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A[beta]42 modified protein and expression and purification method of A[beta]42 modified protein

A purification method and protein technology, which is applied in the fields of biotechnology and genetic engineering, can solve the problems of low expression, protein loss, difficult extraction and purification, etc., and achieve the effect of increasing expression, large expression, and low cost

Active Publication Date: 2018-03-02
百葵锐(深圳)生物科技有限公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Aβ peptides are mainly prepared by solid-phase chemical synthesis (SPPS), but the residual amino acids or peptide fragments in the SPPS synthesis process seriously affect the aggregation and toxicological properties of Aβ.
Due to the high hydrophobicity and easy aggregation characteristics of Aβ42 itself, it is difficult to extract and purify from nerve cell tissue, so it is difficult to obtain a large amount of high-purity peptides
[0005] The genetic engineering method has the advantage of being able to produce a large amount of Aβ. At present, some research groups have designed some expression systems and obtained recombinant Aβ through expression and purification. However, due to the high hydrophobicity of Aβ and the high cytotoxicity of aggregates, the use of traditional The Escherichia coli expression system still has disadvantages such as low expression level, complicated purification steps, and low purity of Aβ
Using the fusion expression of soluble protein and Aβ to increase its solubility and stability can solve the above-mentioned shortcomings of Aβ preparation. However, the Aβ fusion protein obtained by this method needs to be removed by protease enzymatic digestion. This method is not only cumbersome in the purification process Complex, but also cause a large amount of protein loss

Method used

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  • A[beta]42 modified protein and expression and purification method of A[beta]42 modified protein
  • A[beta]42 modified protein and expression and purification method of A[beta]42 modified protein
  • A[beta]42 modified protein and expression and purification method of A[beta]42 modified protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Construction of pET22b-Aβ42 expression vector plasmid and recombinant Aβ42 Escherichia coli expression strain

[0060] The schematic diagram of the construction of the expression vector pET22b-Aβ42 is as follows: figure 2 shown;

[0061] (1) First, optimize the codon preference of Aβ42 expressed in Escherichia coli, and obtain the Aβ42 gene fragment synthesized by a gene company. The sequence is shown in SEQ ID No.3, and the restriction sites at both ends of the target fragment are NdeI and XhoI, respectively;

[0062] (2) Use NdeI and XhoI to excise the Aβ42 fragment from the plasmid provided by the gene company, and after agarose gel electrophoresis, gel purification recovers the target digestion product. Also use NdeI and XhoI to double-digest the pET22b expression vector plasmid, and connect it with the target gene fragment obtained above after gel recovery. After reacting at 16°C for 4-6 hours, the ligation product was added to Escherichia coli JM109 ...

Embodiment 2

[0067] Example 2: Expression and purification of Aβ42-His protein

[0068] (1) Expression of Aβ42-His protein:

[0069] Pick a single colony of the constructed BL21-Aβ42 engineered bacteria and culture it overnight at 37°C in 5mL LB medium, transfer it to 50mL fresh LB medium at 1% inoculum size, cultivate at 37°C until the OD600 is 0.6-0.8, add the final The concentration was 0.5mM IPTG induction, the induction temperature was 37°C, and the induction time was 4h. Finally, the cells were collected by centrifugation at 6000 rpm for 10 min. (2) Purification of Aβ42-His protein:

[0070] Resuspend the bacteria obtained above with buffer solution lysis buffer (20mM Tris-HCl, pH 7.4, 200mM NaCl, 1mM EDTA, 1mMDTT), add lysozyme and 1% PMSF at a final concentration of 30μg / mL, and ultrasonically break after 30min in ice bath. Centrifuge at 12000rpm for 30min, collect the supernatant and precipitate, SDS-PAGE gel electrophoresis found that the target protein exists in the precipita...

Embodiment 3

[0077] Example 3: Aβ42 polypeptide identification

[0078](1) Electrophoretic identification: the purity and molecular weight of the finally obtained Aβ42-His protein were determined by SDS-PAGE electrophoresis, as shown in image 3 As shown, there is an obvious band around 5.6kDa. The protein concentration was measured by the BCA kit, and about 36mg of Aβ42-His polypeptide could be obtained in 1L of bacterial liquid after final calculation, which was much higher than other existing methods for expressing Aβ42 in organisms.

[0079] (2) Mass spectrometry identification: The purified Aβ42-His polypeptide was identified by MALDI TOF / TOF mass spectrometry, such as Figure 4 As shown, the peptide corresponding to the charge-to-mass ratio of 766.33 is the 1-6th amino acid peptide MDAEFR; the peptide corresponding to the charge-to-mass ratio of 1336.6 is the 7th-17th amino acid peptide HDSGYEVHHQK; the charge-to-mass ratio is 1325.69 The corresponding peptide segment is the 18th-2...

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Abstract

The invention belongs to the technical field of biotechnology and gene engineering, and particularly relates to an A[beta]42 modified protein, and an expression and purification method of the A[beta]42 modified protein in escherichia coli. An A[beta]42 expression system constructed by the method provided by the invention provides a construction method of a human amyloid protein A[beta]42 expression vector of prokaryotic expression; the expression vector is introduced into an escherichia coli expression host, and a targeted A[beta]42-His protein with high-purity biological activity is obtainedafter induced expression, two-step purification and identification. A large intestine expression system adopted by the method has the characteristics of high expression efficiency, large expression quantity, low cost, easy operation and the like. Obtained A[beta]42 polypeptide contains an His tag and is convenient to purify; the purity is high; and the yield is high. Compared with chemical synthesis of the A[beta]42, the method provided by the invention has the advantages of better aggregation characteristic, cytotoxicity and the like.

Description

Technical field: [0001] The invention belongs to the technical field of biotechnology and genetic engineering, and in particular relates to a method for expressing and purifying a high-purity human amyloid Aβ42 modified protein using an Escherichia coli expression system, specifically a method for constructing and transforming a recombinant E. Induced expression and purification methods. Background technique: [0002] The aggregation of amyloid into insoluble starch fibers can lead to various neurological diseases, such as Alzheimer's disease (AD), also known as Alzheimer's disease, is a neurodegenerative disease. This disease disproportionately affects millions of elderly patients worldwide. According to the data released by the International Alzheimer's Disease 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 predicted that there will be 130 million AD patients in the world in 2050. Due to ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/36C07K1/34C07K1/30C07K1/22C12N15/62C12N15/70G01N33/68
CPCC07K14/4711C07K2319/21C12N15/62C12N15/70G01N33/68
Inventor 刘夫锋贾龙刚路福平王文娟李玉张会图
Owner 百葵锐(深圳)生物科技有限公司
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