CN1699562A

CN1699562A - Method for separation and purification of gene recombinant protein heme oxygenase

Info

Publication number: CN1699562A
Application number: CN 200510026385
Authority: CN
Inventors: 胡洪波; 张雪洪; 彭华松
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2005-06-02
Filing date: 2005-06-02
Publication date: 2005-11-23
Anticipated expiration: 2025-06-02
Also published as: CN1289666C

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

The method of the separation and purification of gene recombinant protein heme oxygenase

Technical field

What the present invention relates to is the method for a kind of preparation method's of technical field of bioengineering, particularly a kind of gene recombinant protein heme oxygenase separation and purification.

Background technology

Heme oxygenase (Heme Oxygenase, HO) be middle existence the in the mammalian body, oxydase with multiple function relevant with the heme metabolism, it makes the methyl bridge cracking on the protoheme tetrapyrrole ring, finally forms the uteroverdine and the CO of equivalent and discharges iron atom.The eighties in last century middle and advanced stage, Maines etc. take the lead in, and separation and purification obtains in tissue such as the liver of animal and human's body, spleen, lung, brain and testis.So far; obtain three kinds of HO isozyme: HO-1, HO-2 and HO-3 in human and Mammals, HO-1 is induction type HO, as a kind of stress protein; under the condition that some hostile environments stimulate and disease exists, can play the certain protection effect to body, tissue.HO-1 can play the protection transplant organ and reduce the injury that rejection brings in the organ transplantation process, showing good application prospects aspect the pedicterus treatment simultaneously.In general, in actual production, generally need steps such as cytoclasis, centrifugal settling, salt precipitation, anion-exchange chromatography, cation-exchange chromatography and gel permeation chromatography to obtaining highly purified heme oxygenase from lysis.Product yield that various separating step causes low (common product yield about 10%) and production cycle are long, become the bottleneck problem of whole process of production.Therefore, selecting fast and effectively, separation means is the key that addresses the above problem.In recent years, the proposition of integrated isolation technique notion is that bioseparation technology has been opened up new field, and becomes the new focus in the bioseparation technology research gradually, and the expanded bed adsorption technology is exactly one of them.Expanded bed is liquid-solid fluid bed that the solids fluidisation is in stable condition, the back-mixing degree is very little.The expanded bed adsorption technology can be from the coarse fodder liquid that contains cell and cell debris direct Separation and Recovery target product, and save steps such as centrifugal, filtration.

Find by prior art documents, have not yet to see relevant expanded bed technology and be used for the recombinate method of heme oxygenase-1 of separation and purification people.The expanded bed technology is applied to the purge process of recombinant human heme oxygenase-1, can replace the existing purge process that comprises based on centrifugal settling, salt precipitation, hydrophobic interaction chromatography, anion-exchange chromatography, cation-exchange chromatography and gel permeation chromatography, thereby shorten the purifying time greatly, improve the product activity yield, cut down the consumption of energy.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of method of separation and purification of gene recombinant protein heme oxygenase is provided.Make that its method is simple, the cycle is short, the product activity recovery is high is the method for main means with expanded bed.

The present invention is achieved by the following technical solutions, and the present invention carries out the purifying preparation with the fermented liquid of express recombinant heme oxygenase-1 through centrifugal collecting cell, ultrasonic disruption, expanded bed ion exchange chromatography and gel permeation chromatography.

Comprise that step is as follows:

1. expanded bed ion exchange chromatography

With refrigerated centrifuge with the centrifugal 15min of fermented liquid, collecting cell, again suspend with damping fluid, with ultrasonic disruption instrument smudge cells, after cytoclasis liquid dilutes 2～6 times with damping fluid, the expansion column is earlier with Tris-HCl damping fluid balance, feed the fermented liquid that has diluted from the expanded bed bottom then, carry out adsorption operations, damping fluid cleans 1～5 column volume under the expanded bed state, remove foreign protein and cell debris solid substance, at the fixed bed operation state with containing 1mol/L NaCl buffer solution for gradient elution, collect active peak, wash-out finishes when the chromatographic peak response value reaches baseline position.

Step 1. in:

Described fermented liquid is centrifugal under 4 ℃ of conditions, and centrifugal speed is 4000rpm.

Described damping fluid is: 0.001～0.05mol/L pH7.0～9.0.

Described sodium phosphate salt damping fluid is at 20～30 ℃, feeds the fermented liquid that has diluted with the flow velocity of 100～300cm/h from the expanded bed bottom.

Described expanded bed is controlled its bed expansion ratio and is 1.5～3.0 and carries out adsorption operations.

