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Method for purifying protein of enzyme aggregate based on self-aggregation short-peptide induction

A technology for protein purification and enzyme aggregation, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of many operation steps and high costs, and achieve cost reduction and high economy , The effect of overcoming the bottleneck of industrial application

Active Publication Date: 2011-10-26
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the shortcomings of the existing protein purification technology, such as high cost, use of protease, and many operating steps, to provide a fusion tag with self-aggregation and self-cleavage function, and a method for rapid purification of recombinant protein using the fusion tag

Method used

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  • Method for purifying protein of enzyme aggregate based on self-aggregation short-peptide induction
  • Method for purifying protein of enzyme aggregate based on self-aggregation short-peptide induction
  • Method for purifying protein of enzyme aggregate based on self-aggregation short-peptide induction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Expression and purification of a triplet fusion protein using wild-type Bacillus subtilis lipase A (LipA) as the target protein (fused at the N-terminus)

[0039] Strain: Escherichia coli BL21(DE3)

[0040] Vector: pET-30a(+)

[0041] Medium: LB / Kan r , each liter medium contains tryptone 10.0g, yeast extract powder 5.0g, NaCl 10.0g, if it is a solid medium, add agar 15.0g per liter medium, pH7.0, 50μg / ml kanamycin ( Kanamycine).

[0042] 1. Construction of triplet fusion protein expression vectors pET-30a(+)-LipA-Mxe-ELK16 and pET-30a(+)-LipA-Mxe-18A

[0043] The pET-30a(+)-LipA-ELK16 plasmid was extracted using the high-purity plasmid small extraction kit from Tiangen Company (ELK16 is fused to LipA at the C-terminus, and the plasmid structure is as follows Figure 6 shown. Its construction method is to select the pET-30a (+) plasmid of commercial plasmid Novogen Company, use the online tool DNAworks to design Linker and ELK16 nucleotide sequence, utili...

Embodiment 2

[0051] Example 2 Expression and purification of Aspergillus fumigatus Amadoriase II (AMA) as a triplet fusion protein of the target protein (fused at the N-terminus).

[0052] Strain: Escherichia coli BL21(DE3)

[0053] Vector: pET-30a(+)

[0054] Medium: LB / Kan r , each liter medium contains tryptone 10.0g, yeast extract powder 5.0g, NaCl 10.0g, if it is a solid medium, add agar 15.0g per liter medium, pH7.0, 50μg / ml kanamycin ( Kanamycine).

[0055] 1. Construction of triplet fusion protein expression vectors pET-30a(+)-AMA-Mxe-ELK16 and pET-30a(+)-AMA-Mxe-18A

[0056] The pET-30a(+)-LipA-Mxe-ELK16 plasmid was extracted using the high-purity plasmid mini-extraction kit from Tiangen Company (see Example 1 for the construction method, Figure 7 ), pET-30a(+)-LipA-Mxe-18A (see Example 1 for the construction method, Figure 7 ) and pET-30a(+)-AMA-ELK16 plasmid (ELK16 is fused with AMA at the C-terminus, the plasmid structure is as follows Figure 8 As shown, its construction...

Embodiment 3

[0061] Example 3 Expression of Bacillus pumilus xylosidase (XynB) as a triplet fusion protein (fused at the N-terminus) of the target protein and its purification

[0062] Strain: Escherichia coli BL21(DE3)

[0063] Vector: pET-30a(+)

[0064] Medium: LB / Kan r , each liter medium contains tryptone 10.0g, yeast extract powder 5.0g, NaCl 10.0g, if it is a solid medium, add agar 15.0g per liter medium, pH7.0, 50μg / ml kanamycin ( Kanamycine).

[0065] 1. Construction of triplet fusion protein expression vectors pET-30a(+)-XynB-Mxe-ELK16 and pET-30a(+)-XynB-Mxe-18A

[0066] The pET-30a(+)-LipA-Mxe-ELK16 plasmid was extracted using the high-purity plasmid mini-extraction kit from Tiangen Company (see Example 1 for the construction method, and the plasmid structure is as follows Figure 7 shown), pET-30a(+)-LipA-Mxe-18A (see Example 1 for the construction method, the plasmid structure is as follows Figure 7 shown) and pET-30a(+)-XynB-ELK16 plasmid (ELK16 is fused with XynB at t...

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Abstract

The invention provides a method for purifying protein of enzyme aggregate based on self-aggregation short-peptide induction. The method comprises the following steps of: fusing and expressing a target protein, a peptide section (preferably intein) containing a cleavage site and a short peptide with self-aggregation function into a triplet in a sequence from left to right or from right to left, forming an enzyme aggregate in an expressing process, and separating from most soluble impurities in cell lysate by adopting a centrifuging or filtering method; and then releasing the target protein into solution by inducing cutting at a cleavage site so as to achieve the purpose of purification. The method has the characteristics of low cost and simple flow, can be used for laboratory-scale high-throughput protein purification and is also beneficial to industrial-scale protein production.

Description

technical field [0001] The invention relates to the expression and purification of recombinant proteins, in particular to a protein purification method based on enzyme aggregates induced by self-aggregating short peptides. Background technique [0002] The production of recombinant proteins is very important no matter in the industrial field or laboratory scale, and the cost of separation and purification of recombinant proteins accounts for about 60%-80% of the total cost (Chen Hao, Chen Yuhong, Zhu Dexu, Liu Jianning, Purification of recombinant proteins Technology, Chinese Journal of Bioengineering, 2002, 22(5): p.87-92.). Purification of recombinant proteins can be divided into four steps: sample preparation, capture, intermediate purification and fine purification, among which intermediate purification can achieve moderate sample purity, and the processed protein samples can be used for various experimental analysis purposes such as N Terminal sequence analysis, antige...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N9/42C12N9/20C12N9/06C12N15/70C12N15/74C12N15/80C12N15/62C12R1/19C12R1/125
Inventor 林章凛邢磊吴伟
Owner TSINGHUA UNIV
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