Protein fusion design method for fusing two proteins based on alpha helix and maintaining activity of respective subunits

A protein and α-helix technology, which is applied in the direction of fusion polypeptide, chemical instruments and methods, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as cumbersome recombinant expression, difficulty in designing mutation sites, and difficulty in protein expansion

Active Publication Date: 2020-07-28
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has disadvantages such as difficulty in designing mutation sites, cumbersome recombinant expression, and difficulty in protein amplification.

Method used

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  • Protein fusion design method for fusing two proteins based on alpha helix and maintaining activity of respective subunits
  • Protein fusion design method for fusing two proteins based on alpha helix and maintaining activity of respective subunits
  • Protein fusion design method for fusing two proteins based on alpha helix and maintaining activity of respective subunits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Structure and sequence comparison of tubulin from different species

[0028] 1. Through the PDB database, retrieve microtubules from four different sources: Saccharomyces cerevisiae, Eurasian wild boar (Sus scrofa), Homo sapiens and Proteus (Prosthecobacterdejongeii), and collect their structure and sequence information;

[0029] 2. Compare the amino acid sequence homology of microtubule subunits by DNAMAN software: such as figure 1 As shown, the homology degree of α subunit is 70.65%;

[0030] 3. Compare the structural similarity of microtubule subunits by Discovery Studio2.5 software: such as figure 2 As shown, the α subunits of different species are very similar;

[0031] 4. Considering the convenience of experimental operation, convenience of expression and purification, and protein expression, the microtubule α subunit of Protecobacter dejongeii was selected as the basic protein; the D domain of protein A can bind to the antibody of the VHⅢ family. An...

Embodiment 2

[0032] Same amino acid substitution in embodiment 2α-helical structure

[0033]1. Analyze the structure of the 410-435 amino acid sequence of the C-terminal (remove the C-terminal carboxyl structure) of the tubulin α subunit of Protecobacter dejongeii, which is an α-helical structure; analyze the D domain of protein A Structure, found that it is three consecutive α-helical structures;

[0034] 2. Using the α-helical structure for splicing, the C-terminus of the tubulin α-subunit of Prosthecobacterdejongeii is connected to the N-terminus of the D domain of protein A by α-helical connection, and the same amino acid substitution is used. Connection, same amino acid substitution see image 3 hint.

Embodiment 3

[0035] α-helix formation condition screening of embodiment 3 fusion mode

[0036] 1. The conformation of the main chain of the peptide chain can usually be described by Ω, Φ and Ψ, the angle generated by the rotation around the C-N bond in the amide bond is called Ω, and the dihedral angle around the Cα-N bond axis (C-N-Cα-C) is called Φ, and the dihedral angle (N-Cα-C-N) around the Cα-C bond axis is called Ψ.

[0037] Because the C-N bond in the amide bond is affected by the C=O bond, it has a partial double bond property, and because the trans conformation has lower energy, Ω is usually about 180°. The α helix consists of 3.6 residues per turn, the pitch is 0.54nm, Φ is about -57°, and Ψ is about -47°;

[0038] 2. Use the Chimera software to conduct preliminary screening of the same amino acid site splicing at the C-terminal of the tubulin α subunit and the N-terminal of the D domain of protein A, and combine the Ω, Φ and Ψ numerical analysis to screen out a more reasonable...

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Abstract

According to a fusion design method disclosed by the invention, an alpha helical structure is utilized to fuse a tubulin alpha subunit of Proteobacter dejogii with a D structural domain of a protein Aby a same amino acid substitution method; fusion protein with high feasibility is screened out through alpha helix formation conditions, secondary structure analysis, energy calculation and kinetic simulation; experiment results prove that the screened fusion protein really has microtubule assembly characteristics and the activity of a D structural domain affinity VHIII single-chain antibody of protein A; and on the basis of a naturally existing self-assembly system, namely a microtubule site, other active protein subunits are fused on the microtubulin subunits, so that the activity of combining the microtubule self-assembly system with a single-chain antibody is improved under the condition of keeping the self-assembly activity of the microtubule subunits.

Description

technical field [0001] The invention belongs to biological information technology, calculation method and computer virtual reality technology, and specifically relates to a protein fusion design method which utilizes alpha helix to fuse two proteins and the fusion protein maintains the activity of the two proteins. Background technique [0002] Living organisms are composed of biomacromolecules, which play a vital role in life activities. Macromolecular nucleic acids control the activities of cells, while proteins are the embodiment of cellular molecular activities. Various proteins have biological functions that are adapted to the environment. Protein is the most basic structural material and functional material that constitutes an organism, and is the material basis of life activities. It participates in almost all life activities, such as catalysis, immunity, material movement, metabolic engineering, etc. in organisms. Proteins are directly involved and play an important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B15/00G16B25/00G16B30/20G16B35/00C12N15/70C07K14/24
CPCC07K14/24C07K2319/00C12N15/70G16B15/00G16B25/00G16B30/20G16B35/00
Inventor 赵玥张鲁嘉张增辉何晓方波欢
Owner EAST CHINA NORMAL UNIV
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