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Sealing system composed of small anaerobic operation bags and chromatography column in series

A chromatographic column, small-scale technology, applied in the direction of oxidoreductase, peptide preparation, organic chemistry, etc., can solve the problems of inability to obtain effective protection immediately, protein inactivation, high cost, etc., and achieve simple and convenient operation, protection activity, The effect of reducing operating costs

Inactive Publication Date: 2011-09-28
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] The reported anaerobic protein extraction process is completed in a huge anaerobic operation device, which has the following disadvantages: all instruments and reagents are concentrated in the anaerobic device, so that all operation steps need to be operated with gloves, which is very inconvenient; Every time an item is put in or taken out, a large amount of protective gas needs to be injected, which is very costly; if the device leaks, the protein inside will quickly come into contact with oxygen and cannot be effectively protected immediately, resulting in protein inactivation
However, a sealed extraction device that connects a small anaerobic operation bag in series with a chromatographic column has not been reported at home and abroad.

Method used

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  • Sealing system composed of small anaerobic operation bags and chromatography column in series
  • Sealing system composed of small anaerobic operation bags and chromatography column in series

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Separation and Purification of Hydrogenase from Tetragalensis Subcordiformis in Seawater

[0035] In step 1, take the late logarithmic growth period, and the concentration is about 2×10 6 3L of cells / mL Tetraphyllum subcordiformis, concentrated by centrifugation at 1500r / min for 2min. After pouring out the seawater, take 10g of algae cells with a fresh weight, resuspend in 100mL of sterilized seawater, and place them in a closed glass bottle. Nitrogen was passed for 10 minutes, and after the oxygen in the container was excluded, induction was carried out in the dark for 4 hours.

[0036] Step 2 Put the glass bottle into the anaerobic operation bag and fill it with N repeatedly 2 1. After vacuuming three times, divide the algae solution into sealed centrifuge tubes, centrifuge at 1500r / min for 3min, remove the supernatant and resuspend in 10mL of 50mM Tris-Hcl buffer solution with pH7.9. Add 10 g of pre-cooled glass beads (diameter 1 mm) to the resuspension, vortex at...

Embodiment 2

[0043] Isolation and purification of freshwater Chlamydomonas reinhardtii cc124 hydrogenase

[0044] In step 1, the late logarithmic growth is taken, and the concentration is about 2.5×10 6 cells / mL of Chlamydomonas reinhardtii cc124 3L, concentrated by centrifugation at 4000r / min for 5min. After pouring off the supernatant, take 10 g of algae cells with a fresh weight, resuspend them in 100 mL of 50 mM pH7.2 phosphate buffer, and place them in an airtight glass bottle. Nitrogen was passed for 10 minutes, and after the oxygen in the container was excluded, it was induced in the dark for 3 hours.

[0045] Step 2 Put the glass bottle into the anaerobic operation bag and fill it with N repeatedly 2 1. After vacuuming three times, divide the algae solution into sealed centrifuge tubes, centrifuge at 4000r / min for 5min, remove the supernatant and resuspend in 10mL of 50mM Tris-Hcl buffer solution with pH8.5. Add 10 g of pre-cooled glass beads (0.8 mm in diameter) to the resuspen...

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Abstract

The invention relates to a sealing system composed of small anaerobic operation bags and a chromatography column in series, mainly used for isolation and purification of anaerobic protein from plants and microorganisms. The method comprises the following steps: putting reagents, vessels into anaerobic operation bags, sparging all reagents and buffers with a stream of nitrogen, adding sodium hydrosulfite to remove dissolved oxygen, breaking tissues or cells using a high-speed ball mill method, precipitating a crude enzyme solution, isolating and purifying the crude enzyme solution by gel filtration chromatography and ion exchange chromatography, so as to obtain the anaerobic protein. Foreign similar method is realized by putting all useful apparatus, devices into a huge anaerobic device with the disadvantages of occupying large area, high cost of shielding gas and difficult operation; while no relative technologies have been reported in China. The invention has the advantages of easy operation and low cost.

Description

technical field [0001] The invention relates to a sealing system in which a small anaerobic operation bag is connected in series with a chromatographic column, which is mainly used for the separation and purification of anaerobic proteins in plants and microorganisms. Background technique [0002] Many plants and microorganisms in nature can induce anaerobic proteins with different functions in anaerobic environment. Sachs revealed for the first time that under strict anaerobic conditions, the amount of protein synthesized in plant root tips decreased sharply. He used two-dimensional electrophoresis technology to observe that there are about 20 kinds of polypeptides (ANPS) in corn seedlings under hypoxia, of which 10 kinds of molecular weight are larger and 10 kinds are smaller. In contrast, there were more than 90 kinds of polypeptides, and the synthesis of ANPS appeared at 0-1 hour, reached the maximum at 20 hours, and then no longer increased, and the corn seedlings died...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K1/16C12N9/02
Inventor 张卫彦飞陈兆安陆洪斌曹旭鹏薛松
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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