Segmented green fluorescent protein system for protein interaction detection

A green fluorescent protein-protein interaction technology, applied in the field of molecular biology, can solve problems such as increased background and unsatisfactory results

Active Publication Date: 2019-09-27
重庆明道捷测生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

We also used this anti-GFP nanobody later, but the effect is still not ideal, although the signal slightly increased, but the background also increased

Method used

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  • Segmented green fluorescent protein system for protein interaction detection
  • Segmented green fluorescent protein system for protein interaction detection
  • Segmented green fluorescent protein system for protein interaction detection

Examples

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Embodiment 1

[0031] Example 1. Screening of GFP 11 mutants with improved affinity with GFP 1-9 by mutating GFP 11

[0032]The experimental scheme in this example is to perform saturation mutation on amino acids in different positions of GFP 11, and screen for GFP 11 mutants that can enhance the signal intensity of the three-segment sfGFP. We first constructed a three-segment sfGFP detection system based on the hepatitis B virus core protein HBc (two HBc monomers can bind to each other).

[0033] 1. Characterization of HBc monomer interactions using three-segmented sfGFP

[0034] Using HBc as a model, the three-point sfGFP system proposed by Waldo's research group was tested. This test needs to construct 3 kinds of plasmids, namely the plasmid expressing GFP 1-9 (PCH-GFP 1-9), the plasmid expressing GFP 10-HBc fusion protein (pGFP10-HBc), and the plasmid expressing GFP11-HBc fusion protein (pGFP11-HBc). The following is the specific construction process:

[0035] 1.1 Construction of pla...

Embodiment 2

[0069] Example 2. Increasing the signal intensity of the three-segment sfGFP system through high-affinity molecule pairs

[0070] Since the first embodiment is unsuccessful, we continue to try the aforementioned second strategy. We hope that by fusing a pair of high-affinity molecules on GFP1-9 and GFP11, respectively, the affinity of GFP1-9 and GFP11 can be improved, and the signal intensity of the whole system can be increased. The principle is as follows image 3 Shown in A: The final fluorescent signal is enhanced by increasing the affinity between GFP1-9-P1 and GFP11-P2-B by fusing a pair of high-affinity molecules P1 and P2 to GFP1-9 and GFP11, respectively . We selected two molecular pairs with affinity, their molecular weights are different, and the affinity between molecular pairs is also different, Kd is pM level and fM level, which are N11S and C10 respectively (refer to Dixson et al., NanoLuc ComplementationReporter Optimized for Accurate Measurement of Protein I...

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Abstract

The invention discloses a protein fragment. Nucleic acid sequences encoding the protein fragment is shown in SEQ ID No: 56 or SEQ ID No: 57 or SEQ ID No: 58. The invention further discloses a segmented green fluorescent protein system for protein interaction detection, which comprises GFP10 in a three-part sfGFP system, and further comprises a protein encoded by the nucleic acid sequence shown in SEQ ID No: 58, or further comprises proteins encoded by the nucleic acid sequences shown in SEQ ID No: 56 and SEQ ID No: 57. The invention further discloses recombinant vectors, recombinant bacteria, transgenic cell lines or expression cassettes comprising the aforementioned nucleic acid sequences, and also discloses the application of the protein fragment to detection of the interaction among proteins, or the application of the nucleic acid sequences to detection of the interaction among proteins. A fluorescent signal of the improved segmented sfGFP system is significantly improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a segmented green fluorescent protein system for protein interaction detection. Background technique [0002] Probing protein-protein interactions (PPIs) in living cells is very important to reveal the roles of these molecules in physiological or pathological processes. To detect protein interactions, researchers have invented various methods, including affinity chromatography, co-immunoprecipitation, various two-hybrid systems, affinity hybridization, phage display, fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer (BRET), split luciferase complementation assay (SLCA), etc. (Eric M. et al, Microbiological Reviews. 1995; Micheal C. et al, Drug Discovery Today. 2016 et al). These methods have their own characteristics and are suitable for different application s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K19/00G01N21/64
CPCC07K14/43595C07K2319/70G01N21/6486
Inventor 胡接力黄爱龙沈静
Owner 重庆明道捷测生物科技有限公司
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