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Method for inducing protein to form amyloid fiber through fibration segment

A protein and amyloid technology, applied in the fields of molecular biology and biochemistry, can solve the problem of not finding a way to fix proteins and achieve the effect of easy editing and construction

Inactive Publication Date: 2012-10-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no widely used method of immobilizing proteins that has no effect on protein activity has been found so far.

Method used

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  • Method for inducing protein to form amyloid fiber through fibration segment
  • Method for inducing protein to form amyloid fiber through fibration segment
  • Method for inducing protein to form amyloid fiber through fibration segment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Using IFQINS to make Tau protein form long and curved protein amyloid fibrils.

[0022] 1. Using the molecular cloning method to insert the fiber forming fragment IFQINS into Tau 244-372 / ΔPHF6 / ΔPHF6* at the position of PHF6.

[0023] 2. Protein expression and purification: Add the constructed mutant plasmid to 200 μl BL21 competent cells, bathe in ice for 30 minutes, heat shock at 42°C for 90 seconds, add 800 μl LB medium for 1 hour, coat with Plates were incubated overnight at 37°C. On the next day, pick a single colony and activate it overnight at 37°C at a shaking rate of 200 rpm in a 5ml LB tube containing a final concentration of 100μM ampicillin. Inoculate into 2 L of LB medium with a volume of 250 ml per bottle containing 100 μM ampicillin at a final concentration. Cultivate to OD in a shaker at 37°C with a speed of 200 rpm 600 =0.6-1.0, adding a final concentration of 0.4mM IPTG to induce protein expression, culturing for 3 hours under the same c...

Embodiment 2

[0026] Example 2: Using GVATVA to make Tau protein form long and branched protein amyloid fibrils.

[0027] The difference between the specific operation method and Scheme 1 is that the inserted fragment is GVATVA.

Embodiment 3

[0028] Example 3: Use of NNQQNY to form Tau protein into long and bundled protein amyloid fibrils.

[0029] The difference between the specific operation method and scheme 1 is that the inserted fragment is NNQQNY.

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PUM

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Abstract

The invention discloses a method for inducing a protein to grow an amyloid fiber through a fibration segment. A polypeptide sequence capable of independently growing the protein amyloid fiber is selected, and the polypeptide sequence is the fibration segment; and the fibration segment is inserted into a flexible structure of a target protein and an attainable area of a solvent with a molecular clone method, and then the target protein is induced to form the amyloid fiber. The method disclosed by the invention has more important application prospects in the fields of medical science, chemical industry and the like.

Description

technical field [0001] The invention belongs to the related fields of molecular biology and biochemistry, and relates to a method for inducing a target protein to form amyloid fibers by fusing and expressing a polypeptide containing a fiber-forming fragment and the target protein. Background technique [0002] Protein is a biomacromolecule with various biological activities, which can perform functions including catalysis, molecular recognition, antigen, antibody, etc. Enzymes are mostly composed of proteins. The catalytic efficiency of enzymes is very high. If they can be widely used in chemical and medical fields, it will bring very important breakthroughs to related fields. However, there are many factors that limit the widespread use of enzymes. One of the most important factors is that the enzyme preparation cost is high but the stability is not high enough, so the industrial cost is relatively high. [0003] A very core purpose of protein engineering is the direction...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K19/00C12R1/19
Inventor 梁毅朱应竹孟生荣郭童莫重瑛陈杰
Owner WUHAN UNIV
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