Efficient soluble expression and purification method of Abeta42 in escherichia coli

An Escherichia coli, soluble technology, applied in the fields of biotechnology and genetic engineering, can solve problems such as reducing the efficiency of enzymatic cleavage, and achieve the effects of low cost, large expression amount and high yield

Active Publication Date: 2017-10-13
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Application Information

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Problems solved by technology

However, the use of TEV proteolytic digestion has the problem that the target protein after digestion carries amino acid residues. This problem can be modified by modifying the TEV restriction site, that is, the seven amino acid sequence is modified into a six amino acid sequence, and the terminal amino acid of the target protein is used to replace the first amino acid residue. However, the m

Method used

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  • Efficient soluble expression and purification method of Abeta42 in escherichia coli
  • Efficient soluble expression and purification method of Abeta42 in escherichia coli
  • Efficient soluble expression and purification method of Abeta42 in escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Construction of pMAL-Aβ42 expression vector plasmid and Aβ42 Escherichia coli expression strain

[0061] The schematic diagram of the construction of the expression vector pMAL-Aβ42 is as follows: figure 2 shown;

[0062] (1) First according to (NANP) 3 - The amino acid sequence of TEV-Aβ42 was optimized for expression codon preference in Escherichia coli, and it was synthesized by a gene company (NANP) 3 -TEV-Aβ42 fusion gene fragment, the sequence is as shown in SEQ ID No.1, and the restriction sites at both ends of the target fragment are EcoRI and HindIII respectively;

[0063] (2) with EcoRI and HindIII will (NANP) 3 - The TEV-Aβ42 fragment was excised from the plasmid, and after agarose gel electrophoresis, gel purification was performed to recover the target digestion product. The pMALc2x expression vector plasmid was also double digested with EcoRI and HindIII, and ligated with the target fusion gene fragment obtained above after gel recovery. A...

Embodiment 2

[0068] Example 2: Expression and purification of MBP-Aβ42 fusion protein and purification of Aβ42 polypeptide by proteolysis

[0069] (4) Expression of MBP-Aβ42 fusion protein:

[0070] Pick a single colony of the constructed BL21-MBP-Aβ42 engineering bacteria and culture it overnight at 37°C in 5mL LB medium, transfer it to fresh LB medium according to the inoculum size of 1%, cultivate it at 37°C until the OD600 is 0.6-0.8, add the final The concentration is 0.5mM IPTG induction, the induction temperature is 16°C, and the induction time is 16-18h. Finally, the cells were collected by centrifugation at 6000 rpm for 10 min.

[0071] (5) Purification of MBP-Aβ42 fusion protein:

[0072] Resuspend the bacteria obtained above with 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 40min, and collect the supernatant. Add th...

Embodiment 3

[0078] Example 3: Aβ42 polypeptide identification

[0079] (1) Electrophoretic identification: the purity and molecular weight of the finally obtained Aβ42 polypeptide were determined by Tricine-SDS-PAGE electrophoresis, as shown in Figure 5 As shown, there is a distinct band around 4.5kDa. The protein concentration was measured by the BCA kit, and about 20mg of Aβ42 polypeptide could be obtained in 1L of bacterial liquid, which was much higher than the existing methods for expressing Aβ42 in other organisms.

[0080] (2) Mass spectrometry identification: the purified Aβ42 polypeptide was subjected to mass spectrometry to identify the molecular weight, such as Figure 6 As shown, the molecular weight is 4539.15, which is consistent with the molecular weight of human amyloid polypeptide Aβ42.

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Abstract

The invention belongs to the fields of biotechnologies and genetic engineering technologies and in particular relates to an efficient soluble expression and purification method of Abeta42 in escherichia coli. An Abeta42 expression system constructed by the invention provides a construction method of an expression vector of prokaryote for soluble fusion expression of human amyloid protein Abeta42 and the expression vector is introduced into an escherichia coli expression host; target Abeta42 with high-purity bioactivity is obtained through induced expression, protein enzyme digestion, two-step purification and identification. According to the method provided by the invention, an escherichia coli expression system and fusion protein have the characteristics of high expression efficiency, large expression amount, low cost, easiness for operation and the like; an expressed Abeta42 polypeptide has the advantages of no residues, high purity, high productivity 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 preparing human amyloid Aβ42 fusion protein through high-efficiency soluble expression of an Escherichia coli expression system, specifically a method for constructing and transforming a recombinant E. Purification method. Background technique: [0002] 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. The disease disproportionately affects millions of elderly patients worldwide, thereby imposing an enormous social and economic burden on families and society. 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 predicted that there will be 1...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K19/00C07K14/47C12P21/06
CPCC07K14/4711C07K2319/24C07K2319/50C12N15/62C12N15/70C12N2800/22C12P21/06
Inventor 刘夫锋贾龙刚路福平王文娟张会图秦慧民李玉
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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