Target protein labeling or tracing composition and method

A target protein and composition technology, which is applied in the field of target protein labeling or tracer composition, can solve problems such as affecting the function of the target protein, lack of function of the target protein, affecting the structural stability of the target protein, etc.

Pending Publication Date: 2022-07-05
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, directly inserting the fluorescently labeled protein into the target protein often affects the function of the target protein
Because most fluorescently-labeled proteins are spherical proteins of nearly 200 amino acids, the insertion of this larger fluorescently-labeled protein may affect the structural stability of the target protein, resulting in loss of function of the target protein
[0004] In summary, fluorescent labeling of target proteins by traditional methods may destroy the protein structure and its intracellular location, thereby affecting the function of the protein and even affecting normal cell proliferation.

Method used

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  • Target protein labeling or tracing composition and method
  • Target protein labeling or tracing composition and method
  • Target protein labeling or tracing composition and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Dnm1 tracer

[0078] 1.1 The N- or C-terminal fluorescent labeling of Dnm1 affects its function

[0079] In fission yeast, Dnm1 is the only protein known to directly mediate mitochondrial fission. Our study found that the mitochondrial morphology of wild-type yeast usually forms a complex network structure, and the number of mitochondria in each cell is generally multiple. When Dnm1 is deleted in fission yeast, mitochondria do not divide, and the mitochondria are abnormally morphologically and number 1 (see figure 2 A); The statistical graph of the number of mitochondria in each cell is figure 2 B. When the C- and N-termini of Dnm1 were tagged with GFP or HA, the mitochondrial phenotype was identical to that in the absence of Dnm1 (see figure 2 A. figure 2 B). When the GFP tag was replaced with two red fluorescent protein tags, mCherry or tdTomato, the mitochondrial phenotype was also the same as that in the absence of Dnm1 ( figure 2 B). It can b...

Embodiment 2

[0086] Example 2 Mal3 tracer

[0087] Mal3 is a microtubule positive end-binding protein in fission yeast and maintains microtubule stability.

[0088] 2.1 The N- or C-terminal fluorescent labeling of Mal3 affects its function

[0089] We found that C- or N-terminal labeling of Mal3 resulted in very short microtubules, similar to the microtubule phenotype of Mal3-deficient cells (see Figure 5 A, B). Thus, Mal3 C-terminal or N-terminal tagged GFP can affect Mal3 function. When Mal3 is out of function or functionally impaired, microtubules are unstable and prone to premature depolymerization, making it difficult to grow to wild-type microtubule lengths, thereby exhibiting a phenotype with shorter microtubules.

[0090] 2.2 Design of Mal3 surface fluorescent labeling

[0091] Since the labeling of both ends of Mal3 affects protein function, we tried to label Mal3 with a new method. First, we obtained the resolved structure of Schizosaccharomyces cerevisiae Mal3 (PDB: 5m79) ...

Embodiment 3

[0094] Example 3 Nda3 localization observation

[0095] Microtubules are formed by the polymerization of alpha microtubules and beta tubulin. In Schizosaccharomyces cerevisiae, α-tubulin Nda2 and β-tubulin Nda3 are essential genes, and the two ends of the proteins are labeled and lethal. Therefore, most researchers can only label the non-essential gene at the N-terminus of α-tubulin Atb2 (the C-terminus cannot also be labeled) to achieve microtubule observation. We used the novel method invented here to label the beta-tubulin Nda3 and assess changes in microtubule function.

[0096] 3.1 Design of a new method to label β-tubulin

[0097] First, we obtained the resolved fission yeast Nda3 complex structure (PDB: 5mjs) according to the website https: / / www.rcsb.org / ; according to the Nda3 structure (see Figure 8 ), we searched for the loop sequence facing outward on the surface of the target protein at the interface where the target protein does not interact with its interacti...

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Abstract

The invention relates to the field of protein marking or tracing, in particular to a composition and a method for marking or tracing a target protein. The method comprises the following steps: firstly, analyzing the structure of a target protein and determining a ring sequence facing the outside of the surface of the protein, then inserting a small tag (such as an HA tag) with flexible amino acid connecting peptide fragments at two ends into the middle of the ring sequence by a molecular cloning method, and finally expressing the small tag nano antibody fused with the fluorescent protein. Therefore, intracellular tracing of the target protein is realized by utilizing the characteristic that the nano antibody specifically recognizes the tag on the surface of the target protein.

Description

technical field [0001] The present invention relates to the field of protein labeling or tracing, in particular to a target protein labeling or tracing composition and method. Background technique [0002] In order to analyze the dynamic localization of proteins in cells, it is necessary to fluorescently label the target proteins and use live-cell fluorescence microscopy to image them. Traditional fluorescent labeling methods are often achieved by adding fluorescently labeled proteins to the C-terminus and N-terminus of the target protein. However, adding fluorescently labeled proteins to the C-terminus or N-terminus of proteins may affect the stability and function of the target protein. For example, the C-terminus or N-terminus of the target protein may be inside the protein structure, and the protein structure will be destroyed after connecting the fluorescently labeled protein; the C-terminus or N-terminus of the target protein may also interact with other proteins, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/58G01N33/68
CPCG01N33/533G01N33/68G01N33/582G01N2333/39
Inventor 符传孩储永康
Owner UNIV OF SCI & TECH OF CHINA
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