Application of ubiquitin-binding domain fusion protein in detection and separation of ubiquitination protein

A technology for ubiquitinated proteins and binding domains, applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of poor ubiquitination detection effect and low sensitivity of protein ubiquitination detection, Achieve high detection capacity and sensitivity, low cost, and improve efficiency

Inactive Publication Date: 2013-04-24
JIANGSU UNIV
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Problems solved by technology

[0005] In order to solve the problem that the detection sensitivity of protein ubiquitination is not high in the prior art, especially the detection effect of ubiquitination of low-abundance endogenous proteins is very poor, the present invention utilizes the ubi

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  • Application of ubiquitin-binding domain fusion protein in detection and separation of ubiquitination protein

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Embodiment

[0018] Example: Isolation and detection of ubiquitinated proteins using a fusion protein of GST-ACK1Uba.

[0019] 1. Expression and purification of GST-ACK1Uba fusion protein.

[0020] ACK1 (Activated Cdc42-associated Kinase1, also known as Tnk2) is a non-receptor tyrosine kinase whose carboxyl terminus contains a UBA domain. The ACK1UBA domain was cloned into the GST fusion protein expression vector pGEX4T3 (GE Life Sciences) to obtain the plasmid pGEX4T3-GST-ACK1Uba. The sequence of the fusion protein cDNA plasmid was confirmed by DNA sequencing. pGEX4T3-GST-ACK1Uba was transformed into E. coli JM109 for expression of the fusion protein. The transformed bacteria were cultured in LB medium at 37°C to a density of A600 (light absorption at 600nm) of 1.0, and then 1 / 1000 IPTG (0.5mM) was added to induce fusion protein expression for 3-5 hours. The bacteria after fusion protein expression were collected by centrifuging at 8000 rpm for 10 minutes.

[0021] The pelleted bacter...

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Abstract

The invention relates to a method for utilizing ubiquitin-binding domain (UBD) fusion protein to separate and detect ubiquitination protein. The principle is that a fusion protein is constructed by using an affinity label and a ubiquitin-binding domain. The fusion protein can be expressed in colon bacillus, purified and fixed on a gel particle through the ligand of the affinity label. Mixed culture is carried out on the gel particle with the ubiquitin-binding domain fusion protein and lysate of cell or tissue, and the ubiquitination protein in the lysate can be separated out through precipitation. On the aspect of the detection of ubiquitination protein, the method disclosed by the invention is higher in efficiency, detection capacity and flexibility compared with the current antibody immune precipitation detection method. Moreover, on the aspect of economic benefit, the cost of detecting ubiquitination protein in the invention is extremely lower than that of expensive antibody immune precipitation method; and the method can be applied to fundamental research of protein ubiquitination and the detection of ubiquitination protein in medical diagnosis test.

Description

technical field [0001] The invention is applied to the detection and separation of ubiquitinated proteins in biological and medical research and medical diagnosis. The present invention specifically relates to the detection and isolation of ubiquitinated proteins by using a fusion protein containing an affinity tag (Affinity Tag) and a ubiquitin-binding domain (Ubiquitin-binding domain, UBD) and similar methods. Background of the invention [0002] Protein ubiquitination is an important signal transduction pathway in biological cells. Recent studies have shown that ubiquitination is not only involved in regulating protein degradation, but also plays an important role in membrane transport, protein recognition, virus budding, DNA replication, gene transcription and protein translation. Ubiquitin is a polypeptide with 76 amino acids, widely present in biological cells. Ubiquitination is the biochemical process of covalently attaching ubiquitin to target proteins. The comple...

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Application Information

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IPC IPC(8): C07K19/00C12N9/10C12N9/38G01N33/68
Inventor 林琼
Owner JIANGSU UNIV
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