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Aβ42 modified protein with the function of preventing protein aggregation and its expression and purification method

A protein and expression vector technology, applied in the fields of biotechnology and genetic engineering, can solve problems such as aggregation characteristics and toxicity research, and achieve the effects of high yield, high expression efficiency and high purity

Active Publication Date: 2021-07-13
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And a series of Aβ variants, both C-terminal and N-terminal extended residues are derived from APP protein, which is a homologous residue extension, while for the Aβ42 variants with non-homologous residue extension at the N-terminal Aggregation characteristics and toxicity have not been systematically studied

Method used

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  • Aβ42 modified protein with the function of preventing protein aggregation and its expression and purification method
  • Aβ42 modified protein with the function of preventing protein aggregation and its expression and purification method
  • Aβ42 modified protein with the function of preventing protein aggregation and its expression and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: pET22b-His 6 -Aβ42 expression vector plasmid and recombinant His 6 - Construction of Aβ42 Escherichia coli expression strain

[0078] His 6 - Aβ42 expression and purification flow chart as shown figure 1 shown;

[0079] Expression vector pET22b-His 6 -Aβ42 construction schematic diagram as shown in figure 2 shown;

[0080] (1) Perform codon optimization on the Aβ42 gene sequence according to the amino acid sequence of Aβ42 and the codon preference of Escherichia coli to obtain the amino acid sequence of Aβ42 such as SEQ ID No.3 and the nucleotide sequence such as SEQ ID No.4;

[0081] (2) Using primer NdeⅠ-His 6 -Aβ42-F and XhoⅠ-His 6 -Aβ42-R, the primer sequences are shown in SEQ ID No.5 and SEQ ID No.6. The upstream primer NdeⅠ-His 6 -Aβ42-F contains His 6 The gene sequence of the tag, add His at the upstream of the Aβ42 gene sequence by PCR 6 tag, get His 6 -Aβ42 target gene sequence as shown in SEQ ID No.2, His 6 - The amino acid sequence o...

Embodiment 2

[0096] Example 2: His 6 -Expression and purification of Aβ42 protein

[0097] His 6- Aβ42 expression and purification flow chart as shown figure 1 shown.

[0098] (1)His 6 -Aβ42 protein expression: use 1mM isopropyl-β-D-thiogalactopyranoside to induce protein expression, and the engineering bacteria BL21-His obtained in Example 1 6 -Aβ42 was induced at 30°C for 20 hours. After the induction, the bacterial liquid was centrifuged to collect the bacterial cells to obtain 6 -Escherichia coli thalline of Aβ42 fusion protein;

[0099] (2) His 6 -Purification of Aβ42 protein: Resuspend the bacteria obtained above with buffer, add 1% phenylmethylsulfonyl fluoride, sonicate after 30 minutes in ice bath, and centrifuge at 12000 rpm for 30 minutes. 6 -Aβ42 protein is mainly expressed in the form of inclusion bodies, and the inclusion bodies are denatured and dissolved by using buffer A solution containing urea.

[0100] The composition of the denaturing solution (buffer A) is: 20...

Embodiment 3

[0106] Example 3: His 6 -Aβ42 peptide identification

[0107] (1) Electrophoretic identification: the His prepared in Example 2 of the present invention 6 - The purity and molecular weight of Aβ42 protein were determined by SDS-PAGE electrophoresis, such as image 3 As shown, there is an obvious band around 5.47kDa. The protein concentration was detected by the NanoDrop 2000 protein and nucleic acid quantitator, and the final calculation of 1L bacterial solution can obtain about 22mg His 6 - Aβ42 polypeptide.

[0108] (2) Mass spectrometry identification: the purified His 6 -Aβ42 polypeptide was identified by MALDI TOF mass spectrometry, such as Figure 4 As shown, the peak with a charge-to-mass ratio of 5470 is His 6 -Aβ42 polypeptide, so it can be proved that this protein is the target protein His 6 - Aβ42.

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Abstract

The invention discloses an Aβ42 modified protein with the function of resisting protein aggregation and an expression and purification method thereof, belonging to the technical fields of biotechnology and genetic engineering. The present invention utilizes Escherichia coli expression system to express and purify to prepare high-yield and high-purity N-terminal with His 6 Tagged Aβ42 protein. By studying the aggregation properties and cytotoxicity of the polypeptide, it is proved that the Aβ42 with residues at the N-terminus prepared by the present invention has significantly weakened aggregation properties compared with Aβ42 without redundant residues, and has the effect of obviously resisting the aggregation of Aβ42.

Description

Technical field: [0001] The invention belongs to the technical fields of biotechnology and genetic engineering, and particularly relates to Aβ42 modified protein with the function of resisting protein aggregation and its expression and purification method. Background technique: [0002] Alzheimer's disease (AD), also known as senile dementia, is a common neurodegenerative disease. According to the data released by the International Alzheimer's Association in 2016, there were about 46.8 million AD patients in the world in 2015, and the number has doubled in 20 years. It is expected to increase to 132 million by 2050, becoming the world's largest public health problems. The American Alzheimer's Association released statistics including 2016 statistics and forecasts for 2017 on March 8, 2017. Compared with the facts and figures published in the previous year, the number of AD patients in the United States has increased 100,000 people, spending on dementia care in 2017 increas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/4711C07K2319/21C12N15/62C12N15/70
Inventor 刘夫锋位薇王英贾龙刚赵文平张会图
Owner TIANJIN UNIV OF SCI & TECH
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