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Fusion protein containing leucine-rich repetitive sequence, and preparation method and application thereof

A fusion protein and protein technology, applied in the field of fusion proteins, can solve the problems of difficulty in protein purification, cumbersome processes and steps, inaccurate expression of target proteins, etc.

Inactive Publication Date: 2014-10-29
李华顺
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, proteins with histidine can be adsorbed by nickel agarose gel or nickel NTA agarose gel under natural conditions, but because the His tag is very small, if it is folded into the protein during the fusion protein folding process, the His tag will be caused If it is not easy to expose, it is difficult to carry out protein purification; at the same time, the His tag does have the problem of insufficient specificity
[0013] Usually, the purity of the protein separated by His tag technology is about 90%. However, in the prior art, the process and steps for further protein purification are relatively cumbersome, and the added protein or peptide often affects the activity of the target protein. Interference, resulting in inaccurate expression of the target protein or excessive impurities

Method used

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  • Fusion protein containing leucine-rich repetitive sequence, and preparation method and application thereof
  • Fusion protein containing leucine-rich repetitive sequence, and preparation method and application thereof
  • Fusion protein containing leucine-rich repetitive sequence, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1 Construction of pcDNA3.1(-) / Slit2LRR1-2 vector

[0131] Method: Use exonuclease method to construct plasmid. In the process of constructing the plasmid, two fragments (signal peptide sequence + target sequence, restriction site + tag protein sequence) were amplified by co-PCR, and the fragments were inserted twice. The primers used are Primer1 and Primer2 respectively.

[0132] Specific steps of exonuclease method:

[0133] 1. Prepare the reaction system: 50ng each of the vector fragment (recovered by the gel after digestion with BamHI and determine the concentration) and PCR segment (recovered by the gel after PCR and determine the concentration), 1ul10x exonuclease buffer, supplemented with ddH 2 O to 10ul;

[0134] 2. Place on ice for 5 minutes, add 1ul of Exonuclease III (Takara) diluted to 20U / ul, mix well and place on ice for 60 minutes;

[0135] 3. Add 1ul0.5M EDTA (pH8.0) solution to stop the reaction, and then water bath at 65℃ for 5min;

[0136] 4. Place on i...

Embodiment 2

[0144] Example 2 Successful expression of pcDNA3.1(-) / Slit2LRR1-2

[0145] Method: In order to detect whether the constructed plasmid was successfully expressed, the plasmid was transfected into AD293 cells, and then the Western Blot was made with the tag protein 8×His to detect whether the fusion protein was successfully expressed.

[0146] Materials: High glucose DMEM medium and fetal bovine serum were purchased from HyClone; the transfection reagent Magetran was purchased from Origene; AD293 cells were cultured in High glucose DMEM medium containing 10% fetal bovine serum. The inverted microscope is a product of Olympus, and the fluorescent inverted microscope is a product of Nikon.

[0147] step:

[0148] 2.1. Resuscitate and culture the frozen cells, pass them 3 to 4 times to make the cells reach a good growth state, and carry out the plating test. Magetran reagent transfects adherent cells.

[0149] 2.2. Inoculate AD293 cells to a 6-well plate and culture it with High glucose DM...

Embodiment 3

[0152] Example 3 Test of expression level

[0153] A purified HisGFP protein of known concentration (20μg / ml) was used as a control, and the protein expression level was determined by the method of Western Blot.

[0154] The specific method is the same as in Example 2. The cells are transfected first, and then Western Blot is performed. The difference is that when running SDS-PAGE electrophoresis, a certain known amount of HisGFP protein is added as a positive control.

[0155] After the result of exposure is obtained, the expression of protein is calculated by gray analysis of the band.

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Abstract

The invention discloses fusion protein containing a leucine-rich repetitive sequence, and a preparation method and application thereof. Concretely, the invention relates to fusion protein, and the fusion protein possesses the following structure from N terminal to C terminal: A-X-E-Y1-Y2 (formula Ia) or A-Y2-Y1-E-X (formula Ib), wherein A is an optional secretion signal peptide, X is a member rich in leucine repetitive sequence 1-2 (LRRs1-2) of Slit2 protein (neuronal guidance factor 2), E is a restriction enzyme cutting site, Y1 is a first tag peptide member, Y2 is a second tag short peptide member and the second tag short peptide member is His tag member, and '-' represents a peptide bond or a peptide joint for connecting the above members. The fusion protein is beneficial for correct folding of LRR (leucine-rich repeat) and forming active functional polypeptide, and is capable of simply removing two tag members through once resection enzyme cutting, thereby preparing high-purity high-activity LRR sequence.

Description

Technical field [0001] The invention relates to the field of biomolecules, in particular to a fusion protein containing leucine-rich repeat sequences, and a preparation method and application thereof. Background technique [0002] Nerve guidance factor Slit is a highly conserved secretory extracellular matrix glycoprotein in evolution. Its gene was discovered in 1988 to play a role in the formation of the central nervous system. Slit is a group of extracellular secreted proteins with a relative molecular mass of 170-190kDa. It is a member of the axon orientation molecular family that guides axon growth and neuron migration. [0003] Slit protein is a multifunctional guide molecule that acts as a ligand for Roundabout (Robo). It not only regulates the growth direction of nerve axons, guides nerve cell migration, and affects nerve cell morphological differentiation. Recent studies have found that Slit also plays a role in various physiological processes such as angiogenesis, cardiac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12P21/02C12P21/06
Inventor 李华顺
Owner 李华顺
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