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A kind of dialysis method and application of recombinant human tissue plasminogen kinase derivative inclusion body enrichment solution

A technology of plasmin and prokinase, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of less solution treatment, waste of materials, and heavy workload, and achieve shortened dialysis time and improved dialysis efficiency Effect

Active Publication Date: 2022-07-22
SHANDONG EHUA BIOLOGICAL PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The solubilization of inclusion body proteins generally requires the addition of denaturants such as guanidine hydrochloride or urea. Before renaturation, guanidine hydrochloride needs to be removed. The conventional method is to use dialysis bags to dialyze the solubilization solution. For example, the patent CN102367264B provides a method for purifying inclusion body proteins. But this method solution processing capacity is few, and dialysis time is long, and workload is big, waste material, and efficiency is low, and effect is relatively poor compared with the method described in the present invention; The phenomenon that protein folding leads to the precipitation of the target protein

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  • A kind of dialysis method and application of recombinant human tissue plasminogen kinase derivative inclusion body enrichment solution
  • A kind of dialysis method and application of recombinant human tissue plasminogen kinase derivative inclusion body enrichment solution
  • A kind of dialysis method and application of recombinant human tissue plasminogen kinase derivative inclusion body enrichment solution

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Embodiment 1

[0028] The preparation of embodiment 1 solution

[0029] (1) Preparation of solubilizing dialysis internal fluid: prepare a solubilizing solution (6mol / L guanidine hydrochloride, 50mmol / L Tris, 1mmol / LEDTA·Na 2 , pH is 8.6); take rPA inclusion bodies and add them to the enrichment solution, mix the inclusion bodies and the enrichment solution according to the ratio of 1.5kg: 30L, and then add dithiothreitol (DTT) to it to a concentration of 40mmol / L, and use The pH of the NaOH solution was fine-tuned to 8.6, and after stirring for 2 h, the pH was adjusted to 3.0 with dilute hydrochloric acid to obtain the solubilized dialysis internal fluid, which was used for later use.

[0030] (2) Preparation of external dialysis fluid:

[0031] Dialysis external fluid 1 (4mol / L urea, 1mmol / L EDTA·Na 2 , pH3.0);

[0032] Dialysis external fluid 2 (5mol / L urea, 1mmol / L EDTA·Na 2 , pH3.0);

[0033] Dialysis external fluid 3 (6mol / L urea, 1mmol / L EDTA·Na 2 , pH3.0);

[0034] Dialysis ex...

Embodiment 2

[0038] Example 2 Dialysis of recombinant human tissue-type plasmin kinase derivative inclusion body enrichment solution by ultrafilter

[0039] Get 4L of the solubilizing dialysis inner fluid in Example 1, use the membrane package (2.5 square) with a cut-off of 10kD to carry out ultrafiltration phase change, first concentrate to 2L, add 2L of dialysis outer fluid 1 to mix and then concentrate to 2L, then Dialysis phase exchange was carried out with external dialysis fluid 1, and the permeation rate of the ultrafilter and the addition rate of external dialysis fluid were maintained at 2.5 L / h, and the dialysis phase exchange was performed for 4 hours. During 2h25min, 3h15min, and 4h of the dialysis phase exchange process, 10ml of the inner dialysate fluid was taken, respectively, and the samples A1, A2, and A3 were labeled.

Embodiment 3

[0040] Example 3 Dialysis of recombinant human tissue-type plasmin kinase derivative inclusion body enrichment solution by ultrafilter

[0041] Get 4L of solubilizing dialysis inner fluid in Example 1, use the membrane package (2.5 square) that the retention capacity is 10kD to carry out ultrafiltration phase change, first concentrate to 2L, add 2L of dialysis outer fluid 2 to mix and then concentrate to 2L, then Dialysis phase exchange was carried out with external dialysis fluid 2, and the permeation rate of the ultrafilter and the addition rate of external dialysis fluid were both maintained at 2.5 L / h, and the dialysis phase exchange was performed for 4 hours. During 2h25min, 3h15min, and 4h of the dialysis phase exchange process, 10ml of the inner dialysate was taken respectively, and the samples B1, B2, and B3 were marked.

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Abstract

The invention provides a dialysis method and application of a recombinant human tissue-type plasmin kinase derivative inclusion body enrichment solution. Compared with dialysis using a dialysis bag, the dialysis efficiency of the invention is significantly improved, and the dialysis time is shortened to The original 1 / 5, the amount of external dialysis fluid is reduced to 1 / 20 of the original, the rPA solution after dialysis is clear and transparent, and the removal of small molecular substances such as guanidine hydrochloride and reducing agent DTT is more thorough; the rPA solution obtained after ultrafilter dialysis The overall yield of rPA protein produced in subsequent steps increased by 20%. The invention provides a simple and efficient method for the renaturation and purification of large-scale and industrialized inclusion bodies, especially inclusion bodies containing more disulfide bonds.

Description

technical field [0001] The invention relates to the technical field of renaturation and purification of inclusion bodies, in particular to a dialysis method and application of a recombinant human tissue plasmin kinase derivative inclusion body enrichment solution. Background technique [0002] Recombinant human tissue plasmin kinase derivative (rPA) is suitable for thrombolytic therapy for adult myocardial infarction caused by coronary infarction, and can improve ventricular function after myocardial infarction. At present, rPA has become one of the representatives of the third-generation thrombolytic drugs because of its strong thrombolytic effect, rapid onset of action, long maintenance time, high recanalization rate, small adverse reactions, and convenient use. rPA is a deletion mutant of native tissue plasminogen kinase (tPA), which retains only the tPA serine protease domain (related to enzyme activity) and the Kringle2 domain (related to fibrin-oriented properties). B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64
CPCC12N9/6459C12Y304/21068
Inventor 秦绪才解福生郝向慧庞甲佩姜栋吴化斌徐兴贵孙云磊宋义健田立帅付蓝天
Owner SHANDONG EHUA BIOLOGICAL PHARMA
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