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Method for highly sensitive detection of protein-protein interaction

A detection method and protein technology, applied in the field of detection of protein-protein interactions, can solve problems such as inability to maintain intentional activity

Active Publication Date: 2014-11-12
THE UNIV OF TOKYO +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a method of using complementarity to detect protein-protein interactions, fragments of the split reporter protein are usually fused to the protein of interest, but in this case, none of the fragments retain the intended activity by themselves

Method used

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  • Method for highly sensitive detection of protein-protein interaction
  • Method for highly sensitive detection of protein-protein interaction
  • Method for highly sensitive detection of protein-protein interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] In this example, in the presence of rapamycin, the interacting proteins FKBP (NM_054014) and FRB (NM_019906) with binding ability were combined with the peptide having the N-terminal side sequence of luminescent tap beetle luciferase, respectively. lucN and the peptide lucC with the C-terminal side sequence are fused, and the luminescent ability changes due to the different combinations of these fusion proteins; amino acid sequence) and lucCmax (such as the amino acid sequence shown in the 394th-542nd of SEQ ID NO: 1), it shows that the luminescent activity is significantly enhanced.

[0060] First, using the cDNA of the luminescent beetle luciferase (sequence shown in Figure 1A) as a template, PCR was performed with the primers shown in Figure 1B to obtain 14 genes with coding sequences from N-terminus to 404th to 417th. DNA fragments of 14 kinds of peptides with amino acid sequences (here, use each pair of primers N-PtGR-F001 and N-PtGR-R404~R417) and 25 kinds of pept...

Embodiment 2

[0071] In this example, the somatostatin receptor (SSTR2; somatostatin type 2 receptor) (NM_000794) (NM_000794) which is a GPCR (G-protein coupled receptor) and β- Arrestin (beta 2) (NM_004313) replaced FKBP and FRB. SSTR2 is a membrane protein present on the cell membrane. When somatostatin binds to the extracellular domain of GPCR, the intracellular domain of SSTR2 binds to β-arrestin, which is an adapter molecule present in the cytoplasm, and sends the signal downstream. Therefore, the C-terminus of SSTR2 was bound to the C-terminus of Eluc, and the N-terminus of β-arrestin was bound to the N-terminus of Eluc, the fusion protein was expressed in cultured cells, and somatostatin was injected into the cultured cells to investigate the effects of light emitted in.

[0072] First, using the human brain cDNA library (TAKARA) as a template, PCR was performed with the following primers to obtain DNA fragments encoding arrestin and SSTR2.

[0073] ARRB2-nestF2: AAAGGATCCATGGGGGAG...

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Abstract

The present invention intends to provide an assay system using split luciferase that has a remarkably high detection sensitivity. In an embodiment, binding of mutually binding first and second proteins is detected by preparing a first fusion protein comprising the first protein fused with a peptide having the amino acid sequence of amino acid SEQ ID NO: 1 and a second fusion protein comprising the second protein fused with a peptide having an amino acid sequence selected from the group consisting of amino acid SEQ ID NOS: 2 to 6, and allowing the first fusion protein to bind with the second fusion protein to form a complex, and detecting luminescence emitted from the complex.

Description

technical field [0001] The present invention relates to a method for detecting the interaction between proteins. Background technique [0002] Recently, using the complementarity of split luciferase fragments, people have developed a system to detect the interaction between two target proteins (Kim, S.B., Ozawa, T., Watanabe, S., Umezawa, Y., 2004.Proc. Natl. Acad. Sci. USA. 101, 11542-11547). As a method of using complementarity to detect protein-protein interactions, fragments of a split reporter protein are usually fused to the protein of interest, but in this case, none of the fragments retain the intended activity by themselves. If the protein of interest interacts, the two inactive reporter protein fragments complement each other, restoring activity and generating a readable signal that can be used to indirectly track the protein-protein interaction. [0003] For various reporter proteins such as dihydrofolate reductase and β-lactamase green fluorescent protein, a me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09
CPCG01N33/542C12N9/0069C12Q1/6804C12Q1/66
Inventor 小泽岳昌三泽直美三浦研二冈本将西井重明增田兼治
Owner THE UNIV OF TOKYO