ultrasonic cell disruption on the proinsulin-containing thallus cell, washing and dissolving the buffer solution in strong denaturant (such as 8mol/L or 6-7 mol/L guanidine hydrochloride) thereby obtaining the recombinant human proinsulin subjected to crude separation; (2) directly feeding the proinsulin-containing denaturated leaching liquor under the optimized chromatographic condition, wherein the purity of target fraction rhPI obtained through one-step chromatography by employing urea concentration linear gradient elute can be over 96 percent, and the mass recovery rate is 56.8 percent; and (3) further desalting the proinsulin-containing original fraction, thereby obtaining the renaturated and purified high-purity and activity fraction. The recombinant human proinsulin obtained by the method can be converted into insulin through digestion on a chromatographic column or digestion in a solution, and the chromatographic process is easy to operate, high in repeatability and easy for large-scale production.
Description
Technical field
The present invention relates to a kind of efficient Size Exclusion Chromatograph SEC (HPSEC or SEC) method renaturation and insulinogenic method of purification of recombinant human utilized, belong to metaprotein renaturation technical field in biological medicine.
Background technology
The strand peptide that proinsulin (Proinsulin, PI) is comprised of 86 amino acid, the two ends of its C peptide are connected with the C-terminal of B chain with the N-terminal of INSULIN A chain by two alkaline amino acid residues separately.The folding curling correct docking that guarantees three pairs of disulfide linkage of proinsulin molecule.In the Regular Insulin preparation, obtaining activated Regular Insulin after employing proinsulin method direct enzyme cutting has been a kind of method of maturation.Therefore, the active recombinant insulinum primary that has that obtains the high quality rate of recovery (is called for short: the prerequisite that is rhPI) the preparation recombinant human insulin.
Robert B. M etc. [Robert B. M,
et al, Protein Expres Purif,1999,15:308-313] once utilized ion-exchange chromatography (IEC) method and affinity chromatography (AFC) method to carry out purifying to the rhPI inclusion body protein, cut and obtain RPLC method analysis for product with dilution method renaturation, enzyme afterwards.And Gusarova V etc. [Gusarova V,
et al, J Chromatogr A.2007,1176:157-162] reported and first utilized dilution method renaturation rhPI, then, after carrying out two-step purifying by IEC method and SEC method respectively, by enzyme, cut and be converted to Regular Insulin.Aforesaid method all utilizes the dilution method renaturation, is difficult to carry out on a large scale the preparation of protein drug.
Summary of the invention
The object of the present invention is to provide a kind of efficiently, renaturation and the insulinogenic method of purification of recombinant human fast; can improve mass recovery and the purity of recombinant insulinum primary; and can be directly used in enzyme and be cut to Regular Insulin, be applicable to the large-scale production of Regular Insulin.
Implementation procedure of the present invention is as follows:
The renaturation of recombinant insulinum primary and purification process comprise the following steps:
(1) intestinal bacteria (
e.coli) in the somatic cells of the recombinant insulinum primary (rhPI) of expressing after centrifugal, ultrasonication respectively with damping fluid I and II wash, centrifugal being dissolved in strong sex change damping fluid obtain the sex change extract that contains recombinant insulinum primary;
Described damping fluid I is 10～20 mmol/L PBS, 0.5～1 mmol/L EDTA, and pH 7.0～7.5;
Described damping fluid II is 0.5～2.0 mol/L urea, 0.5～1 mmol/L EDTA, and 0.5～1.0 mol/L NaCl, 10～20 mmol/L PBS, pH 7.0～7.5;
Described strong sex change damping fluid is 6.0～8.0 mol/L ureas, 10～20 mmol/LDTT, 0.5～1 mmol/LEDTA, pH7.0～8.0; Or 6～7 mol/L Guanidinium hydrochlorides, 10～20mmol/LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0;
(2) will contain the sex change extract direct injection of recombinant insulinum primary to mobile phase A (20～40 mmol/LPBS, 0～8.0 mol/L urea, pH6.0～7.0) chromatographic column of balance, then with containing 20～40 mmol/LPBS, the Mobile phase B linear gradient elution of pH6.0～7.0, flow velocity is 0.3～0.7mL/min, collects the target peak chromatographic fraction and obtains the chromatographic fraction that renaturation and purifying contain recombinant insulinum primary;
(3) the chromatographic fraction direct injection containing the proinsulin human by step (2), with 0～10 mmol/LPBS, the further desalination of chromatographic column of the moving phase balance of pH6.0～7.0, continue wash-out 20～40 min, flow velocity 0.5～1 mL/min, obtain recombinant insulinum primary.
