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Process for separation and purification of gene recombinant protein heme oxygenase

A technology of heme oxygenase and gene recombination, which is applied in the field of preparation in the field of bioengineering technology, can solve the problems such as separation and purification of human recombinant heme oxygenase-1, etc. The effect of broad application prospects

Inactive Publication Date: 2005-11-23
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After searching the existing technical literature, it is found that there is no method for the separation and purification of human recombinant heme oxygenase-1 using expanded bed technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The fermentation broth was centrifuged at a speed of 4000 rpm at 4° C. for 15 min in a refrigerated centrifuge, and the cells were collected, resuspended in 0.005 mol / L Tris-HCl buffer at pH 7.0, and disrupted with an ultrasonic disruptor. The cell disruption solution was diluted 2 times with 0.005mol / L Tris-HCl buffer pH7.0. StreamlineDEAE is packed into the XK 26 column with a packing height of 11cm. Adjust the fluid distributor on the chromatographic column to 22.5cm and fix it. Pass 0.005mol / L pH7.0 Tris-HCl buffer from the lower part of the chromatographic column to expand the bed twice and equilibrate for 30min. Pass the diluted raw material liquid through the lower part of the chromatographic column, adjust the flow rate of the material liquid, maintain the expansion ratio of the expanded bed, and collect the solution flowing out of the chromatographic column. After loading the sample, keeping the expansion rate unchanged, pass in 0.005mol / L pH7.0 Tris-HCl buffer, wa...

Embodiment 2

[0027] The fermentation broth was centrifuged at a speed of 4000 rpm at 4°C for 15 min in a refrigerated centrifuge, and the cells were collected, resuspended in 0.01 mol / L pH 8.0 Tris-HCl buffer, and disrupted with an ultrasonic disruptor. The cell disruption solution was diluted 3 times with 0.01mol / L Tris-HCl buffer pH8.0. StreamlineDEAE is packed into the XK 26 column with a packing height of 11cm. Adjust the fluid distributor on the chromatographic column to 28 cm and fix it. Pass 0.01mol / L pH8.0 Tris-HCl buffer from the lower part of the chromatographic column to expand the bed by 2.5 times and equilibrate for 30 minutes. Pass the diluted raw material liquid through the lower part of the chromatographic column, adjust the flow rate of the material liquid, maintain the expansion ratio of the expanded bed, and collect the solution flowing out of the chromatographic column. After loading the sample, keeping the expansion rate constant, pass in 0.01mol / L pH8.0 Tris-HCl buffer, w...

Embodiment 3

[0030] The fermentation broth was centrifuged at a speed of 4000 rpm at 4° C. for 15 min in a refrigerated centrifuge, and the cells were collected, resuspended in 0.02 mol / L pH 8.5 Tris-HCl buffer, and disrupted with an ultrasonic disruptor. The cell disruption solution was diluted 2 times with 0.02mol / L Tris-HCl buffer of pH 8.5. StreamlineDEAE is packed into the XK 26 column with a packing height of 11cm. Adjust the fluid distributor on the chromatographic column to 28 cm and fix it. Pass 0.02mol / L pH8.5 Tris-HCl buffer from the bottom of the chromatographic column to expand the bed by 2.5 times and equilibrate for 30min. Pass the diluted raw material liquid through the lower part of the chromatographic column, adjust the flow rate of the material liquid, maintain the expansion ratio of the expanded bed, and collect the solution flowing out of the chromatographic column. After loading the sample, keeping the expansion rate unchanged, pass in 0.02mol / L pH8.5 Tris-HCl buffer, was...

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Abstract

The invention relates to a process for separation and purification of gene recombinant protein heme oxygenase, which comprises subjecting the expression recombination protoheme oxygenase-1 fermentation liquor to centrifugal collection of cells, ultrasonic wave fragmenting, expanded-bed ion exchange chromatography and gel filtration chromatography for purification preparation. Compared with the existing separation process, provided method has the advantages of substantially reduced time of separation process and increased yield of recombination human heme oxygenase-1.

Description

Technical field [0001] The invention relates to a preparation method in the technical field of bioengineering, in particular to a method for separating and purifying genetic recombinant protein heme oxygenase. Background technique [0002] Heme Oxygenase (HO) is a multi-functional oxidase that exists in mammals and is related to heme metabolism. It cleaves the methyl bridge on the heme tetrapyrrole ring. Eventually, equal amounts of biliverdin and CO are formed and iron atoms are released. In the middle and late 1980s, Maines and others took the lead in separating and purifying the liver, spleen, lung, brain and testis of animals and humans. So far, three HO isozymes have been obtained in humans and mammals: HO-1, HO-2 and HO-3. HO-1 is an inducible HO. As a stress protein, it can stimulate and disease in some harmful environments. Under existing conditions, it can protect the body and tissues to a certain extent. HO-1 can protect the transplanted organs and reduce the damage cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
Inventor 胡洪波张雪洪彭华松
Owner SHANGHAI JIAO TONG UNIV
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