Molecular probe for detecting malonyl coenzyme A

A technology of malonyl coenzyme and DNA molecules, which is applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc. It can solve the problems of reporter gene transcription efficiency influence, inability to detect cells, inability to distinguish, etc.

Active Publication Date: 2018-11-06
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The problem with the above two types of methods is that tissue extracts must be used to achieve detection
[0008] The disadvantages of this method are: 1. Due to the transcription process involved, it is affected by the transcription efficiency of the reporter gene
2. It can only detect the amount of Malonyl-CoA in the whole cell, but cannot distinguish the amount of Malonyl-CoA in each organelle
3. Due to the reaction in the cell system, extracellular Malonyl-CoA cannot be detected, such as the detection of cell-free areas such as intercellular matrix or blood

Method used

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  • Molecular probe for detecting malonyl coenzyme A
  • Molecular probe for detecting malonyl coenzyme A
  • Molecular probe for detecting malonyl coenzyme A

Examples

Experimental program
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Effect test

Embodiment 1

[0104] Example 1. Preparation and Identification of Molecular Probes for Detection of Malonyl-CoA

[0105] 1. Preparation of molecular probes for detection of malonyl-CoA

[0106] In the present invention, the molecular probe used to detect malonyl-CoA is specifically structured as figure 1 Fusion proteins indicated. From N-terminal to C-terminal, the fusion protein is NanoLuc luciferase large subunit (Lg), linker peptide I (Linker1), FapR△43 protein (FapR△43), linker peptide II (Linker2) and NanoLuc luciferase Small subunit (Sm). The amino acid sequence of the fusion protein is shown in SEQ ID No.1, wherein the 1-159th position is the NanoLuc luciferase large subunit (Lg), the 160-174th position is the connecting peptide I (Linker1), and the 175-318th position The position is FapR△43 protein (FapR△43), the 319-331 position is linker peptide II (Linker2), and the 332-342 position is NanoLuc luciferase small subunit (Sm). The fusion protein was named NanoLuc-FapRΔ43.

[0...

Embodiment 2

[0117] Example 2, detection of malonyl-CoA with the molecular probe prepared in Example 1

[0118] 1. In vitro experiment

[0119] 1. Demonstrate the reactivity of molecular probes to Malonyl-CoA

[0120] The reaction was carried out in a 96-well microplate, and the total reaction volume was 100 microliters. The reaction was divided into 8 groups, and each group replicated 5 wells. First, the purified fusion protein NanoLuc-FapRΔ43 prepared in Example 1 was added to each well so that the final concentration was 0.1 μg / ml. After that, different concentrations of Malonyl-CoA were added, and the final concentrations of each group were 0, 0.1, 1, 10, 50, 250, 1250 and 6250 μm / L. After incubating at room temperature for 10 minutes, NanoLuc's substrate Furimazine was added to a final concentration of 1 μm / L. After 10 minutes at room temperature, the fluorescence value of each well was measured with a multifunctional microplate reader.

[0121] The results showed that it was obs...

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Abstract

The invention discloses a molecular probe for detecting malonyl coenzyme A. The moklecular probe contains fusion protein of NanoLuc luciferase large subunit, FapRdelta43 protein and NanoLuc luciferasesubunit from the N end to the C end. The molecular probe can both detect the malonyl coenzyme A in the extract of sample in vitro and detect and perform detection and dynamic observation on the malonyl coenzyme A in living cells; most importantly, the malonyl coenzyme A can be detected in different organelles of the living cells, simpleness in operation is achieved, and therefore, the fusion protein has the potential to become a commercial product.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a molecular probe for detecting malonyl-CoA. Background technique [0002] Malonyl-CoA (Malonyl-CoA) is an important intermediate metabolite in the process of fatty acid synthesis and metabolism, and also a regulatory molecule in the process of fatty acid oxidative decomposition. The increase of malonyl-CoA in cells promotes its combination with mitochondrial surface fatty acid transport-related protein CPT1 to inhibit the transport of fatty acids to mitochondria, thereby reducing the oxidative decomposition of fatty acids. Recent studies have found that malonyl-CoA is also a group donor in the process of protein post-translational modification malonylation. Under certain conditions, malonyl-CoA can spontaneously modify proteins by malonylation. The function of Malonylation modification is still under study. [0003] Currently, there are three main types of detection methods for M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/65C12N15/62C12Q1/66
CPCC07K14/195C07K2319/00C12N15/65C12Q1/66
Inventor 卫涛涛杜贻鹏
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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