A protein chip for analyzing interaction between protein and substrate peptide thereof

A protein chip and substrate peptide technology, applied in the field of protein chips, can solve the problems of low economic benefit, difficulty in practical application, and difficulty in inducing interaction between peptides and reactive proteins, etc.

Inactive Publication Date: 2006-02-08
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the various protein chip technologies mentioned above have been developed, the low-molecular-weight peptides used in the current protein chip technology generally consist of less than 50 amino acids. Structural issues that make it difficult for existing protein chips to induce interactions between immobilized peptides and reactive proteins
Furthermore, the practical application of this technique is difficult due to the many drawbacks of using fluorescently labeled antibodies to detect interactions
In addition, this technique requires high concentrations of peptides to be immobilized on the chip, making it currently less economical

Method used

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  • A protein chip for analyzing interaction between protein and substrate peptide thereof
  • A protein chip for analyzing interaction between protein and substrate peptide thereof
  • A protein chip for analyzing interaction between protein and substrate peptide thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: Construction of recombinant plasmid

[0035] (1) Construction of recombinant plasmids pLKM and pLKD

[0036] Construction of leptin-kemptide protein expressing specificity against protein kinase A ( figure 1 ) recombinant plasmids pLKM and pLKD. In order to fuse the substrate peptide specific for protein kinase A, that is, Kemptide (SEQ ID NO: 1) and human leptin in a monomeric form, perform PCR amplification, and the template DNA used is a recombinant gene containing 414bp leptin Plasmid pEDOb5 (Jeong et al., Appl.Environ.Microbiol., 65(7):3027-32, 1999), the primer 1 used contains the digestion site of restriction enzymes NdeI and BamHI, and the primer 2 used contains the kemptide gene sequence.

[0037] Primer 1 (SEQ ID NO: 2): 5'-CGGAATTCATATGGTGCCCATCCAAAAAGTCCA-3'

[0038] Primer 2 (SEQ ID NO: 3): 5'-GCGGATCCTTAGCCCAGGCTCGCACGACGCAGGCACCCAGGGCTGAGG-3'

[0039] In addition, when fused in a dimer form, PCR amplification was performed using the abo...

Embodiment 2

[0064] Example 2: Analysis of the interaction between leptin-kemptide protein and protein kinase A on a protein chip immobilized with leptin-kemptide protein

[0065] (1) Prepare a protein chip on which leptin-kemptide protein is immobilized

[0066] Recombinant E.coli transformed with recombinant plasmids pLKM and pLKD were inoculated into 200ml LB medium and cultured at 37°C, wherein the recombinant plasmids pLKM and pLKD contained genes encoding leptin-kemptide protein. When the optical density at 600 nm wavelength reached 0.7, 1 mM IPTG was added to induce the expression of leptin-kemptide protein. After 4 hours, the culture broth was centrifuged at 4°C, 6000 rpm for 5 minutes, and the resulting pellet was washed with 100 ml of TE buffer (Tris-HCl 10 mM; EDTA 1 mM, pH 8.0). The washed material was centrifuged at 6000 rpm at 4°C for 5 minutes, and then suspended in 100 ml of TE buffer. The resulting cells were disrupted with a sonicator (Branson Ultrasonics Co., USA).

...

Embodiment 3

[0074] Example 3: Analysis of the interaction between malic enzyme-kemptide and protein kinase A using a protein chip immobilized with malic enzyme-kemptide protein

[0075] (1) Preparation of a protein chip on which malic enzyme-kemptide protein is immobilized

[0076] The recombinant E.coli transformed by the recombinant plasmid pTLMK3 was cultivated under the same conditions as in Example 2-(1), wherein the recombinant plasmid pTLMK3 contained the gene encoding the malic enzyme-kemptide protein, and then the cultured cells were disrupted with a sonicator . The crushed solution was centrifuged at 4°C and 6000rpm for 30 minutes, the supernatant was collected, and the equilibrium solution (50mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0), followed by purification with a nickel-chelating resin (Quiagen, USA), dialyzed against PBS, and filtered through a 0.2 μm filter. The protein contained in the filtrate was quantified by the same method as in Example 2-(1), and then...

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Abstract

A protein chip of a S-L-SP form wherein a substrate peptide (SP) is immobilized on a solid substrate (S) by the mediation of a linker protein (L). Such chip is usefully employed in a method for analyzing the interaction between a reactive protein and its substrate peptide involving the steps of: adding to the protein chip a reactive protein showing a specific interaction with the substrate protein immobilized on the protein chip; and detecting the interaction between the reactive protein and the substrate peptide. The use of such protein chip and methodology allows an increase in the reactivity between a peptide with low molecular weight and an enzyme with high molecular weight and between the peptide and a reactive antibody on the protein chip, so that the interaction between the peptide and the protein can be analyzed rapidly and massively.

Description

technical field [0001] The invention relates to a protein chip for analyzing the interaction between protein and substrate peptide. In particular, the present invention relates to a protein chip of the S-L-SP type, wherein the substrate peptide (SP) is immobilized on a solid substrate through the mediation of connexin (L), and the present invention also relates to using the protein chip to analyze Methods of interaction between proteins and substrate peptides. Background technique [0002] Protein chips are a key technology in the research of discovering the function of biomolecules that can specifically interact with specific proteins, and developing methods for treating and preventing diseases. These studies were not possible using traditional methods based on data obtained from protein analysis and network analysis. [0003] The protein chip technology developed so far can be roughly divided into the following four categories. [0004] (1) A technology that uses DNA mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543C40B30/04G01N33/68
CPCG01N33/54353C07K1/1077C40B30/04G01N33/6803C07K14/5759C12N9/0006G01N33/543
Inventor 李相烨李锡宰
Owner KOREA ADVANCED INST OF SCI & TECH
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