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Transmembrane Dps (starvation-induced DNA binding protein) and application

A protein-binding and starvation-induced technology, applied in the field of starvation-induced DNA-binding proteins, can solve the problems of improper protein assembly, improper assembly, solubility and protein stability, and achieve good water solubility, uniform size, Easy to purify effect

Active Publication Date: 2019-09-27
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The wild-type Dps protein can complete self-assembly in vivo. If other short peptides are fused at its N-terminus or C-terminus to have transmembrane function, it may not be assembled correctly due to protein conformation problems; The N-terminal of wild-type Dps plus lysosomal escape peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), HA2 (GLFEAIEGFIENGWEGMIDGWYG), HA2TAT (GLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRRR), melittin (NH2-QQRKRKIWSILAPLGTTLVKLVAGIG-COOH) or synthetic short peptide ), the protein cannot be assembled correctly; even if some have transmembrane function, their solubility and protein stability will be affected

Method used

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  • Transmembrane Dps (starvation-induced DNA binding protein) and application
  • Transmembrane Dps (starvation-induced DNA binding protein) and application
  • Transmembrane Dps (starvation-induced DNA binding protein) and application

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A transmembrane starvation-induced DNA-binding protein, the protein is sequentially fused with 1 Avitag and X His at the N-terminal or C-terminal of Dps; or sequentially fused with Y GGGGS and 6 His at the N-terminal or C-terminal of Dps His; said X is any natural number from 1 to 7; said Y is 1 or 2 or 3.

[0035] In the embodiment of the present invention, one Avitag and two His fusion proteins are sequentially fused to the N-terminus of Listeria-derived Dps 2 BDps is taken as an example for illustration, the H 2 The DNA sequence of BDps is shown in SEQ ID NO.1.

[0036] The Dps protein can be Dps protein from different species, and can also be some protein nanocages with a spherical structure.

Embodiment 2

[0038] A method for preparing a transmembrane starvation-induced DNA-binding protein, comprising the steps of:

[0039] The embodiment of the present invention is illustrated by taking the Dps protein derived from prokaryotes as an example, and the Dps protein derived from other species can also complete the present invention;

[0040] Using Listeria genomic DNA as a template, amplify the wtDps nucleotide sequence (the nucleotide sequence is shown in SEQ ID NO.2), and the primers for amplifying the wtDps nucleotide sequence are: 5'CATATGAAAACAATCAACTCAGTAG3' and 5'CTCGAGTTATTCTAATGGAGCTTTT3' .

[0041] Design primers to sequentially add 1 Avitag and X His nucleotide sequences to the N-terminal of Dps, or design primers to sequentially add Y GGGGS and 6 His nucleotide sequences to the N-terminal of Dps; Add enzyme cleavage sites and corresponding protective bases, select double enzyme cleavage sites NdeI and Xho I, perform PCR amplification, detect by agarose gel electrophores...

Embodiment 3

[0060] Detection of the transmembrane ability of starvation-induced DNA-binding proteins:

[0061] First, modify the Alex555 fluorescent dye on the surface of the protein. Specific steps: take wtDps as the reference concentration at 2 mg / ml, adjust the molar concentration of the fusion protein prepared in Example 2 to be consistent with wtDps, and the feed ratio of protein and Alex555 is corresponding to one protein ball 12 fluorescent molecules, the reaction buffer is 0.1M sodium bicarbonate, shake at 600rpm / min at 25°C for 1h, then stand overnight at 4°C, dialyze into PBS solution to remove free Alex555, then pass through SDS-PAGE, optical density instrument GS900 For imaging, the image J software was used for grayscale analysis to quantify the protein concentration, and the fluorescence spectrophotometer was used to detect the modification of the dye in the sample, which was used to correct the results of flow cytometry.

[0062] Choose skin fibroblast HFF-1 and African gre...

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Abstract

The invention belongs to the field of bioscience, and particularly relates to transmembrane Dps (starvation-induced DNA binding protein) and an application. By means of rational design of a protein nanocage derived from prokaryotes, the protein nanocage is enabled to be an antioxidant protein nanomaterial which can be applied to mammalian cells. The protein nanomaterial is characterized in that transportation across mammalian cell membranes of Dps is realized by fused expression of His-tag and Avitag (LNDIFEAQKIEWHE) at the N-terminal of Dps; the cells can be protected from being damaged by ROS (Reactive Oxygen Species); meanwhile, the protein nanomaterial is good in biocompatibility, simple to design, easy to prepare, high in yield and suitable for large-scale production.

Description

technical field [0001] The invention belongs to the field of biological sciences, and specifically relates to a transmembrane starvation-induced DNA binding protein and its application. Background technique [0002] Reactive oxygen species (Reactive Oxygen Species, ROS) are natural by-products of cellular oxidative metabolism, mainly derived from mitochondria, including singlet oxygen, superoxide ions, hydrogen peroxide, and hydroxyl radicals. Although ROS play an important role in regulating cell differentiation, proliferation, signal transduction, and inflammatory response, the body is in complex environmental conditions, and many chemical, physical, and biological factors can cause the increase of active oxygen in the body. When the oxygen content exceeds the clearance threshold of the body, excessive ROS in the cell can damage biological macromolecules, such as lipid peroxidation, protein inactivation, DNA mutation, etc., resulting in loss of cell function. Among them, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61P39/06
CPCC07K14/195C07K14/245A61P39/06C07K2319/21C07K2319/20A61K38/00
Inventor 李峰朱伟伟方倜张先恩
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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