Described damping fluid cleans under the expanded bed state with the flow velocity of 100～300cm/h.

2. gel permeation chromatography

Top elutriant is joined in the Sephacryl S-200 gel chromatography column, finish to going out the peak, collect active peak, preserve after the lyophilize with the phosphate buffered saline buffer washing.

Step 2. in:

Described elutriant adds with the flow velocity of 10～50cm/h.

Described phosphate buffered saline buffer, for: 0.001～0.05mol/L pH6.0～8.0.

Described phosphate buffered saline buffer finishes to going out the peak with the washing of the flow velocity of 10～50cm/h.

Purifying Heme Oxygenase-1's of the present invention process is compared with existing separation method, its tangible advantage is: after the somatic cells fragmentation, need not centrifugal at low temperatures, saltout, dissolution precipitation and the dissolving after centrifugal process, be directly used in the expanded bed adsorption chromatographic separation after can suitably diluting, shortened the time of sepn process greatly, improve the yield of recombinant human heme oxygenase-1 significantly, and reduced energy consumption.This technology has broad application prospects in the purge process of recombinant human heme oxygenase-1.

Embodiment

Below in conjunction with specific embodiment technical scheme of the present invention is elaborated

Embodiment 1

With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 15min of the speed of 4000rpm, collecting cell suspends again with the Tris-HCl damping fluid of 0.005mol/L pH7.0, with ultrasonic disruption instrument smudge cells.Cytoclasis liquid dilutes 2 times with the Tris-HCl damping fluid of 0.005mol/L pH7.0.StreamlineDEAE is filled in XK 26 chromatographic columns, and packing height is 11cm.Fluid distributor on the chromatographic column is adjusted to the 22.5cm place, fixing.Feed the Tris-HCl damping fluid of 0.005mol/L pH7.0 from the chromatographic column bottom, make 2 times of bed expansions, balance 30min.With diluted stock liquid feed from the chromatographic column bottom, regulate the flow velocity of feed liquid, the ratio of expansion of keeping expanded bed is constant, collects simultaneously from the effusive solution of chromatographic column.Behind the last sample, keep rate of expansion constant, feed the Tris-HCl damping fluid of 0.005mol/L pH7.0, clean 2～3 column volumes, collect the effusive solution of wash phase chromatographic column.To go up fluid distributor and reduce to the 11.5cm place, adopt the fixed bed operation mode, flow velocity with 60cm/h carries out gradient elution from the Tris-HCl damping fluid that the feeding of chromatographic column top contains the 0.005mol/LpH7.0 of 1mol/L NaCl, collect active peak, wash-out finishes when the chromatographic peak response value reaches baseline position.

The flow velocity of top elutriant with 40cm/h joined in the Sephacryl S-200 gel chromatography column, finish to going out the peak with the flow velocity washing identical, collect active peak, preserve after the lyophilize with sample introduction with the phosphate buffered saline buffer of 0.02mol/L pH7.5.

Embodiment 2

With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 15min of the speed of 4000rpm, collecting cell suspends again with the Tris-HCl damping fluid of 0.01mol/L pH8.0, with ultrasonic disruption instrument smudge cells.Cytoclasis liquid dilutes 3 times with the Tris-HCl damping fluid of 0.01mol/L pH8.0.StreamlineDEAE is filled in XK 26 chromatographic columns, and packing height is 11cm.Fluid distributor on the chromatographic column is adjusted to the 28cm place, fixing.Feed the Tris-HCl damping fluid of 0.01mol/L pH8.0 from the chromatographic column bottom, make 2.5 times of bed expansions, balance 30min.With diluted stock liquid feed from the chromatographic column bottom, regulate the flow velocity of feed liquid, the ratio of expansion of keeping expanded bed is constant, collects simultaneously from the effusive solution of chromatographic column.Behind the last sample, keep rate of expansion constant, feed the Tris-HCl damping fluid of 0.01mol/L pH8.0, clean 3～4 column volumes, collect the effusive solution of wash phase chromatographic column.To go up fluid distributor and reduce to the 11.5cm place, adopt the fixed bed operation mode, flow velocity with 60cm/h carries out gradient elution from the Tris-HCl damping fluid that the feeding of chromatographic column top contains the 0.01mol/LpH8.0 of 1mol/L NaCl, collect active peak, wash-out finishes when the chromatographic peak response value reaches baseline position.

The flow velocity of top elutriant with 20cm/h joined in the Sephacryl S-200 gel chromatography column, finish to going out the peak with the flow velocity washing identical, collect active peak, preserve after the lyophilize with sample introduction with the phosphate buffered saline buffer of 0.05mol/L pH7.0.