In above-mentioned steps (2), chromatographic column is TSK gel G2000SWXL type (7.8mm * 300mm) or Superde * 200 10/300GL types (10 mm * 300 mm).
After described in above-mentioned steps (2), type of elution can adopt the mobile phase A balance, with Mobile phase B linear gradient elution 20～40 min, extend 5～10 min.
Chromatographic column described in above-mentioned steps (3) is HiTrap Desalting column.
Advantage of the present invention: the method for urea concentration gradient Size Exclusion Chromatograph SEC to recombinant insulinum primary renaturation and purifying of utilizing provided by the invention, solved protein molecular directly from the high density denaturing agent be diffused into moving phase, and can not provide the problem of the albumen refolding environment of a mitigation, also overcome other chromatographic process costs high, be difficult to the shortcoming of amplifying simultaneously.After the present invention has added the small molecules urea in moving phase, in elution process, for the albumen refolding provides the environment of a gentleness, promote that protein folding becomes structure closely, retention time extends, and annealing efficiency and mass recovery also are improved.And more be beneficial to follow-up enzyme and cut and obtain Regular Insulin after desalting column, maturation simple to operate, stable, the stationary phase cost is lower, is easy to control and amplify
The accompanying drawing explanation
Before and after Fig. 1 renaturation and purifying, the SDS-PAGE of rhPI analyzes
Band M: protein molecular weight standard; Band 1: the recombinant insulinum primary of abduction delivering not; Band 2: after recombinant insulinum primary abduction delivering 5 h; Band 3:8.0 mol/L urea extract; Band 4: purifying and the renaturation of urea concentration gradient SEC method to rhPI; Band 5: secondarily purified to a step cut desalination; Band 6: standard Regular Insulin; Band 7: enzyme is cut the Regular Insulin of conversion;
When Fig. 2 adds different urea concentrations to renaturation and purifying color atlas (A) and the mass recovery (B) of rhPI
In figure (A), X-coordinate is retention time (time), and ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU);
In figure (B), X-coordinate is for adding urea concentration (Urea concentration), and ordinate zou is mass recovery (Mass recovery);
Curve 1:0.0 mol/L urea; Curve 2:2.0 mol/L urea; Curve 3:4.0 mol/L urea; Curve 4:6.0 mol/L urea; Curve 5:8.0 mol/L urea;
Fig. 3 is to renaturation and the purifying of rhPI under different elution mode
In figure, X-coordinate is retention time (time), and ordinate zou is wavelength 280nm place ultraviolet absorption value (mAU);
Curve 1: add 0.0 mol/L urea isocratic elution in mobile phase A; Curve 2: add 2.0 mol/L urea isocratic elutions in mobile phase A; Curve 3: add 2.0 mol/L urea gradient elutions in mobile phase A.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with embodiment and accompanying drawing, but protection domain is not by this restriction.In embodiment, equipment used or raw material all can obtain from market.
(1) preparation of recombinant insulinum primary sex change extract
With
e.colias host cell expression recombinant insulinum primary (rhPI) albumen, again by zymocyte liquid through centrifugal, cytoclasis and use respectively damping fluid I(20 mmol/L PBS, 1 mmol/L EDTA, pH 7.4) and II(2.0 mol/L urea, 1 mmol/L EDTA, 1.0 mol/L NaCl, 20 mmol/L PBS, pH 7.4) washing, and after centrifugal thick purifying, the inclusion body obtained is dissolved in the 8.0mol/L urea, 20 mmol/LDTT, 1.0 mmol/LEDTA, pH 8.0(or 6～7 mol/L Guanidinium hydrochlorides, 10～20mmol/LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0) in denaturing agent, sex change extract concentration is 9.92 mg/mL, rhPI purity is more than 65.7 %, electrophoresis is shown in band 3 in Fig. 1.
