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Separation and purification method of wild-type p53 protein

A separation and purification, wild-type technology, applied in the field of wild-type p53 protein separation and purification, to achieve the effect of improving yield and maintaining activity

Inactive Publication Date: 2011-10-12
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can partially restore the activity of prokaryotically expressed proteins, there are still certain problems if the target protein is to be applied to BIACORE molecular interaction or to perform some in vivo experiments.

Method used

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  • Separation and purification method of wild-type p53 protein

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Effect test

Embodiment 1p5

[0021] The separation and purification of embodiment 1p53 protein

[0022] 1. Materials and methods

[0023] 1. Materials and Reagents

[0024] 1.1 Materials: human liver cDNA, DH5α strain, JM109 competent bacteria, H1299 cells were preserved by our laboratory; eukaryotic expression vector pCMV-Myc, mouse (mouse) anti-p53 monoclonal antibody was purchased from Clontech Company; PCR amplification kit; PCR product purification kit was purchased from Boehringer Company; cell culture medium and fetal bovine serum were purchased from Hangzhou Sijiqing Bioengineering Materials Company; cells were transiently transfected with Invitrogen liposome transfection kit; p53 monoclonal antibody (DO1) was purchased from In SANTACRUZ Company: All primers used were synthesized by Shanghai Boya Biotechnology Co., Ltd.

[0025] 1.2 Reagents:

[0026] 50×TAE: Dissolve 242g of Tris base in 600ml of ddH2O, add 57.1ml of glacial acetic acid, 100ml of 0.5M EDTA (pH 8.0), and dilute to 1L.

[0027]...

Embodiment 2

[0072] Example 2 The effect of p53 recombinant protein on H1299 tumor cell apoptosis measured by flow cytometry

[0073]H1299 cells induced by p53 recombinant protein at a concentration of 180ng / ul were selected, and the cells were collected at four time points of 12h, 24h, 48h, and 72h, washed once with 1×PBS (pH7.4), and placed in citrate buffer After 1 h, centrifuge to remove the supernatant. Appropriate amounts of trypsin digestion solution and trypsin inhibitor were added successively, and PI was added to stain in the dark for 15 minutes, and then a single cell suspension was formed for detection on the machine. The H1299 tumor cells treated with p53 recombinant protein at four time gradients can detect apoptosis peaks in flow cytometry, and the apoptosis rate is greatly improved compared with the control group. It can be seen that with the prolongation of the action time of P53 recombinant protein, the apoptosis rate increased significantly. The apoptotic rate of the e...

Embodiment 3

[0074] Example 3 Using the BIACORE molecular interaction instrument to detect the interaction between the purified p53 protein and small molecules

[0075] BIACORE molecular interaction instrument is based on surface plasmon resonance technology to track the interaction between biomolecules. Since it does not require any markers and truly reflects the various interactions between biomolecules, it has been widely used by researchers in recent years. BIACORE molecular interaction instrument was used to detect the interaction between p53 recombinant protein and Hbx. The concentration of recombinant protein is determined in advance, and protein coupling can be carried out when a certain concentration requirement is reached. Dilute the target protein to be analyzed to 50ug / ml with sodium acetate buffer solution of pH 4.0, 4.5, 5.0 and 5.5, respectively, and then inject the sample, so that the above protein dilutions flow through the surface of the CM5 sensor chip successively. Th...

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Abstract

The invention belongs to the related fields of biomedical products and clinical detection and the like, and mainly relates to a new method for separating and purifying wild-type p53 protein. The separation and purification method of the wild-type p53 protein comprises cloning of a p53 gene, construction of recombinant expression plasmid, protein induced expression and purification, prokaryotic system-based inducible expression, ultrasonic cracking of thalli, and separation and purification of the p53 protein in the obtained cracked supernate by using nickel column affinity chromatography. High-concentration active target protein is separated and purified by the prokaryotic system-based inducible expression and the nickel column affinity chromatography in the ultrasonic cracked thalli supernate. Denaturation and renaturation of inclusion bodies are not performed, and the protein is directly purified from the ultrasonic cracked supernate, so that the activity of the target protein is furthest kept; and compared with the conventional method, the method has the advantage that: the yield of the target protein is greatly improved by optimizing the inducible expression and using a purification scheme.

Description

technical field [0001] The invention belongs to the relevant fields of biomedical products and clinical testing, and specifically relates to a novel method for separating and purifying wild-type p53 protein. Background technique [0002] The p53 gene is so far found to be the most relevant gene to human tumors, and more than half of human tumors have p53 gene changes. The human p53 gene is located at 17p13.1, contains 11 exons and 10 introns, and encodes a nucleophosphoprotein with a molecular weight of 53KD, so it is called p53. The p53 gene is divided into two types: wild type and mutant type. The half-life of wild-type p53 protein is very short (20-30min), and it is not easy to accumulate in tissues, so the level of p53 in normal human body is low. In tumor patients, the p53 gene is mutated, and the half-life of the mutant p53 protein is as long as 20-40 hours, which can accumulate in the tumor tissue, and at the same time, a large amount of p53 protein is released into...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/47C07K1/22
Inventor 王佳
Owner FUDAN UNIV
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