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Method for realizing purification of recombinant protein by using antifreeze protein-intein as purification tag and application

An antifreeze protein and purification tag technology, applied in the field of separation and purification of exogenous recombinant proteins, can solve the problems of increased cost of protease, separation steps, complicated process, difficulty in scale, etc., and achieve easy molecular cloning operation, simple operation and labor saving. Effect

Pending Publication Date: 2021-10-08
李宪臻
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method and application of using the antifreeze protein-intein sequence as a purification tag to realize the separation and purification of exogenous recombinant protein, thereby solving the need for expensive chromatographic fillers and chromatographic columns in the existing recombinant protein purification technology, Removing the tag sequence requires the use of protease to further increase the cost and separation steps, resulting in high cost of separation and purification, complicated process, and difficulty in scaling up.

Method used

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  • Method for realizing purification of recombinant protein by using antifreeze protein-intein as purification tag and application
  • Method for realizing purification of recombinant protein by using antifreeze protein-intein as purification tag and application
  • Method for realizing purification of recombinant protein by using antifreeze protein-intein as purification tag and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of pET-24a-GST-Mxe GyrA-linker-AFP expression vector

[0048] (1) Synthesize the following primers respectively:

[0049] SEQ ID NO.4: Primer P1:

[0050] 5'-GGAATTC CATATG ATGTCCCCTATACTAGGTTATTG-3', where the underlined part is QuickCut TM Nde I recognition site.

[0051] SEQ ID NO.5: Primer P2:

[0052] 5'TGCATCTCCCGTGATGCA CATTCGCAT TTTTGGAGGATGGTCGCCACCACCAAAC-3', wherein the underlined part is the base sequence corresponding to the three amino acid MRMs.

[0053] SEQ ID NO.6: Primer P3:

[0054] 5'-GTTTGGTGGTGGCGACCATCCTCCAAAAA ATGCGAATG TGCATCACGGGAGATGCA-3', wherein the underlined part is the base sequence corresponding to the three amino acid MRMs.

[0055] SEQ ID NO.7: Primer P4:

[0056] 5'-ATCACTC AAGCTT AGCGTGGCTGACGAACCCGTTC-3', where the underlined part is QuickCut TM Hind III recognition site.

[0057] (2) Under the guidance of the above-mentioned P1 and P2 primers, the GST gene was amplified by PCR using the pGEX-4...

Embodiment 2

[0059] Example 2 The heterologous expression of GST-Mxe GyrA-linker-AFP

[0060] The recombinant expression plasmid pET24a-GST-Mxe GyrA-linker-AFP obtained in Example 1 was transferred into Escherichia coli BL21 (DE3) competent cells by the heat shock method, and a single colony was picked in a cell containing 50 μg / mL kanamycin Incubate in 5 mL of LB medium at 37°C for 12 hours, then transfer to TB medium containing 50 μg / mL kanamycin at a ratio of 2% (v / v), and continue culturing at 37°C until OD 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.5mM. After induction at 16°C for 20 h, the bacterial cells were collected and resuspended in 50 mM Tris-HCl buffer at pH 8.5 for ultrasonication and centrifugation, and the supernatant was collected for SDS-PAGE electrophoresis.

Embodiment 3

[0061] Example 3 Separation and purification of GST using antifreeze protein-intein tag

[0062] Prepare the ice shell: Measure 100mL of pre-cooled deionized water into a 500mL round bottom flask, immerse the round bottom flask in the freezing liquid and rotate rapidly for 1min so that part of the deionized water in the round bottom flask forms a close fit to the round bottom flask For the ice shell on the inner wall, pour out the remaining deionized water, continue to immerse the round-bottomed flask with the ice shell in the ice-adsorption freezing liquid and rotate for 2 minutes, until the ice shell has evenly distributed cracks, and then follow-up experiments can be carried out.

[0063] Dilute the supernatant of broken cells to 1 mg / mL, pre-cool with ice-water mixture, and slowly drop on the surface of the ice shell in the round-bottom flask, such as figure 2 As shown, connect the round-bottomed flask to the rotary evaporator and immerse the round-bottomed flask in the i...

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Abstract

The invention discloses a method for realizing separation and purification of an exogenous recombinant protein by using an antifreeze protein-intein sequence as a purification tag and application, and belongs to the technical field of biology. The method comprises the following steps: (1) constructing a recombinant plasmid with an antifreeze protein-intein purification tag and a recombinant protein gene, and transferring the recombinant plasmid into an expression host cell to obtain a recombinant engineering bacterium; (2) culturing the recombinant engineering bacterium, and inducing exogenous protein expression; and (3) after cell breakage, separating and purifying a fusion protein with the antifreeze protein-intein purification tag in one step from a cell breakage supernatant by using ice as an adsorption medium, then inducing the N end of the intein to generate a breaking reaction to release the target protein, and after dialysis, removing the purification tag by ice adsorption so as to obtain the target protein from which the purification tag is removed. The invention provides the method for realizing separation and purification of the exogenous recombinant protein, which has the advantages of low cost, no need of a chromatographic column and a chromatographic filler, simplicity in operation and removal of a purification tag, and application.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method and application for realizing the separation and purification of exogenous recombinant protein by using the antifreeze protein-intein sequence as a purification tag. Background technique [0002] At present, heterologously expressed recombinant proteins are usually separated and purified by affinity chromatography. This method usually uses gene recombination technology to fuse and express an affinity tag with the target protein, and then use a chromatographic filler coupled with a specific ligand to obtain a higher purity protein in one step. Recombinant protein has the advantages of mild conditions, good specificity, and wide application range. Peptides or proteins commonly used as affinity tags mainly include polyhistidine, maltose-binding protein, small ubiquitin-related modified proteins, glutathione sulfhydryl transferase, and the like. However, the use...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C07K19/00C07K1/14C12N9/10
CPCC12N15/70C12N15/62C07K14/35C07K14/461C12N9/1088C12Y205/01018C07K2319/20C07K2319/60
Inventor 王从纲孙佳明李明玉郑延蓉庞焦李宪臻
Owner 李宪臻
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