(2) efficient Size Exclusion Chromatograph SEC renaturation and purification of Recombinant proinsulin human
With mobile phase A (20 mmol/ LPBS, 4.0mol/L urea, pH6.5) balance HPSEC post (TSK gel G2000SWXL, 300 * 7.8 mm I.D.), sex change extract direct injection 200 μ L carry out the linear gradient elution of 30 min, until Mobile phase B (20 mmol/L PBS under flow velocity 0.3 mL/min, pH 6.5) be 100%, extend 10 min.The rhPI mass recovery is 42.5%, and purity reaches more than 95%, and color atlas is shown in the curve 3 of Fig. 2 (A), and mass recovery is shown in Fig. 2 (B).
(3) with the secondarily purified recombinant insulinum primary of desalination chromatographic column
Use 0-10 mmol/LPBS, the desalination chromatographic column of the moving phase balance of pH6.0-7.0 (HiTrap Desalting column), the chromatographic fraction I direct injection containing the proinsulin human that renaturation and purifying are obtained, continue wash-out 30 min, flow velocity 1.0ml/min.The purity of rhPI has reached more than 99%.See band 5 in Fig. 1.
Similar to Example 1, different is that step (2) is with not adding urea 100% mobile phase A balance TSK gel G2000SWXL type HPSEC post (300 * 7.8 mm I.D.), sex change extract direct injection 200 μ L, carry out 30 min linear gradient elutions under flow velocity 0.5 mL/min, until Mobile phase B (20.0 mmol/L PBS, pH 6.5) be 100%, extend 10min.The rhPI mass recovery is 39.7%, more than purity to 95%.Color atlas is shown in the curve 1 of Fig. 2 (A), and mass recovery is shown in Fig. 2 (B).
Similar to Example 1, under the condition of step (2) that different is urea concentration and optimize chromatography condition in moving phase, with 100% mobile phase A (20.0 mmol/ LPBS, 2.0 mol/L urea, pH7.0) balance TSK gel G2000SWXL type HPSEC post (300 * 7.8 mm I.D.), sex change extract direct injection 200 μ L, carry out 30 min urea concentration linear gradient elutions under flow velocity 0.5 mL/min, until Mobile phase B (20.0 mmol/L PBS, pH 7.0) be 100%, extend 10 min.The rhPI mass recovery is 56.8%, more than purity to 96%.Color atlas is shown in the curve 2 of Fig. 2 (A), and mass recovery is shown in Fig. 2 (B).
Similar to Example 1, under the condition of step (2) that different is urea concentration and optimize chromatography condition in moving phase, with 100% mobile phase A (20.0 mmol/ LPBS, 2.0 mol/L urea, pH7.0) the SEC post of balance Superde * 200 10/300GL types (10 * 300 mm I.D.), sex change extract direct injection 200 μ L, carry out 30 min urea concentration linear gradient elutions under flow velocity 0.5 mL/min, until Mobile phase B (20.0 mmol/L PBS, pH 7.0) be 100%, extend 10min.The rhPI mass recovery is 51.7%, more than purity to 96%.
Similar to Example 1, that different is (the 20.0 mmol/ LPBS of 100% mobile phase A for step (2), 2.0 mol/L urea, pH7.5) balance TSK gel G2000SWXL type HPSEC post (300 * 7.8 mm I.D.), sex change extract direct injection 200 μ L, when the change flow velocity is 0.5 mL/min, carry out respectively urea concentration isocratic elution or urea concentration linear gradient elution, latter rhPI mass recovery can improve 10%.Color atlas is shown in Fig. 3 curve 2 and curve 3.
The high purity recombinant insulinum primary that obtains in 37 ℃ of solution or control under the condition of 37 ℃ of column temperatures of HPSEC post, is cut to 30 min with trypsinase and protaminase coordinated enzyme, make rhPI be converted into hI.Electrophoresis is shown in band 7 in Fig. 1.
