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Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote

A protein expression, eukaryotic technology, applied in the field of protein purification, can solve problems such as the influence of CBM cellulose affinity

Inactive Publication Date: 2011-09-07
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the influence of post-translational modification in yeast on the affinity of CBM cellulose, and the spontaneous splicing of intein during yeast expression are all problems that must be solved before the practical use of CBM-intein in yeast.

Method used

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  • Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote
  • Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote
  • Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Fusion expression and purification of the cellulose-binding domain to EGFP.

[0081] EGFP is a green fluorescent protein, which is often used for molecular markers and protein localization. It is a common protein. In this example, this protein is used as an example to express in yeast by fusion with the cellulose-binding domain CBM3. Purification to demonstrate the ability and advantages of CBM3 expression and purification in affinity chromatography

[0082] Reagents and Strains:

[0083] All the reagents in the present invention are commercial reagent grade reagents. Microcrystalline cellulose Avicel PH105 (20 μm) was purchased from FMC Co (Philadelphia, PA). Escherichia coli Escherichia coli XL10 gold (purchased from Stratagene) was used as the host bacteria for DNA manipulation, and the Luria-Bertani (LB) medium containing 100 μg / mL ampicillin (purchased from Bio.Basic.Inu) was used to cultivate E.coli . Pichia pastoris (Pichia pastoris) KM71H (purchased from Inv...

Embodiment 2

[0195] Fusion expression and purification of cellulose-binding domain with yeast intein VMA and EGFP, and pure EGFP protein without affinity tag was obtained by self-cleavage of intein.

[0196] In this example, EGFP is still taken as an example, and the cellulose-binding domain CBM3 and intein are fused and expressed in yeast, and purification is performed to demonstrate the advantages of intein in affinity chromatography for expressing target proteins fused with CBM3.

[0197] Reagents and Strains:

[0198] All reagents in this example are commercially available reagent grade reagents. Microcrystalline cellulose Avicel PH105 (20 μm) was purchased from FMC Co (Philadelphia, PA). Escherichia coli XL10 gold was used as the host bacteria for DNA manipulation, and Luria-Bertani (LB) medium containing 100 μg / mL ampicillin was used to cultivate E.coli. Pichia pastoris KM71H (purchased from Invitrogen) was used to express recombinant proteins. YPD (yeast extract, peptone, glucose...

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Abstract

The invention belongs to the field of recombinant protein expression and purification. The invention provides a method for expressing and purifying recombinant protein in a eukaryote by using a family 3 cellulose binding domain CBM3 serving as an affinity tag for recombinant protein expression. The method comprises the following steps of: (1) constructing a nucleotide sequence of coded CBM3 optimized by a eukaryote codon; (2) constructing a recombinant construct of the nucleotide sequence of the coded CBM3 optimized by the eukaryote codon in the step (1) and a nucleotide sequence of coded target protein; (3) cloning the recombinant construct constructed in the step (2) to an expression vector suitable to be expressed in the eukaryote; (4) expressing the recombinant expression vector obtained in the step (3) in the eukaryote; and (5) purifying the expressed recombinant protein by using the CBM3 as the affinity tag and using cellulose. The invention also provides a method for expressingand purifying the recombinant protein in the eukaryote by using the CBM3 serving as the affinity tag for the recombinant protein expression and internal protein, wherein the method comprises a step of constructing a target protein-internal protein-CBM3 recombinant construct so as to cut out and purify the recombinant protein of fusion expression without additional amino acid.

Description

technical field [0001] The invention relates to a protein purification method in recombinant protein expression, especially a rapid purification method using an affinity tag. More specifically, the present invention relates to methods for protein purification using amorphous cellulose and other celluloses as chromatographic materials. The invention also relates to the construction of affinity tags for protein purification. The present invention also relates to a method for fused expression of the cellulose binding domain with the recombinant protein in eukaryotic cells and rapid purification of the fused recombinant protein. More specifically, the present invention also relates to a method for cutting out and purifying fusion-expressed recombinant proteins without extra amino acids by using family 3 cellulose-binding domain CBM3 and intein in eukaryotes. Background technique [0002] With the rising demand for recombinant proteins and enzymes in the pharmaceutical and enzy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/79C07K1/22
Inventor 洪泂宛雯高晓连
Owner UNIV OF SCI & TECH OF CHINA
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