Diethylaminoethylated dextran modified agarose gel-based chromatographic medium, preparation method and application

A technology of diethylaminoethylated dextran and agarose gel, which is applied in the field of protein chromatographic separation, can solve the problems of complex synthesis process and limited improvement, and achieve good biocompatibility, convenient sterilization, and preparation The effect of simple method

Active Publication Date: 2017-09-19
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the one hand, the synthesis process is relatively complicated; on the other hand, although the ion-exchange chromatographic medium modified by neutral low-molecular-weight dextran can improve the adsorption capacity and mass transfer rate of proteins to a certain extent, the degree of improvement is very limited.

Method used

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  • Diethylaminoethylated dextran modified agarose gel-based chromatographic medium, preparation method and application
  • Diethylaminoethylated dextran modified agarose gel-based chromatographic medium, preparation method and application
  • Diethylaminoethylated dextran modified agarose gel-based chromatographic medium, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0038] (1) Take 1g of agarose gel drained by the G3 funnel and put it into a 50mL Erlenmeyer flask, add 2mL of dimethyl sulfoxide and 1.3mL of epichlorohydrin in sequence, and mix in a shaker at 25°C and 170rpm 0.5h. Then add 2mL of sodium hydroxide (1mol / L) and react for 2.5h. Rinse repeatedly with deionized water until the cleaning solution is washed with phenolphthalein–Na 2 S 2 o 3 The solution does not change color after detection, and the agarose gel medium with active epoxy groups on the surface is prepared. The epoxy group modification density of the agarose gel medium with active epoxy groups on the surface is 51mmol / L.

[0039] (2) Add 1 g of agarose gel medium with active epoxy groups on the surface to 1 mL of DEAE Dextran (Mw 500kDa) aqueous solution (200 mg / mL), and place it in a constant temperature shaker at 20 °C , 120rpm diffusion for 1h, so that DEAE Dextran fully diffused into the medium channel, then add 2mL of sodium hydroxide (0.1mol / L), 20 ℃, 120rpm...

Embodiment 2

[0041] The agarose gel medium with active epoxy groups on the surface of the 3g embodiment 1 step (1) drained by the G3 funnel was added to the DEAE Dextran (Mw 500kDa) aqueous solution (300mg / mL) of 3mL, placed in 25 In a constant temperature shaker at 170rpm for 2.5h to fully diffuse DEAE Dextran into the media pores, add 3mL of sodium hydroxide (1mol / L), react at 25°C and 170rpm for 48h to couple DEAE Dextran to the agarose gel Rinse repeatedly with deionized water until the cleaning solution is detected by phenolphthalein and does not change color, then place the medium in 0.5g / L sodium borohydride solution, react at room temperature for 12h to reduce the residual epoxy group on the surface of the medium, and the reduced medium again Rinse repeatedly with deionized water to prepare a DEAE Dextran grafted agarose gel medium with an ion exchange capacity of 90 mmol / L of the structural formula (I).

Embodiment 3

[0043] The agarose gel medium with active epoxy groups on the surface of the 5g embodiment 1 step (1) drained by the G3 funnel was added to the DEAE Dextran (Mw 500kDa) aqueous solution (600mg / mL) of 5mL, placed in 25 ℃ in a constant temperature shaker, diffuse at 200rpm for 2.5h, so that DEAE Dextran can fully diffuse into the medium channel, add 5mL of sodium hydroxide (1mol / L), and react at 25℃, 170rpm for 60h to couple DEAE Dextran to the agarose gel Rinse repeatedly with deionized water until the cleaning solution is detected by phenolphthalein and does not change color, then place the medium in 0.5g / L sodium borohydride solution, react at room temperature for 12h to reduce the residual epoxy group on the surface of the medium, and the reduced medium again Rinse repeatedly with deionized water to prepare a DEAE Dextran grafted agarose gel medium with an ion exchange capacity of 199 mmol / L of the structural formula (I).

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Abstract

The present invention relates to the chromatographic medium based on agarose gel modified by diethylaminoethylated dextran and its preparation method and application; firstly, the selection of the molecular weight of DEAE Dextran for grafting; secondly, the control of the concentration of DEAE Dextran aqueous solution; thirdly , the determination of the diffusion time before coupling DEAE Dextan; finally, the determination of the reaction time, sodium hydroxide concentration and volume of coupling DEAE Dextran. The agarose gel-based chromatographic medium modified by DEAE Dextran of the present invention has strong adsorption characteristics for proteins at different modification densities, exhibits high adsorption capacity and adsorption rate, and improves separation efficiency. The medium is easy to clean and sterilize, easy to regenerate, has good biocompatibility, simple preparation method and low toxicity, and will have broad application prospects in efficient and rapid separation and purification of proteins.

Description

technical field [0001] The invention relates to a chromatographic medium based on agarose gel modified by diethylaminoethylated dextran and its preparation method and application. It can significantly improve the protein adsorption capacity and mass transfer rate, and belongs to the protein in the field of biotechnology. Chromatographic separation techniques. Background technique [0002] Diethylaminoethylated dextran (DEAE Dextran) is a long-chain cationic polyelectrolyte modified with diethylaminoethyl (DEAE) on dextran. DEAE Dextran is positively charged and can reversibly adsorb negatively charged substances. DEAEDextran has good biocompatibility and is mostly used in gene transfection and sustained sustained release of proteins. In recent years, researchers have found that DEAE Dextran can also be applied to cell immobilization in biosensors. [0003] DEAE Dextran has a larger molecular weight and more hydroxyl groups, and it is easy to couple the spacer arm with the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/291B01J20/286B01J20/26B01J20/30C08B37/12C08B37/02C07K1/22
Inventor 余林玲白姝龚玲莉孙彦
Owner TIANJIN UNIV
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