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Polyethylene glycol chemically modified compound of human fibroblast growth factor-21 (FGF-21) and preparation method thereof

A human fibroblast, polyethylene glycol chemical technology, applied in the field of protein modification, can solve the problem of unpredictable protein PEGylation

Active Publication Date: 2012-04-11
WENZHOU MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013]Therefore, PEGylation of proteins is unpredictable

Method used

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  • Polyethylene glycol chemically modified compound of human fibroblast growth factor-21 (FGF-21) and preparation method thereof
  • Polyethylene glycol chemically modified compound of human fibroblast growth factor-21 (FGF-21) and preparation method thereof
  • Polyethylene glycol chemically modified compound of human fibroblast growth factor-21 (FGF-21) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of polyethylene glycol-FGF-21

[0030] 1. Modification reaction: Mix 10mg of FGF-21 dissolved in 10ml of PB buffer (10mM, PH=6.0) with mPEG-maleimide (PEG molecular weight: 5000Da) at a molar ratio of 1:10, place it at 4°C, and react in the dark for 2 hours . Add 1 ml of 1M glycine to terminate the modification reaction.

[0031] 2. Separation and purification of modified products:Pass the reaction solution in step 1 through a Sephedax G-25 column (20cm×1.5cm) to desalt: use 10mM PB buffer (pH=6.0) to equilibrate the column until the baseline is stable, load the sample at a flow rate of 1ml / min, and use 10mM PB buffer Wash the column with 10mM PB buffer until the baseline is stable, and then remove the salt collected after desalting Load the protein and elute with 10mM PB buffer containing 200mM NaCl at a flow rate of 1ml / min. Collect the eluted peak, which is the PEG modification of FGF-21; elute with 10mM PB buffer containing 500mM NaCl at ...

Embodiment 2

[0032] Example 2 Preparation of polyethylene glycol-FGF-21

[0033] 1. Modification reaction: Mix 10mg of FGF-21 dissolved in 10ml of PB buffer (10mM, PH=7.0) with mPEG-maleimide (PEG molecular weight: 10000Da) at a molar ratio of 1:20, place at 4°C and avoid light for reaction 2 hours. Add 1 ml of 1M glycine to terminate the modification reaction.

[0034] 2. Separation and purification of modified products: Pass the reaction solution in step 1 through a Sephedax G-25 column (20cm×1.5cm) to desalt: use 10mM PB buffer (pH=7.0) to equilibrate the column until the baseline is stable, load the sample at a flow rate of 1ml / min, and use 10mM PB buffer Wash the column with 10mM PB buffer until the baseline is stable, and then remove the salt collected after desalting Load the protein and elute with 10mM PB buffer containing 200mM NaCl at a flow rate of 1ml / min. Collect the eluted peak, which is the PEG modification of FGF-21; elute with 10mM PB buffer containing 500mM NaCl at ...

Embodiment 8

[0050] Example 8 Preparation of polyethylene glycol-FGF-21

[0051] 1. Modification reaction: Mix 10mg of FGF-21 dissolved in 10ml of sodium acetate buffer (20mM, PH=6.0) and mPEG-butyraldehyde (PEG molecular weight: 20000Da) at a molar ratio of 1:30, place it at 25°C, and react in the dark for 4 hours . Add 1 ml of 1M glycine to terminate the modification reaction.

[0052] 2. Separation and purification of modified products: Pass the reaction solution in step 1 through a Sephedax G-25 column (20cm×1.5cm) to desalt: use 20mM sodium acetate buffer (pH=6.0) to equilibrate the column until the baseline is stable, load the sample at a flow rate of 1ml / min, 20mM acetic acid Elute with sodium buffer, collect the first elution peak; after desalting through Sephedax G-25 column, apply to QAE-Sephadex A50 anion exchange column: wash the column with 20mM sodium acetate buffer until the baseline is stable, then desalt The collected protein was loaded and eluted with 20mM sodium ac...

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Abstract

The invention belongs to the field of protein modified compound, and particularly discloses a polyethylene glycol chemically modified compound of a human fibroblast growth factor-21 (FGF-21). The activity of the modified compound is over 40 percent compared with that of an equal molar amount of unconjugated FGF-21. The invention further discloses a preparation method of a modification product. According to the method, the modification product can be prepared, and unmodified FGF-21 can be recovered. Moreover, the invention further discloses application of the modification product to preparation of a medicament for regulating saccharometabolism.

Description

technical field [0001] The invention belongs to the field of protein modifications, in particular, the invention relates to a modified product of human fibroblast growth factor-21 modified with polyethylene glycol, a preparation method and application thereof. Background technique [0002] Fibroblast growth factor (FGF for short)-21 is a new member of the FGF family, a secreted protein, and was first isolated from mouse embryos. Human FGF-21 cDNA encodes 209 amino acids, which has about 75% homology with the amino acid sequence of mouse FGF-21. So far, the biological activity of FGF-21 is mainly reflected in lowering blood sugar and improving blood lipids (lowering blood sugar without causing hypoglycemia). It can be used to treat type 2 diabetes, and has a certain therapeutic effect on drug-induced early liver cancer. In addition, it can also play a role in embryonic development. [0003] The wide range of biological activities of FGF-21 indicates that it has broad applic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K38/18A61P3/10
Inventor 黄志峰李校堃王会岩王晓杰冯文科陆美斐倪春燕孙传传叶朝辉
Owner WENZHOU MEDICAL UNIV
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