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Fluorescence complementary system based on green fluorescent protein sfGFP

A fluorescent and protein technology, applied in the field of fluorescent complementary systems, can solve problems such as easy self-activation and false positives, and achieve the effects of low false positive rate, good effect and broad application prospects

Inactive Publication Date: 2010-09-15
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest disadvantage of superfolder GFP is that it is easy to self-activate BiFC, resulting in serious false positive results.

Method used

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  • Fluorescence complementary system based on green fluorescent protein sfGFP
  • Fluorescence complementary system based on green fluorescent protein sfGFP
  • Fluorescence complementary system based on green fluorescent protein sfGFP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, inventive concept

[0034] (1) if figure 1 As shown, the green fluorescent protein super fold GFP was split into GFP1-10βstrands (sfGFPN) and GFP11βstrands (sfGFPC) from 214 / 215 amino acid residues; Xl), fusion of protein B (such as Bak+) to be detected; if protein A and protein B interact, the separated sfGFPN and sfGFPC will reassemble into GFP that can emit fluorescence; if protein A and protein B do not interact, then Separated sfGFPN and sfGFPC do not reassemble and cannot fluoresce. (2) The present invention further performs point mutation on the sfGFPC fragment, and then detects the interaction between protein A and protein B according to the method described in (1). (3) From sfGFPC non-mutated and sfGFPC fragments with different point mutations, select sfGFPC fragments with strong fluorescence effect and low false positive to apply to the interaction detection of other proteins to be tested, thus completing the present invention.

[0035] It can...

Embodiment 2

[0043] Embodiment 2, the composition of the fluorescent complementary system constructed by the present invention

[0044] Each group of fluorescent complementary systems constructed by the present invention is composed of protein fragment I and protein fragment II, specifically as follows:

[0045] 1. Protein fragment I: shown in SEQUENCE ID: 1 (ie sfGFPN); protein fragment II: shown in SEQUENCE ID: 3 (ie sfGFPC);

[0046] 2. Protein fragment I: shown in SEQUENCE ID: 1 (ie sfGFPN); protein fragment II: shown in SEQUENCE ID: 5 (namely sfGFPC with point mutation, denoted as sfGFPC(m6));

[0047] 3. Protein fragment I: shown in SEQUENCE ID: 1 (ie sfGFPN); protein fragment II: shown in SEQUENCE ID: 7 (namely sfGFPC with point mutation, denoted as sfGFPC(m12));

[0048] 4. Protein fragment I: shown in SEQUENCE ID: 1 (ie sfGFPN); protein fragment II: shown in SEQUENCE ID: 9 (namely sfGFPC with point mutation, denoted as sfGFPC(m15));

Embodiment 3

[0049] Embodiment 3, the effect verification of each group of fluorescence complementary systems of the present invention

[0050] The primers used in the following experiments are shown in Table 1 and Table 2.

[0051] Plasmid pcDNA3.1 was purchased from Invitrogen, catalog number V860-20.

[0052] 1. Preparation of recombinant expression vectors expressing various fusion proteins

[0053] (1) Preparation of recombinant expression vector expressing fusion protein sfGFPC-Bcl-xL

[0054] 1. Preparation of sfGFPC gene

[0055] The sfGFPC gene was synthesized by annealing with primers Fold03c, Fold04n, Fold-05c and Fold-06n, and the two ends of the synthetic gene had NotI and ClaI restriction sites.

[0056] The basic process of annealing synthesis:

[0057] (1) Fragment treatment: primer concentration 0.1nmol / ul, Fold03c 10ul, Fold04n 10ul, Fold05c10ul, Fold06n 10ul, Ligase buffer (10×) 5ul, H 2 O 5ul, a total of 50ul. Boil for 5 minutes, cool to room temperature

[0058]...

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Abstract

The invention discloses a fluorescence complementary system based on green fluorescent protein sfGFP. The protein fragments are shown in 1), 2) or 3): 1) the protein fragment as shown in an amino acid sequence such as SEQUENCE ID: 5; 2) the protein fragment as shown in the amino acid sequence such as SEQUENCE ID: 7; 3) the protein fragment as shown in the amino acid sequence such as SEQUENCE ID: 9. In the invention, two fluorescent protein fragments (sfGFPN and SFFGPC) are obtained by separating a 214th amino acid residue and a 215th amino acid residue based on the superfolder GFP (sfGFP) fluorescent protein, wherein, the sfGFPC fragment is improved into a protein fragment with fewer false positive through a point mutation method. An experience proves that the sfGFPC fragment and the super fold GFPN fragment obtained by mutation have good effect and much lower false positive than control groups on configuring and testing interaction of the protein in the fluorescence complementary system. The super fold GFP BiFC configured by the invention overcomes the defect of the false positive result generated by self-activation of super fold GFP. Thus, the fluorescence complementary system has broad application prospect in the field of testing interaction of the protein.

Description

technical field [0001] The invention relates to a fluorescent complementation system based on green fluorescent protein sfGFP. Background technique [0002] Studying protein-protein interaction (PPI) is the basis for studying signal channels and transduction in cells. Cell homeostasis, internal changes, and cell physiological functions are largely regulated by protein-protein interaction signaling networks. Detecting their interactions and revealing their biological functions is a hotspot in the field of contemporary biomedicine. [0003] Through the method of molecular biology, the regulation of specific protein-protein interaction also provides the possibility of a new treatment method for some intractable diseases. Therefore, both academic research institutions and pharmaceutical companies have shown strong interest in this field, hoping to regulate protein-protein interactions as a new therapeutic method. [0004] There are many methods for studying protein-protein in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/53G01N21/64
Inventor 林坚夏斌周军
Owner PEKING UNIV
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