Claims (4)
1. the renaturation of recombinant insulinum primary and purification process is characterized in that comprising the following steps:
(1) the somatic cells of the recombinant insulinum primary of expression in escherichia coli after centrifugal, ultrasonication respectively with damping fluid I and II washing, centrifugally obtain the sex change extract that contains recombinant insulinum primary in being dissolved in strong sex change damping fluid;
Described damping fluid I is 10～20 mmol/L PBS, 0.5～1 mmol/L EDTA, and pH 7.0～7.5;
Described damping fluid II is 0.5～2.0 mol/L urea, 0.5～1 mmol/L EDTA, and 0.5～1.0 mol/L NaCl, 10～20 mmol/L PBS, pH 7.0～7.5;
Described strong sex change damping fluid is 6.0～8.0 mol/L ureas, 10～20mmol/LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0; Or 6～7 mol/L Guanidinium hydrochlorides, 10～20mmol/LDTT, 0.5～1mmol/LEDTA, pH7.0～8.0;
(2) will contain the chromatographic column of the sex change extract direct injection of recombinant insulinum primary to the mobile phase A balance, then with containing 20～40 mmol/LPBS, the Mobile phase B linear gradient elution of pH6.0～7.0, flow velocity is 0.3～0.7mL/min, collects the target peak chromatographic fraction and obtains the chromatographic fraction that renaturation and purifying contain recombinant insulinum primary;
Mobile phase A is: 20～40 mmol/LPBS, 0～8.0 mol/L urea, pH6.0～7.0;
(3) the chromatographic fraction direct injection containing recombinant insulinum primary by step (2), with 0～10 mmol/LPBS, the further desalination of chromatographic column of the moving phase balance of pH6.0～7.0, continue wash-out 20～40 min, flow velocity 0.5～1 mL/min, obtain recombinant insulinum primary.
2. the renaturation of recombinant insulinum primary according to claim 1 and purification process is characterized in that:
In step (2), chromatographic column is TSK gel G2000SWXL type or Superde * 200 10/300GL types.
3. the renaturation of recombinant insulinum primary according to claim 1 and purification process, is characterized in that: after type of elution described in step (2) adopts the mobile phase A balance, with Mobile phase B linear gradient elution 20～40 min, extend 5～10 min.
4. the renaturation of recombinant insulinum primary according to claim 1 and purification process, it is characterized in that: chromatographic column described in step (3) is HiTrap Desalting column.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104479029A (en) * | 2014-12-22 | 2015-04-01 | 中国人民解放军第四军医大学 | Purification and renaturation method of Notch ligand Delta-like1 fusion protein |
| CN104841375A (en) * | 2015-05-08 | 2015-08-19 | 西北大学 | Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon |
| CN105732820A (en) * | 2016-03-17 | 2016-07-06 | 通化东宝药业股份有限公司 | Renaturation method of restructured human insulin prokaryotic-fusion protein |
| CN114994192A (en) * | 2022-04-29 | 2022-09-02 | 南京汉欣医药科技有限公司 | Liquid phase analysis method for quantitatively detecting precursor content in insulin and analogues thereof |
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2013
- 2013-03-04 CN CN2013100658153A patent/CN103172727A/en active Pending
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| 李晓红 等: "重组人胰岛素制备工艺", 《四川大学学报（工程科学版）》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104479029A (en) * | 2014-12-22 | 2015-04-01 | 中国人民解放军第四军医大学 | Purification and renaturation method of Notch ligand Delta-like1 fusion protein |
| CN104841375A (en) * | 2015-05-08 | 2015-08-19 | 西北大学 | Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon |
| CN105732820A (en) * | 2016-03-17 | 2016-07-06 | 通化东宝药业股份有限公司 | Renaturation method of restructured human insulin prokaryotic-fusion protein |
| CN114994192A (en) * | 2022-04-29 | 2022-09-02 | 南京汉欣医药科技有限公司 | Liquid phase analysis method for quantitatively detecting precursor content in insulin and analogues thereof |
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Application publication date: 20130626 |