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Recombinant human-like elastin and composition thereof

A technology of elastin and recombinant protein, applied in the field of recombinant protein, can solve the problems of protein folding error, small protein molecular weight and easy degradation, and inability to effectively prevent elastin degradation

Pending Publication Date: 2020-09-11
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the large molecular weight of full-length human elastin, cloning and expressing the full-length protein in a prokaryotic system can easily lead to protein folding errors, or the generation of inclusion body proteins, resulting in the inability of elastin to form an active spatial structure and the inability to prepare active recombinants in vitro Human elastin, therefore, can try to construct a catalytically active recombinant human elastin by extracting part of the active polypeptide sequence in the full-length elastin to overcome the above-mentioned difficulties of recombinant human elastin
However, the construction of recombinant human elastin by extracting polypeptide fragments has the problems of small protein molecular weight, easy degradation, and low catalytic activity. Therefore, further design and construction of recombinant human elastin is needed to overcome these problems.
[0004] For the elastin that needs to be used frequently, the elastin is generally stored at -20 degrees. In order to avoid repeated freezing and thawing of the elastin, the general practice is to add a certain amount of glycerin to the elastin, but only adding glycerin can avoid elastin. Repeated freezing and thawing, but can not effectively prevent the degradation of elastin

Method used

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  • Recombinant human-like elastin and composition thereof
  • Recombinant human-like elastin and composition thereof
  • Recombinant human-like elastin and composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: The technical scheme for the construction of engineering bacteria expressing recombinant human-like elastin is as follows:

[0031] 1. Design of the target protein sequence: Human elastin-like protein (ELP) is a recombinant protein obtained by heterologous expression with an artificial sequence designed based on the amino acid sequence of tropoelastin. Usually in the form of (VAPGVG) 3 The peptide chain composed of S amino acid sequence repeat sequence is better than tropoelastin in animals in terms of structure and function. In this embodiment, the protein sequence is designed to repeat 5 times, namely [(VAPGVG) 3 S] 5 , as shown in SEQ NO.1.

[0032] 2. Acquisition of the target gene: the recombinant human elastin gene (rhELP) was artificially synthesized by General Biosystems (Anhui) Co., Ltd., and the target sequence was optimized according to the codon preference of Escherichia coli. Insert restriction sites BamH I (GGATCC) and Hind III (AAGCTT) respe...

Embodiment 2

[0038] Embodiment 2: all the other are identical with embodiment 1, and difference is that (VAPGVG) 3 The number of repetitions of the S amino acid sequence was designed to be 1, 3, 5, 7, 9, 11, 15, and 32 times, and the recombinant bacteria were constructed respectively, and recombinant human-like elastin with different repetition times was purified, and the purified protein stock solution (Concentration: 0.1mg / mL) was used as the test sample, and the superoxide anion scavenging rate (ie, catalytic activity) of different recombinant proteins under different storage days was compared at a constant temperature of 37°C. The results are shown in Table 1.

[0039] Table 1 Comparison results of catalytic activity of recombinant human elastin with different repetition times

[0040]

[0041] The catalytic activity of a recombinant protein depends on the number of catalytic active sites on its surface. In theory, the more repetitions of the polypeptide active fragment on the prima...

Embodiment 3

[0044] Example 3: Dilute the recombinant human-like elastin stock solution of known concentration (concentration should be greater than or equal to 0.1 mg / ml) prepared in Example 1 to 0.1 mg / ml elastin with Tris-Nacl buffer containing 2% glycerol stock solution.

[0045] 1. Use 0.1mg / ml elastin stock solution to prepare protective solutions containing specific contents of arginine and bovine serum albumin BSA respectively. The contents of arginine and bovine serum albumin are:

[0046] 1) Arginine: 50mM / L, 100mM / L, 150mM / L, 200mM / L;

[0047] 2) Bovine serum albumin BSA (mass fraction): 0.2%, 0.5%, 1.0%, 1.5%, 2.0%.

[0048] 2. The prepared reagent was subjected to an accelerated destruction test at 37°C for a period of 7 days.

[0049] The detection operation steps are as follows: put all the samples into a 37°C incubator for accelerated destruction, take out the samples on the 0th, 1st, 3rd, 5th, and 7th day, and use the superoxide anion scavenging ability detection kit (So...

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Abstract

The invention discloses recombinant human-like elastin and a composition thereof. The recombinant human-like elastin is formed through series connection of short repeat amino acid sequences, wherein the short repeat amino acid sequences are target protein polypeptide sequences which are formed through 3-7 times of tandem repeat of (VAPGVG)3S, and the target protein polypeptide sequences are shownin SEQ No. 1. The recombinant human-like elastin which is obtained through in vitro expression of a prokaryotic expression system has high activity for removal of superoxide radicals. Good resistanceto degradation is still maintained through a small number of tandem repeat of the active peptide fragments, and the catalytic activity can be kept longer than the catalytic activity of a single original fragment; and the service cycle of the recombinant human-like elastin can be improved significantly through assistance of a selected protective agent.

Description

technical field [0001] The invention relates to the field of recombinant proteins, in particular to a recombinant human-like elastin and its composition. Background technique [0002] Elastin is the main component of elastic fibers in hide tissue. The peptide chain of elastin contains more than 713 amino acid residues. Unlike collagen and keratin, there is no continuous repeating periodic structure throughout the entire peptide chain in the amino acid sequence of elastin, but there are alternating hydrophobic and hydrophilic peptide segments. Desmosin and desmosin formed by oxidizing lysyl are unique cross-linking structures of elastin. The elastin prepared from the bovine cervical ligament has been proved to have superoxide radical scavenging activity, while the human elastin expressed through in vitro recombination experiments has no relevant biological activity reports. [0003] Due to the large molecular weight of full-length human elastin, cloning and expressing the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K14/765
CPCC07K14/78C12N15/70C07K14/765C07K2319/00C12N2800/22
Inventor 王梦刘洁刘家炉徐文俊单雪芹凡玉芳王亚男李诚茹吴蕾金巢
Owner ANHUI JIUCHUAN BIOTECH
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