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Method for constructing non-denatured protein hydrogel through molecular self-assembly technology

A technology of self-assembly technology and construction method, which is applied in the field of preparing non-denatured protein hydrogel materials to achieve the effect of broadening application scenarios

Active Publication Date: 2022-04-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the technology of using self-assembly technology to prepare a non-denatured protein hydrogel structure in vitro has not been reported.

Method used

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  • Method for constructing non-denatured protein hydrogel through molecular self-assembly technology
  • Method for constructing non-denatured protein hydrogel through molecular self-assembly technology

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preparation example Construction

[0024] A kind of non-denatured protein hydrogel material prepared by molecular self-assembly technology, the preparation method comprises the following steps:

[0025] (a) Design and preparation of protein mutants: Using the sequence of archaeal ferritin from Thermotoga maritima (GenBank: AKE30776.1) as a template, a single point mutation was performed on the sequence by PCR gene amplification technology, and in A stop codon was added after glycine at position 150, named TmFtn△E. Then, using the sequence of TmFtn△E as a template, a single point mutation was performed on the sequence by PCR gene amplification technology to mutate the glycine at position 40 of TmFtn△E into glutamic acid / aspartic acid, which was named TmFtn△E40D / Ferritin mutants of E. The plasmid of the mutant was introduced into BL21(DE3) competent, and E. coli was amplified in LB medium containing a final concentration of 50 μg / mL Amp. When the OD600 reached about 0.8, the target was induced by adding 200 μM ...

Embodiment 1

[0044] The steps of protein expression and purification of Thermotoga maritima ferritin mutant in the present invention.

[0045] (1) Plasmid preparation and protein expression of thermotoga maritima ferritin mutant

[0046] Design and preparation of protein mutants: Using the sequence of archaeal ferritin from Thermotoga maritima as a template (GenBank: AKE30776.1), a single point mutation was performed on the sequence by PCR gene amplification technology, and the glycine at position 150 After adding a stop codon, named TmFtn△E. Then, using the sequence of TmFtn△E as a template, a single point mutation was performed on the sequence by PCR gene amplification technology to mutate the glycine at position 40 of TmFtn△E into glutamic acid / aspartic acid, which was named TmFtn△E40D / Ferritin mutants of E. The mutant plasmid was introduced into BL21(DE3) competent cells. The specific operation was to add 5 μL of the mutant plasmid to 100 μL of competent cells. After standing on ice...

Embodiment 2

[0050] In the present invention, the construction of the thermotoga maritima one-dimensional protein assembly, the formation of the protein network structure and the preparation of the protein hydrocoagulant are carried out in various steps.

[0051] (1) Construction of one-dimensional and network structural protein assemblies of Thermotoga maritima

[0052] Thermotoga maritima ferritin TmFtn△E 40D / E After separation and purification, dialyze into a buffer containing 10-50mM EDTA (pH8.0, 25mM HEPES, 250mM NaCl) to completely remove free metal ions. The protein solution was then dialyzed again into buffer (pH 8.0, 25 mM HEPES, 250 mM NaCl) to remove EDTA and its formed metal complexes. The protein concentration was measured by the Lowery method, and a protein stock solution with a final concentration of 1 mg / mL was prepared. Use acidified water to prepare a zinc / cobalt / nickel / copper ion solution with a mother liquor concentration of 100mM, dilute the corresponding multiples a...

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Abstract

The invention relates to a method for constructing non-denatured protein hydrogel by a molecular self-assembly technology, which comprises the following steps: a) mutating thermotoga maritime ferritin 40-site glycine into glutamic acid / aspartic acid by a point mutation technology to obtain a ferritin mutant; and b) after the protein mutant is expressed and purified, a protein one-dimensional assembly is formed through zinc / cobalt / nickel / copper ion coordination. And c) mutating 111-site serine of the mutant into histidine to prepare a second protein mutant. D) after the mutant is expressed and purified, a protein network structure is formed through zinc / cobalt / nickel / copper ion coordination, and the protein concentration is further improved to prepare the protein hydrogel. According to the protein hydrogel structure prepared by the method, a natural protein structure is reserved, the protein biological activity of the protein hydrogel is maintained, and meanwhile, the protein hydrogel has injectability and a self-healing function and has a good application prospect in the fields of food industry and medical treatment.

Description

technical field [0001] The invention relates to a non-denatured protein hydrogel material prepared by molecular self-assembly technology. The prepared protein hydrogel can be reversibly regulated by metal ions, and has injectability and self-healing function. Background technique [0002] Hydrogel is a high molecular polymer that takes into account the two-phase characteristics of solid and liquid. It usually forms a three-dimensional network structure through physical or chemical cross-linking. It can swell rapidly in water and hold a large amount of water without dissolving. Hydrogel materials have attracted the attention of scientists in recent years because of their unique porous structure, good viscoelasticity, and ductility. At present, hydrogels have broad application prospects in the fields of cell scaffolds, environmental engineering, and equipment coatings. Some natural hydrogel materials, such as agarose, gelatin, and methylcellulose, have been used in agricultur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/70C12N15/31C07K1/30C07K1/18C07K1/22C07K1/14C08J3/075C08L89/00
CPCY02A50/30
Inventor 吕晨艳刘宇赵广华
Owner CHINA AGRI UNIV
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