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Method and Application of Heat Treatment to Improve Biological Activity of Pegylated Staphylokinase

A staphylokinase biology, polyethylene glycol technology, applied in the field of biomedicine, can solve the problems of protein biological activity reduction, loss, weakening protein and its substrate or receptor interaction, etc.

Active Publication Date: 2016-10-05
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the perspective of steric hindrance and thermodynamics, PEG attached to the surface of the protein molecule will cause a spatial shielding effect on the active region of the protein, weakening the interaction between the protein and its substrate or receptor, thereby reducing the biological activity of the protein. , or even completely lose

Method used

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  • Method and Application of Heat Treatment to Improve Biological Activity of Pegylated Staphylokinase
  • Method and Application of Heat Treatment to Improve Biological Activity of Pegylated Staphylokinase
  • Method and Application of Heat Treatment to Improve Biological Activity of Pegylated Staphylokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of PEGylated SAK with site-directed single modification of N-terminus

[0026] (1) De-dimerization of SAK

[0027] Since cysteine ​​residues are introduced into the C-terminus of SAK, it is easy to generate SAK dimers through disulfide bonds during storage, so a reducing agent is used to open the disulfide bonds before PEG modification. Recombinant staphylokinase and reducing agent tris(2-carbonylethyl)phosphate hydrochloride (TCEP) were mixed at a molar ratio of 1:5, the reaction buffer system was 20mM acetic acid-sodium acetate buffer, pH 5.0, and placed at room temperature for 3 hours, and then centrifuged to remove excess TCEP by membrane ultrafiltration with a molecular weight cut-off of 3 kDa.

[0028] (2) PEG site-directed modification of the N-terminus of SAK

[0029] The buffer system of the reaction was 20 mM acetic acid-sodium acetate buffer, pH 5.0. PEG aldehydes with molecular weights of 5kDa and 20kDa in NaCNBH 3 Reaction with SAK...

Embodiment 2

[0031] The reaction mixture was first purified by SP Sepharose HP column (1.6cm×25cm, GE Healthcare, USA). The SP Sepharose HP column was equilibrated with 20mM sodium acetate-acetic acid buffer (pH5.0). Continue to use this buffer for sufficient elution after loading the sample at a flow rate of 1.0ml / min to remove unreacted PEG aldehyde and NaCNBH 3 . Continue elution with 20 mM sodium acetate-acetic acid buffer (pH 5.0) containing 0.5 M sodium chloride at a flow rate of 1.0 ml / min, and collect the eluted peaks. The eluted peak was further purified by a Superdex200 column (2.6cm×60cm, GE Healthcare, USA). The Superdex200 column is equilibrated with 0.01M phosphate buffer (pH 7.4), and the buffer is used for elution after sample loading at a flow rate of 3.0ml / min. The PEG single-modification product (Ald5K) corresponding to the molecular weight of 5kDa and the 20kDa PEG single-modification product (Ald20K) were collected. Example 2 Preparation of PEGylated SAK whose link...

Embodiment 3

[0040] Example 3 Preparation of PEGylated SAK whose connecting bridge is pentyl and site-directed single modification of C-terminus

[0041] (1) De-dimerization of SAK

[0042] Since cysteine ​​residues are introduced into the C-terminus of SAK, it is easy to generate SAK dimers through disulfide bonds during storage, so a reducing agent is used to open the disulfide bonds before PEG modification. Recombinant staphylokinase and reducing agent tris(2-carbonylethyl)phosphate hydrochloride (TCEP) were mixed at a molar ratio of 1:5, the reaction buffer system was 20mM sodium phosphate buffer (pH7.2), and reacted at room temperature for 3 hours , and then centrifuged to remove excess TCEP by membrane ultrafiltration with a molecular weight cut-off of 3 kDa.

[0043] (2) Preparation of PEG maleimide with pentyl groups

[0044] The molar ratio of PEG amines with molecular weights of 5kDa and 20kDa to EMCS is 1:2, react at room temperature for 3 hours, and dialyze through a dialysis...

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Abstract

The invention relates to the field of biological medicines, in particular to a heat treatment method for improving the biological activity of polyethylene glycol (PEG) staphylokinase. The heat treatment method comprises the steps that (1) PEG staphylokinase is heated by a water bath; PEG staphylokinase is subjected to ice bath treatment immediately after heating stop; (2) the selected heating temperature is 50-80 DEG C; the optimal heating temperature is 70 DEG C; and (3) the selected heating time is 1-6h; and the optimal heating time is 2h. The heat treatment method can improve the biological activity of PEG staphylokinase.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to improving the in vitro biological activity of PEGylated staphylokinase by means of heat treatment. Background technique [0002] Cardiovascular and cerebrovascular thrombotic diseases are common diseases in my country and one of the most important fatal diseases. Thrombosis is the main cause of many cardiovascular and cerebrovascular diseases. Staphylokinase (staphylokinase, SAK) is a national first-class new drug for the treatment of cardiovascular and cerebrovascular thrombotic diseases, and has become one of the hot spots in the development of new thrombolytic agents in recent years. SAK is a non-enzyme protein secreted by some Staphylococcus aureus, single-chain, with a molecular weight of 15.5kDa and no disulfide bond in the molecule. SAK has a structural domain, and five β-sheets tightly wrap one α-helix and one β-sheet to form a tight structure. SAK has a thrombolytic effect, and...

Claims

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Application Information

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IPC IPC(8): C12N9/96C12N9/48A61K38/48A61K47/48A61P7/02
CPCC07K14/31
Inventor 胡涛薛晓莹季韶洋
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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