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Process for preparing potein molecule imprinted polymer and its application in biological engineering

A technology for imprinting polymers and protein imprinting, which is applied in the field of bioengineering and can solve the problems of difficult renaturation and increased cost.

Inactive Publication Date: 2005-05-04
NANJING UNIV SCI PARK DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its shortcoming is: a. Must be able to obtain a large amount of high-purity substrates, inhibitors, ligands, receptors, etc. (not all known substrates, inhibitors, ligands, receptors, etc.; It is known that not all of them can be obtained in large quantities with high purity), which brings new difficulties and increases costs; b. Most of the recombinant proteins that are difficult to refold are macromolecules, multi-domains or even multimers protein
How to solve this problem is still unclear

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0009] 1. Mix the monomer 3-aminopropyltriethoxysilane and ethyl orthosilicate in a certain proportion, and add it to human urokinase or bovine serum albumin solution (dissolved in 0.1M, pH7.0 Potassium phosphate buffer), mixed rapidly for 3 minutes, placed at 25°C for 5-6 hours, and then placed at 4°C overnight. After the obtained polymer product was crushed, a certain volume of potassium phosphate buffer solution was added, mixed for 1 minute, allowed to stand for 20 minutes, centrifuged (3500 rpm) for 30 minutes, and washed 5 times repeatedly. The protein content in the polysiloxane imprinted polymer supernatant was determined by Coomassie brilliant blue staining quantitative method, and the enzyme activity of urokinase in the supernatant was determined by plate assay and urokinase fluorescent substrate G2386 assay .

[0010] 2. Centrifuge the imprinted polymer stored in potassium phosphate buffer at 3500 rpm, remove the supernatant, add 4 mL proteinase K reaction solution...

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PUM

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Abstract

The invention belongs to the technical field of bioengineering. The method for synthesizing protein imprinted polymers and improving protein renaturation is characterized in that a polymer imprinted polymer with a target protein as an imprint is synthesized, and the protein imprinted polymer can improve protein renaturation. The renaturation efficiency of the product improves the specific activity and purity of the product, which is beneficial to the separation and purification; after the imprinted polymer is treated, it can be used as a nano or nano therapeutic drug, or a separation medium, catalyst, sensor, etc.

Description

1. Technical field: [0001] The invention belongs to the technical field of biological engineering. 2. Background technology: [0002] Escherichia coli expression system is the preferred expression system for genetic engineering, which has many advantages such as short growth cycle, simple method, low price and high expression level. However, when exogenous genes are expressed in Escherichia coli, they often exist in the form of insoluble, non-biologically active inclusion bodies. Although the product form of inclusion body provides a lot of convenience for separation and purification, the product must undergo in vitro denaturation and renaturation to obtain biological activity. The process of protein renaturation in vitro is complicated and the yield is low. For proteins with polysulfide bonds (especially pharmaceutical proteins), there are isomers with mismatched disulfide bonds, which increases the difficulty and brings difficulties for downstream separation and purifica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C12N9/00
Inventor 华子春周敏
Owner NANJING UNIV SCI PARK DEV
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