Embodiment 3

With refrigerated centrifuge with fermented liquid under 4 ℃ of conditions with the centrifugal 15min of the speed of 4000rpm, collecting cell suspends again with the Tris-HCl damping fluid of 0.02mol/L pH8.5, with ultrasonic disruption instrument smudge cells.Cytoclasis liquid dilutes 2 times with the Tris-HCl damping fluid of 0.02mol/L pH 8.5.StreamlineDEAE is filled in XK 26 chromatographic columns, and packing height is 11cm.Fluid distributor on the chromatographic column is adjusted to the 28cm place, fixing.Feed the Tris-HCl damping fluid of 0.02mol/L pH8.5 from the chromatographic column bottom, make 2.5 times of bed expansions, balance 30min.With diluted stock liquid feed from the chromatographic column bottom, regulate the flow velocity of feed liquid, the ratio of expansion of keeping expanded bed is constant, collects simultaneously from the effusive solution of chromatographic column.Behind the last sample, keep rate of expansion constant, feed the Tris-HCl damping fluid of 0.02mol/L pH8.5, clean 3～4 column volumes, collect the effusive solution of wash phase chromatographic column.To go up fluid distributor and reduce to the 11.5cm place, adopt the fixed bed operation mode, flow velocity with 60cm/h carries out gradient elution from the Tris-HCl damping fluid that the feeding of chromatographic column top contains the 0.02mol/LpH8.5 of 1mol/L NaCl, collect active peak, wash-out finishes when the chromatographic peak response value reaches baseline position.

Claims

1. A method for the separation and purification of gene recombinant protein heme oxygenase, characterized in that: the fermentation broth expressing recombinant heme oxygenase-1 is centrifuged to collect cells, ultrasonic crushing, expanded bed ion exchange chromatography Purified and prepared by gel filtration chromatography, and stored after freeze-drying.

2. the method for the separation and purification of gene recombinant protein heme oxygenase according to claim 1, is characterized in that, comprises steps as follows:

① Expanded bed ion exchange chromatography

Centrifuge the fermentation broth with a refrigerated centrifuge for 15 minutes, collect the cells, resuspend them with Tris-HCl buffer, and break the cells with an ultrasonic breaker. -HCl buffer balance, and then pass the diluted fermentation broth from the bottom of the expanded bed for adsorption operation, the buffer solution is washed in the expanded bed state for 1 to 5 column volumes to remove impurity proteins and cell debris solids, and in the fixed bed In the operating state, use gradient elution with a buffer containing 1mol/L NaCl, collect the active peak, and complete the elution when the response value of the chromatographic peak reaches the baseline position;

②Gel filtration chromatography

The above eluate was added to the gel chromatography column, washed with phosphate buffer until the peak was eluted, and the active peak was collected.

3. The method for separating and purifying gene recombinant protein heme oxygenase according to claim 2, characterized in that, in step ①, the fermentation broth is centrifuged at 4°C at a centrifugation speed of 4000rpm.

4. The preparation method for the separation and purification of gene recombinant protein heme oxygenase according to claim 2, characterized in that, in step ①: the buffer is: 0.001～0.05mol/L pH7.0～ 9.0.

5. The method for separating and purifying gene recombinant protein heme oxygenase according to claim 2, characterized in that, in step ①, the phosphate buffer solution is heated at 20-30°C at 100-300cm/ The flow rate of h is passed into the diluted fermentation broth from the bottom of the expanded bed.

6. The method for separating and purifying gene recombinant protein heme oxygenase according to claim 2, characterized in that, in step ①, the expanded bed is controlled to have a bed expansion ratio of 1.5 to 3.0 for adsorption operate.

7. The method for the separation and purification of genetically recombinant protein heme oxygenase according to claim 2, characterized in that, in step 1.: the buffer solution is in an expanded bed state at a flow rate of 100 to 300 cm/h down to wash.

8. The method for separating and purifying the gene recombinant protein heme oxygenase according to claim 2, characterized in that, in step ②: the eluent is added at a flow rate of 10-50 cm/h.

9. The method for separating and purifying the genetically recombinant protein heme oxygenase according to claim 2, characterized in that, in step ②: the phosphate buffer is: 0.001～0.05mol/LpH6.0 ~8.0.

10. The preparation method for the separation and purification of genetically recombinant protein heme oxygenase according to claim 2, characterized in that, in step ②, the phosphate buffer solution is used at a flow rate of 10-50 cm/h Wash until peak elution is complete.