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Method for foam separation of beta-glucosidase fusion protein

A glucosidase and fusion protein technology, which is applied in the directions of glycosylase, fusion polypeptide, chemical instruments and methods, etc., can solve the problems of inability to complete the separation and purification of β-Glu, low yield and purification multiple, complicated operation process, etc. Achieve the effect of low cost, simple operation and improved separation efficiency

Pending Publication Date: 2021-12-21
SUZHOU CHIEN SHIUNG INST OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of these methods is that multiple methods need to be used in combination, and a single method cannot complete the separation and purification of β-Glu
These methods are complex in operation, high in cost, low in yield and purification factor, and thus have certain limitations.

Method used

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  • Method for foam separation of beta-glucosidase fusion protein
  • Method for foam separation of beta-glucosidase fusion protein
  • Method for foam separation of beta-glucosidase fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] (1) Induced expression of fusion protein GLEGB

[0078] Coded and designed ELP amino acid sequence, denoted as ELP(VPGVG)50, inserted into GB polypeptide, linked to Glu with connecting peptide linker, and constructed recombinant plasmid pET-GLEGB(Glu-linker-ELP-GB).

[0079] figure 1 It is a schematic diagram of the structure of the recombinant plasmid pET-GLEGB (containing ELP and GB tags).

[0080] The amino acid sequence of the Glu is shown in SEQ.ID.NO.1; the amino acid sequence of the linker is shown in SEQ.ID.NO.2; the amino acid sequence of the ELP is shown in SEQ.ID.NO.3 ; The amino acid sequence of GB is shown in SEQ.ID.NO.4. Each amino acid sequence is as follows:

[0081] SEQ.ID.NO.1(Glu):

[0082]MDDVDNDTLVTFPDDFKLGAATASYQIEGGWDADGKGPNIWDTLTHERPHLVVDRSTGDVADDSY HLYLEDVRLLKDMGAEVYRFSISWARILPEGHDNNVNEAGIEYYNKLIDALLRNGIEPMVTMYHWDLPQ KLQDLGGWPNRILAKYAENYARVLFSNFGDRVKQWLTFNEPLTFMDAYASDTGMAPSVDTPGIGDYLTA HTVILAHANIYRLYEREFREEQQGQVGIALNIHWCEPETGSPKDVEACERYQQFNL...

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Abstract

The invention belongs to the technical field of recombinant protein separation and purification, and mainly relates to a method for separating and purifying beta-glucosidase fusion protein, in particular to a method for constructing a foam separation system by using hydrophobic label performance and separating and purifying beta-Glu fusion protein by combining the temperature-sensitive performance of fusion enzyme. The invention aims to overcome the defects of complex extraction process, low separation and purification efficiency, high cost and the like in the existing protein separation and purification method, and provides a process for separating and purifying beta-glucosidase fusion protein by combining foam separation and inverse transition cycling (ITC). The process has the advantages of simple equipment, low investment, low energy consumption, no pollution and the like, is good in separation effect and low in enzyme activity loss, and is of great practical significance.

Description

technical field [0001] The invention belongs to the field of recombinant protein separation and purification, and specifically discloses a method for foam separation of β-glucosidase fusion protein. Background technique [0002] β-glucosidase (β-Glu) is a hydrolase that can hydrolyze the non-reducing β-D-glucosidic bond bound to the terminal, and it can decompose some flavor precursors in plants into natural odorant Aroma substances can also assist other cellulase enzymes in degrading cellulose. In addition, it also participates in the glucose metabolism of organisms and plays an important role in maintaining the normal physiological functions of organisms. Therefore, it is of great practical significance to study the separation and purification of β-Glu. [0003] At present, the commonly used separation and purification methods of β-Glu mainly include: ammonium sulfate / acetone fractional precipitation, gel filtration, Sephadex column chromatography, high performance liqui...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/62C12N15/70
CPCC12N9/2445C12N15/70C12Y302/01021C07K2319/00
Inventor 解雪乔方思涵苗向阳黄文睿
Owner SUZHOU CHIEN SHIUNG INST OF TECH
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