A kind of thermosensitive polypeptide and its encoding gene and its preparation method and application

A coding gene and temperature-sensitive technology, which is applied in the field of temperature-sensitive polypeptides and their coding genes, can solve problems such as difficult synthesis and difficult acquisition of temperature-sensitive polypeptides, and achieve the effects of high recovery efficiency, wide application range and broad application prospects

Active Publication Date: 2022-08-05
SHAANXI SCI TECH UNIV
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  • Abstract
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Problems solved by technology

First, it is difficult to obtain a temperature-sensitive polypeptide with a phase transition critical temperature suitable for protein separation and purification labels; second, a temperature-sensitive polypeptide is generally composed of multiple repeating units, which is not easy to synthesize

Method used

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  • A kind of thermosensitive polypeptide and its encoding gene and its preparation method and application
  • A kind of thermosensitive polypeptide and its encoding gene and its preparation method and application
  • A kind of thermosensitive polypeptide and its encoding gene and its preparation method and application

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Embodiment Construction

[0038] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

[0039] 1. Materials and Reagents

[0040] Restriction endonuclease, T4 polynucleotide kinase, bovine intestinal alkaline phosphatase and T4 DNA ligase were purchased from NEB Company. DNA markers were purchased from Nearshore Protein Technology Co., Ltd., and prestained protein markers were purchased from Thermo Scientific. The plasmid extraction kit was purchased from OMEGA BIO-TEK, and the gel recovery kit was purchased from Beijing Boda Tek Bio-Gene Technology Co., Ltd. Expression plasmid pET-28a, strain E.coli Top10 and BL21(DE3) were kept by our laboratory.

[0041] 2. Design of monomeric genes for thermosensitive polypeptides

[0042] During our research, we found that the polypeptides formed by continuous repeats of GVGVP (SEQ...

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Abstract

The present invention relates to a temperature-sensitive polypeptide, comprising a plurality of short peptide repeating units, and the sequence of each of the short peptide repeating units is shown in SEQ ID NO: 1; it also relates to a gene encoding the temperature-sensitive polypeptide; The temperature-sensitive polypeptide encoding gene is used as a protein expression vector of a purification tag; it also relates to a preparation method of the protein expression vector; it also relates to a protein purification method. The temperature-sensitive polypeptide synthesized in the present invention has the property of reversible phase transition, and can realize the separation and purification of polypeptide molecules through the reversible phase transition cycle process. Using temperature-sensitive polypeptides as tags to endow fusion proteins with reversible phase transition properties, a simple and fast protein separation and purification method with low purification cost, high recovery efficiency and wide application range was developed.

Description

technical field [0001] The present invention relates to the field of protein separation and purification, more particularly, to a temperature-sensitive polypeptide and its encoding gene, a preparation method and application thereof. Background technique [0002] In the process of protein separation and purification, although chromatographic column chromatography is the most commonly used separation technology, this technology is time-consuming and labor-intensive, requires special separation equipment and reagents, and has high production costs. [0003] In order to simplify the separation steps, people began to explore different separation methods. The researchers found that some short peptides are temperature-sensitive and undergo reversible phase transitions with changes in ambient temperature. At a certain concentration, it is soluble at low temperature; when the ambient temperature increases, the randomly coiled and stretched structure gradually forms a β helix, which ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/70C12N15/66C12N15/62
Inventor 王珊珊
Owner SHAANXI SCI TECH UNIV
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