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Polypeptide probe and application of polypeptide probe to identification of post-translational modification binding protein

A post-translational modification and protein-binding technology, which is applied in the direction of material inspection products, measuring devices, instruments, etc., can solve problems such as difficult identification and identification

Active Publication Date: 2020-02-21
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The present invention aims to address protein post-translational modifications (including histone post-translational modifications and non-histone post-translational modifications) binding proteins (including reader proteins and their complexes and demodification enzyme proteins and Its complex) is difficult to identify and identify. Combining nano-metal particle self-assembly technology and affinity photocrosslinking technology, a new polypeptide probe is developed, the preparation method of the probe is established, and the probe is used for efficient and highly sensitive , Highly selective protein post-translational modification binding protein identification and analysis method

Method used

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  • Polypeptide probe and application of polypeptide probe to identification of post-translational modification binding protein
  • Polypeptide probe and application of polypeptide probe to identification of post-translational modification binding protein
  • Polypeptide probe and application of polypeptide probe to identification of post-translational modification binding protein

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Experimental program
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Effect test

Embodiment 1

[0111] The preparation of embodiment 1 polypeptide probe

[0112] The numbers and sequences of the five polypeptides are: ①: ARTKQTARKSC, ②: ARTK(me3)QTARKSC, ③: ARTKQTARKSTGGKAC, ④: ARTKQTARK(cr)STGGKAC, ⑤: ART(ph)K(me3)QTARKSC. The polypeptides were synthesized by Beijing Yaguang Biotechnology Co., Ltd. For the convenience of expression, the numbering of the polypeptide probes used in the following examples is consistent with the numbering of the polypeptides carried on it, and the above-mentioned corresponding relationship of numbering is used.

[0113] Mercapto polyethylene glycol coupled benzophenone, the molecular formula is HS-(CH 2 CH 2 O) 13 -C 6 h 4 COC 6 h 5 , synthesized by Shanghai Tuoyang Biotechnology Co., Ltd.

[0114] Gold nanoparticles (particle size 5nm) were purchased from BBI Company.

[0115] Preparation of polypeptide probe: Mix 2 μL (1 mM) polypeptide with 1 μL (1 mM) mercaptopolyethylene glycol-coupled benzophenone and add to 50 μL (80 nM) gol...

Embodiment 2

[0117] Example 2 Recognition and Efficient Enrichment of Target Proteins by Polypeptide Probes

[0118] Using BPTF (which has a PHD domain, which is known to be able to bind and modify H3K4me3) as the target protein, the verification of the effect of using the polypeptide probe in Example 1 was carried out. The BPTF domain protein is self-expressed with reference to the literature (Angew Chem Int EdEngl.2016Jul 4,55(28):7993-7), and the identified structure is shown in the appendix Figure 6 .

[0119] Experimental group: Incubate the target protein (10μg) and the peptide probe ② (4pmol) in binding buffer solution (50mM Tris-HCl, pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride) overnight at 4°C . Then the sample tube was transferred to ice, and irradiated under a 365 nm ultraviolet lamp for 15 min to initiate a photocrosslinking reaction. Uncrosslinked proteins are then washed with binding buffer. The captured target protein was separat...

Embodiment 3

[0123] Example 3: Under the background condition of cell lysate or BSA, the selective enrichment of target protein by polypeptide probe.

[0124] Also using BPTF as the target protein, the effect of using the polypeptide probe ② in Example 1 was verified.

[0125] Experimental group: mix target protein (10μg) with cell lysate (50μg) (or bovine serum albumin (BSA), 50μg) in a certain ratio, and then mix with polypeptide probe (4pmol) in buffer solution (50mM Tris-HCl , pH 7.8, 200mM sodium chloride, 2.5mM magnesium chloride and 2.5mM potassium chloride) were incubated overnight at 4°C. Then the sample tube was transferred to ice, and irradiated under a 365 nm ultraviolet lamp for 15 min to initiate a photocrosslinking reaction. Next, the uncrosslinked protein was washed three times with PBS buffer solution (50 mM, pH 7.4) containing 4M urea. The captured protein was separated by centrifugation, and the target protein was added to the sample buffer (250mM Tris-HCl pH 6.8, 10% ...

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Abstract

The invention provides a polypeptide probe based on photo-crosslinking and nano-metal particle self-assembly technologies. The polypeptide probe is modified with protein-post-translational modified polypeptide. According to the polypeptide probe, affinity recognition and enrichment capture processes are integrated, so that the convenient and efficient recognition and identification of a protein-post-translational modified binding protein or a compound of the binding protein. According to the polypeptide probe of the invention, a photo-crosslinking group is introduced into nano metal particles,so that a weak acting force between the polypeptide and the binding protein is converted into a strong covalent bond effect formed by the photo-crosslinking group, and therefore, the polypeptide probe can bear strong elution conditions and can capture proteins with low abundance and transient interaction; the influence of the modification of the photo-crosslinking group on a polypeptide frameworkis avoided, and therefore, the original structure and recognition characteristic of the polypeptide framework are well maintained, and influence on the binding specificity of the corresponding binding protein is greatly reduced. The invention further provides a preparation method of the polypeptide probe, and the application of the polypeptide probe to the identification of post-translational-modified binding proteins, the identification of interaction between protein modifications and the discovery of new protein-modified binding proteins.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a polypeptide probe based on photocrosslinking and nano-metal particle self-assembly technology, its preparation method and its post-translationally modified binding protein identification, protein modification interaction identification, Applications in the discovery of novel protein-modified binding proteins. Background technique [0002] Post-translational modification of proteins refers to the process of covalently modifying individual amino acid residues on proteins after mRNA is translated into proteins. More than 400 protein post-translational modifications have been found in eukaryotic cells, the most common of which are ubiquitination, phosphorylation, glycosylation, lipidation, methylation, and acetylation. Protein post-translational modification plays a very important role in living organisms. It makes the structure of the protein more complex, the function more ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6845Y02P20/55
Inventor 张锴翟贵金
Owner TIANJIN MEDICAL UNIV
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