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Novel tag protein for protein enrichment expression and intracellular localization as well as application thereof

A technology for labeling proteins and proteins, applied in the field of labeling proteins, can solve the problems of reducing cell activity, limiting protein yield, increasing production costs, etc., to achieve the effects of increasing final output, maintaining activity, and reducing loss

Active Publication Date: 2019-12-10
NAT UNIV OF DEFENSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, there is a significant limiting factor in the process of heterologous expression: the massive expression of foreign proteins in host cells may affect the physiological metabolic process of the cells themselves, reduce cell viability or even kill them, and limit the final protein yield , which increases the cost of production

Method used

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  • Novel tag protein for protein enrichment expression and intracellular localization as well as application thereof
  • Novel tag protein for protein enrichment expression and intracellular localization as well as application thereof
  • Novel tag protein for protein enrichment expression and intracellular localization as well as application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1. Expression of pEGFP-C1 and pEGFP-C1-RA1 in 293T cells

[0060] figure 1 . Confocal microscopy results of 293T cells transfected with pEGFP-C1 for 24 hours.

[0061] A) Excitation wavelength 488, signal collection channel 507, eGFP green fluorescence signal;

[0062] B) Excitation wavelength 364, signal collection channel 461, DAPI blue fluorescence signal;

[0063] C) Experimental results after fusion of eGFP and DAPI signaling channels;

[0064] D) Partial magnification of 5 times.

[0065] from figure 1 In A, it can be seen that there are a large number of green fluorescent signals (derived from the expression of eGFP) in the 293T cells successfully transfected with the pEGFP-C1 plasmid. figure 1 The blue signal in B comes from DAPI. Due to the use of a membrane-breaking agent (TritonX-100), no matter whether the cells are successfully transfected and express eGFP, the nuclei can be stained by DAPI, so the number of cells is greater than figure 1 a. ...

Embodiment 2

[0073] Example 2. Expression of pEGFP-C1 and pEGFP-C1-RA1 in Hela cells

[0074] from image 3 In A, it can be seen that there are a large number of green fluorescent signals (derived from the expression of eGFP) in the Hela cells successfully transfected with the pEGFP-C1 plasmid. image 3 The blue signal in B comes from DAPI. Due to the use of a membrane-breaking agent (TritonX-100), no matter whether the cells are successfully transfected and express eGFP, the nuclei can be stained by DAPI, so the number of cells is greater than image 3 a. Will image 3 After the images of A and 3B are fused, it can be found that the cells emitting eGFP green fluorescence overlap with some DAPI-stained nuclei. After partially amplifying the overlapping part (5 times), it can be found that the green fluorescent signal is evenly distributed in Hela cells successfully transfected with pEGFP-C1 plasmid, and the DAPI signal in the nucleus is blocked to a certain extent.

[0075] image 3 ....

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Abstract

The invention relates to novel tag protein for protein enrichment expression and intracellular localization as well as application thereof. The tag protein is derived from membrane protein-reflectin A1(RA1) of a squid iridophore. The application of the novel tag protein for protein enrichment expression and intracellular localization comprises the following steps: (a) providing a construction object, wherein in an open reading frame of a gene expression vector, the construction object, from 5' to 3' segments, sequentially comprises the following operable connecting components: an encoding geneof eGFP and an encoding gene of the RA1; (b) transferring the construction object in (a) into a host cell so as to express eGFP and RA1 tags; (c) providing a group of recombinant protein formed by the eGFP and RA1 tags, wherein the recombinant protein contains functional areas of the eGFP and the RA1, and the eGFP exerts under the action of the RA1 functional area; and (d) performing intracellular enrichment and spatial orientation to be favorable for the subsequent separation and purification.

Description

technical field [0001] The invention belongs to the technical field of tagged proteins in biotechnology, and in particular relates to a novel tagged protein for protein enrichment expression and intracellular positioning and application thereof. Background technique [0002] In biological research in the post-genome era, protein functional analysis is an important aspect of in vitro experiments. Achieving rapid, convenient and economical expression of recombinant proteins has become a prerequisite. Currently commonly used protein expression systems include Escherichia coli, yeast, insect cells, and mammalian cell systems. [0003] In the process of heterologous expression, it is often necessary to detect the expression of the target protein, so a series of protein tags need to be used for labeling and assistance. Protein expression tags play a vital role in promoting protein expression, assisting protein detection, and realizing protein purification. [0004] For example,...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/85
CPCC07K14/43504C07K2319/01C07K2319/60C07K2319/735C12N15/62C12N15/85C12N2800/107
Inventor 宋俊祎吴文健胡碧茹
Owner NAT UNIV OF DEFENSE TECH
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