Dialysis method of recombinant human tissue type plasminogen kinase derivative inclusion body enhancement solution, and application

A technology of fibrinolytic enzyme and inclusion body, which is applied in the direction of biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of less solution treatment, long dialysis time, waste of materials, etc., shorten the dialysis time and improve the dialysis efficiency , Improve the effect of clarity

Active Publication Date: 2021-03-19
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

Method used

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  • Dialysis method of recombinant human tissue type plasminogen kinase derivative inclusion body enhancement solution, and application
  • Dialysis method of recombinant human tissue type plasminogen kinase derivative inclusion body enhancement solution, and application
  • Dialysis method of recombinant human tissue type plasminogen kinase derivative inclusion body enhancement solution, and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 solution

[0029] (1) Preparation of solubilized dialysis inner fluid: prepare solubilizing solution (6mol / L guanidine hydrochloride, 50mmol / L Tris, 1mmol / LEDTA·Na 2 , pH is 8.6); take rPA inclusion body and join in the enhancement solution, inclusion body and enhancement solution are mixed according to the ratio of 1.5kg: 30L, then add dithiothreitol (DTT) to it to concentration to be 40mmol / L, use Finely adjust the pH of the NaOH solution to 8.6, stir for 2 hours, and then adjust the pH to 3.0 with dilute hydrochloric acid to obtain the solubilized dialysis inner solution, which is set aside.

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

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

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

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

[0034] Dialysis external fluid 4 (7mol / L urea, 1mmol / L EDTA·Na 2 , pH3.0);

[0035...

Embodiment 2

[0038] Example 2 Using an ultrafilter to dialyze the recombinant human tissue-type plasmin kinase derivative inclusion body enhancement solution

[0039] Take 4L of the solubilized dialysis internal solution in Example 1, use a membrane bag (2.5 square meters) with a cut-off of 10kD to perform ultrafiltration and phase change, first concentrate to 2L, add 2L of dialysis external solution 1 to mix and then concentrate to 2L, and then Use dialysis fluid 1 for dialysis phase change, keep the ultrafilter permeation rate and dialysis fluid addition rate at 2.5 L / h, and dialysis phase change for 4 hours. During 2h25min, 3h15min, and 4h of the dialysis phase change process, 10ml of dialysis fluid was taken respectively, and samples A1, A2, and A3 were marked.

Embodiment 3

[0040] Example 3 Using an ultrafilter to dialyze the recombinant human tissue-type plasmin kinase derivative inclusion body enhancement solution

[0041] Take 4L of the solubilized dialysis internal solution in Example 1, use a membrane bag (2.5 square meters) with a cut-off of 10kD to perform ultrafiltration and phase change, first concentrate to 2L, add 2L of dialysis external solution 2 to mix and then concentrate to 2L, and then Use dialysis fluid 2 for dialysis phase change, keep the ultrafilter permeation rate and dialysis fluid addition rate both at 2.5 L / h, and dialysis phase change for 4 hours. During 2h25min, 3h15min, and 4h of the dialysis phase change process, 10ml of the dialysis fluid was taken respectively, and samples B1, B2, and B3 were marked.

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Abstract

The invention provides a dialysis method of a recombinant human tissue type plasminogen kinase derivative inclusion body enhancement solution, and application. The invention adopts ultrafilter dialysis, compared with dialysis bag dialysis, dialysis efficiency is obviously improved, dialysis time is shortened into 1/5 of original dialysis time, a use amount of dialysis external liquid is reduced to1/20 of an original use amount, the dialysed rPA solution is clear and transparent, and small molecule substances, including guanidine hydrochloride, reductant DTT and the like, can be more thoroughly removed; and after the ultrafilter dialysis, the obtained rPA solution is processed by subsequent steps, and the integral yield of produced rPA protein is improved by 20%. The invention provides a convenient and efficient method for renaturation purification of a large-scale industrial inclusion body, especially the inclusion body containing more disulfide bonds.

Description

technical field [0001] The invention relates to the technical field of inclusion body renaturation and purification, in particular to a dialysis method and application of a recombinant human tissue-type plasmin kinase derivative inclusion body enhancement solution. Background technique [0002] Recombinant human tissue plasmin kinase derivative (rPA) is suitable for thrombolytic therapy of myocardial infarction caused by coronary artery infarction in adults, 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 effect, long maintenance time, high recanalization rate, small adverse reactions, and convenient use. rPA is a deletion mutant of natural tissue plasminogen kinase (tPA), which only retains the tPA serine protease region (related to the activity of the enzyme) and the Kringle2 region (related to the fibr...

Claims

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Application Information

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Patent Type & Authority Applications(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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