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Method for efficiently detecting interaction of in vivo proteins

A technology for detecting in vivo and internal proteins, applied in the biological field, can solve problems such as complex loss, restriction, and too large tag peptides, and achieve the effect of time-consuming and cost-effective detection

Inactive Publication Date: 2009-11-18
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the TAP technology still has the following disadvantages: 1) the amount of protein naturally expressed in mammalian cells is very small, requiring very large-scale cultured cells (generally up to 10 9 order of magnitude) to obtain the amount of protein used for mass spectrometry identification; 2) weak and transient interaction complexes will be lost during multi-step washing; 3) the tag peptide is too large, which may hinder the interaction of the target protein with other proteins
However, since this method uses affinity-purified peptide tags based on antibody interactions, it still requires a large number of starting cells (approximately 10 9 or more) to enrich target proteins, which restricts the use of this method for large-scale high-throughput protein interaction detection, especially the interaction detection and research of low-abundance proteins in signaling pathways

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  • Method for efficiently detecting interaction of in vivo proteins
  • Method for efficiently detecting interaction of in vivo proteins
  • Method for efficiently detecting interaction of in vivo proteins

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Embodiment 1

[0039] 1. Construction of plasmid vector

[0040] Based on the plasmid pAP-N that expresses the biotinylated artificial small peptide tag, the plasmid pAP-N-1433 containing 14-3-3ε was constructed. The gene sequence of 14-3-3ε was inserted into the pAP-N Xbal and EcoRV restriction sites. Invitrogen medium extraction kit (Cat. No.: K210004) was used for the medium extraction of plasmids for cell transfection.

[0041] 2. Cell culture

[0042] HEK293T cells (commercially available) are human embryonic kidney cells that grow adherently. The stable cell line was cultured in a 37°C, 5% carbon dioxide incubator, and its medium was normal or d3-Leu-labeled high-glycemic DMEM. 293T cells were labeled with AACT technology and cultured in DMEM medium containing 10% dialyzed fetal bovine serum and labeled amino acids (called 293T-d3), while unlabeled 293T cells were used ordinary DMEM containing 10% fetal bovine serum Culture medium (referred to as 293T-d0). For related AACT labeling technol...

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Abstract

The invention belongs to the biotechnology field and relates to a method for efficiently detecting interaction of in vivo proteins, which is built by utilizing an efficient immediate transfection and expression system, improving a biotin-streptavidin one-step affinity purification technique, applying a label only having 15 amino acids and integrating an isotope labeling technique. The method adopts initiator cells with consumption of 10<7> order of magnitudes, is applied to the detection of the interaction of the proteins of primary cells, can identify more interacted proteins, especially weak and immediate interaction, has obviously higher specificity and efficiency than the prior art and can greatly reduce the detection time and the detection cost. The invention is beneficial to know the signal conductive process of the cells and has an important reference value for the development of relevant medicines for pointedly treating diseases and the treatment method.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to protein interactions, in particular to a method for efficiently detecting protein interactions in vivo Background technique [0002] The prior art reveals that most of the biological functions of cells are mediated through the interaction of multiple proteins. Although some proteins can function in the form of monomers, most proteins form complexes with other proteins. The form of things comes into play. Cells receive exogenous or endogenous signals (physiological, pathological, and drugs, etc.), and regulate the expression of their genes through corresponding signal transmission pathways to maintain their biological characteristics and perform corresponding biological functions. In the whole process mentioned above, the interactions between proteins are involved and change with time and space (1). Due to the huge number of proteins in cells, in the post-genomic era, to better understand th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/15
Inventor 贺宇飞陈先
Owner FUDAN UNIV
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