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A kind of Aβ42 modified protein and its expression and purification method

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

Active Publication Date: 2020-05-12
百葵锐(深圳)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 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 Aβ in large quantities. 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, using traditional methods The Escherichia coli expression system still has disadvantages such as low expression level, complicated purification steps, and low purity of Aβ
Using soluble protein and Aβ fusion expression to increase its solubility and stability can solve the above-mentioned shortcomings of Aβ preparation. However, the fusion protein containing Aβ 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 kind of Aβ42 modified protein and its expression and purification method
  • A kind of Aβ42 modified protein and its expression and purification method
  • A kind of Aβ42 modified protein and its expression and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0060] The construction diagram of expression vector pET22b-Aβ42 is as follows figure 2 Shown

[0061] (1) First, optimize the codon preference of E. coli expression for Aβ42, and synthesize the Aβ42 gene fragment by the 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 cut the Aβ42 fragment from the plasmid provided by the gene company, and after agarose gel electrophoresis, gel purification to recover the target digestion product. Similarly, the pET22b expression vector plasmid was double-cut with NdeI and XhoI, and after the gel was recovered, it was connected with the target gene fragment obtained above. After 4-6 hours of reaction at 16°C, the ligation product was added to E. coli JM109 competent cells, and the mixture was ice-bat...

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 into 5mL LB medium and culture overnight at 37℃, transfer to 50mL fresh LB medium according to 1% inoculum, and cultivate at 37℃ until the OD600 is 0.6-0.8. Concentration of 0.5mM IPTG induction, induction temperature 37℃, induction time 4h. Finally, the cells were collected by centrifugation at 6000 rpm for 10 min. (2) Purification of Aβ42-His protein:

[0070] Resuspend the obtained bacteria in lysis buffer (20mM Tris-HCl, pH 7.4, 200mM NaCl, 1mM EDTA, 1mM DTT), add lysozyme and 1% PMSF at a final concentration of 30μg / mL, and sonicated after 30 minutes of ice bath. Centrifuge at 12000rpm for 30min, collect the supernatant and precipitate, SDS-PAGE gel electrophoresis found that the target protein is present in the precipitate, that is, the Aβ42-His protein is mainly expressed in the form of ...

Embodiment 3

[0077] Example 3: Identification of Aβ42 polypeptide

[0078] (1) Electrophoresis identification: The final Aβ42-His protein is determined by SDS-PAGE electrophoresis to determine the purity and molecular weight, such as image 3 As shown, there is a clear band around 5.6kDa. The protein concentration is determined by the BCA kit, and the final calculation is that 1L of bacterial solution can obtain about 36mg of Aβ42-His polypeptide, which is much higher than the existing methods of other biological expression of Aβ42.

[0079] (2) Mass spectrometry identification: subject the purified Aβ42-His polypeptide to MALDI TOF / TOF mass spectrometry identification, such as Figure 4 As shown, the peptide corresponding to a charge-to-mass ratio of 766.33 is the 1-6 amino acid peptide MDAEFR; the peptide corresponding to a charge-to-mass ratio of 1336.6 is the 7-17 amino acid peptide HDSGYEVHHQK; the charge-to-mass ratio is 1325.69 The corresponding peptide is the 18th-29th amino acid peptid...

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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 and preparing high-purity human amyloid Aβ42 modified protein by using an E. coli expression system, specifically a method for constructing and transforming a recombinant E. coli expression vector, and protein Induced expression and purification methods. Background technique: [0002] The aggregation of amyloid into insoluble starch fibers can cause various neurological diseases, such as Alzheimer's disease (AD), also known as Alzheimer's disease, which is a neurodegenerative disease. This disease severely affects millions of elderly patients in the world. According to data released by the International Alzheimer's Disease Association in 2016, there were approximately 46.8 million AD patients worldwide in 2015, and their number has doubled in 20 years. It is predicted that there will be 130 million AD pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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