Purification method of fluorescent protein

A technology of fluorescent protein and purification method, which is applied to the preparation method of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problems of environmental pollution, complex process, low purification factor of fluorescent protein, etc., and achieve simple and efficient purification method, high purification low cost effect

Active Publication Date: 2021-08-31
生工生物工程(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for purifying fluorescent protein, so as to alleviate technical problems such as low purification factor, complex process, and easy environmental pollution in the prior art.

Method used

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  • Purification method of fluorescent protein
  • Purification method of fluorescent protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Fluorescent protein expression vector construction

[0052] With EGFP-F (5'-cggcagc catatg gtgagcaagggcgagg-3', the underline is the Nde I restriction site) is the upstream primer, EGFP-R (5'-ccg ctcgag ttagtggtgatggtgatggtg-3', the underline is the Xho I restriction site) as the downstream primer, and the commercially available plasmid pEGFP-N1 was used as the template to amplify the EGFP gene by PCR. After recovery, the PCR product was digested with Nde I and XhoI, and the double digested product was recovered and connected to the commercialized plasmid pET-24a that had undergone the same double digestion to obtain the pET-24a-EGFP vector, which is a fluorescent protein Expression vector of EGFP.

Embodiment 2

[0053] Example 2. Fluorescent protein expression

[0054] The expression vector pET-24a-EGFP was transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB plates (containing 50 mg / L kanamycin), and cultivated overnight in a 37°C incubator.

[0055] Pick a single colony from the overnight cultured LB plate into a 250mL shake flask filled with 50mL LB medium, place it on a constant temperature shaker at 37°C at 200rpm / min, and cultivate it for 20h as a seed solution.

[0056] Inoculate the cultivated seed solution into 1000mL shake flasks with 100mL self-induction medium, inoculate 8 bottles altogether, the inoculum size of each bottle is 5% (v / v), place in a constant temperature shaker, 200rpm / min, After incubating at 37°C for 3h, transfer to 25°C for 20h. Among them, the formula of self-induction medium is: lactose 2g / L; peptone 10g / L; yeast powder 5g / L; NaCl 10g / L; glycerin 8ml / L.

[0057] Collect all the culture fluid, centrifuge and remove the supernatant...

Embodiment 3

[0058] Example 3. Fluorescent protein purification

[0059] Add 100 mL of Tris (50 mmol / L, pH 8.0) buffer solution to the centrifuge bottle where the bacteria were collected to suspend the bacteria, and break the cells with a high-pressure homogenizer until the suspension becomes clear. Place the bacteriostasis solution in a water-bath shaker at 65°C, and incubate at 100rpm / min for 15min. After heat treatment, centrifuge (8000rpm / min, 10min), take the supernatant, and discard the precipitate.

[0060] Add 2mol / L ZnCl to the supernatant 2 Mix the solution until its final concentration is 20mmol / L, and a large amount of protein precipitation can be observed, centrifuge again (8000rpm / min, 10min), take the precipitate, and discard the supernatant.

[0061] Add 100mL Tris (50mmol / L, pH 8.0) buffer solution to the precipitate to fully suspend the precipitate, centrifuge (8000rpm / min, 10min), and take the supernatant. Dissolve the precipitate with 100mL EDTA (5mmol / L, pH8.0) solu...

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Abstract

The invention relates to the field of biotechnology, in particular, it provides a method for purifying fluorescent protein. In the method for purifying fluorescent protein provided by the invention, the cell wall solution is subjected to precipitation and reconstitution in sequence, and then purified by ion exchange chromatography to obtain fluorescent protein. The purification method is simple and efficient, and is suitable for the purification of fluorescent proteins in all expression systems. It is safe and does not pollute the environment, and the purification cost is low. The purified fluorescent protein has high purity and high concentration. The method is suitable for industrial production and application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying fluorescent proteins. Background technique [0002] In 1962, Osamu Shimomura, a Japanese scientist living in the United States, became interested in a luminous jellyfish because he discovered that the jellyfish would emit green fluorescence when disturbed by the outside world. After salvaging about 50,000 luminous jellyfish from the west coast of the United States, through arduous extraction and purification work, hundreds of milligrams of the protein were finally obtained, which emitted green fluorescence at 508nm by itself, called green fluorescent protein. Later studies found that by modifying the amino acid sequence of green fluorescent protein and mutating at one or several sites, fluorescent proteins of different colors such as yellow fluorescent protein, blue fluorescent protein and cyan fluorescent protein can be obtained. [0003] Fluorescent proteins...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/30C07K1/18
CPCC07K14/43595
Inventor 傅向阳
Owner 生工生物工程(上海)股份有限公司
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