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Method for increasing protein adsorption capacity and adsorption rate by using high-density polyethylenimine (PEI) modified medium

A polyethyleneimine, high-density technology, applied in the field of protein chromatographic separation, can solve the problems of high price of dextran, high cost of medium synthesis, complex synthesis process, etc., and achieves low price, high adsorption rate, and biocompatibility. Good results

Inactive Publication Date: 2014-02-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of medium modification method requires two-step coupling reaction, and the synthesis process is relatively complicated.
At the same time, dextran is expensive, and the synthesis cost of the medium is relatively high, while the monomer of polymethacrylate is more toxic and more dangerous to synthesize
In addition, these grafted media have a narrow operating range and are generally only usable in environments with lower salt concentrations

Method used

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  • Method for increasing protein adsorption capacity and adsorption rate by using high-density polyethylenimine (PEI) modified medium
  • Method for increasing protein adsorption capacity and adsorption rate by using high-density polyethylenimine (PEI) modified medium
  • Method for increasing protein adsorption capacity and adsorption rate by using high-density polyethylenimine (PEI) modified medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Take 1g Sepharose FF (average particle size 90μm) drained from the G3 funnel and put it into a 50mL Erlenmeyer flask. Add 2mL dimethyl sulfoxide, 1mL epichlorohydrin, and 2mL NaOH (1mol / L). 25℃, 170rpm shaker reaction for 2.5h, rinse repeatedly with deionized water until the cleaning solution is phenolphthalein-Na 2 S 2 O 3 Solution detection does not change color, and an activation medium with active epoxy groups on the surface is prepared. The epoxy modification density of the activation medium is 60mmol / L.

[0025] Add 1mL of PEI (MW1200Da) aqueous solution (25%w / w) to the activation medium (1g), 25℃, 170rpm, 4h, so that PEI can fully diffuse into the medium pores, add 0.5mL NaOH (2mol / L) , 25℃, 170rpm reaction for 48h to couple PEI to agarose, rinse repeatedly with deionized water, until the cleaning solution is detected with phenolphthalein and do not change color, then put the medium in 0.5g / L sodium borohydride solution at room temperature After 12 hours of reactio...

Embodiment 2

[0027] Add 1g of the activation medium in Example 1 drained with a G3 funnel to 1mL of PEI (MW750000Da) aqueous solution (6%w / w), 25℃, 170rpm, 4h, so that PEI can fully diffuse into the pores of the medium, add 1mL of NaOH (1mol / L), 25℃, 170rpm for 48h to couple PEI to agarose, rinse repeatedly with deionized water until the cleaning solution is detected by phenolphthalein and do not change color, and then put the medium at 0.5g / L In the sodium borohydride solution, the residual epoxy group on the surface of the reduction medium was reacted at room temperature for 12 hours, and the ion exchange capacity of the prepared medium was 520 mmol / L by repeated washing with deionized water.

Embodiment 3

[0029] Add 1g of the activation medium in Example 1 drained with a G3 funnel to 1mL of PEI (MW750000Da) aqueous solution (12%w / w), 25℃, 170rpm, 8h, so that PEI can fully diffuse into the medium pores, add 1mL of NaOH (1mol / L), 25℃, 170rpm for 48h to couple PEI to agarose, rinse repeatedly with deionized water until the cleaning solution is detected by phenolphthalein and do not change color, and then put the medium at 0.5g / L In the sodium borohydride solution, the residual epoxy groups on the surface of the reduction medium were reacted at room temperature for 12 hours, and the ion exchange capacity of the prepared medium was 740 mmol / L by repeated washing with deionized water.

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Abstract

The invention discloses a method for increasing protein adsorption capacity and adsorption rate by using a high-density polyethylenimine (PEI) modified medium. The medium is a medium formed by modifying the PEI on the surfaces of sepharose gel particles with the average particle size of 50 to 170 Mum by an epoxy spacer arm. A preparation method comprises the steps of activating a chromatographic matrix by using epoxy chloropropane, and carrying out reaction between the activated medium and the amino group of the PEI to finish ligand coupling. The chromatographic medium has strong adsorption capability on protein in the range of 0.01 to 1 mol / L and shows high adsorption capacity and adsorption rate; the protein adsorption shows high salt concentration tolerance, so that material liquid can be directly contacted with the chromatographic medium, without being pretreated, to quick capture the target protein. The medium has a wide application prospect in high-efficiency and quick separation and purification of the protein.

Description

Technical field [0001] The invention relates to the application of a high-density polyethyleneimine modified medium in improving the adsorption capacity and the adsorption rate of a protein, and belongs to a protein chromatographic separation technology in the field of biotechnology. Background technique [0002] Poly(ethylenimine) (PEI) is a long-chain cationic polyelectrolyte with low cost, low toxicity and branched structure. The theoretical ratio of primary, secondary, and tertiary amines in PEI polymer molecules is 1:2:1, and the charge density can reach 23.3mEq / g when fully protonated, which is currently the highest charge density substance. PEI is positively charged in a wide pH range, and can reversibly adsorb negatively charged substances. PEI has good biocompatibility and has been widely used in gene transfer and separation and purification of biological products, such as extracting heparin and removing bacterial endotoxins. In recent years, researchers have found tha...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01J20/30
Inventor 孙彦余林玲史清洪
Owner TIANJIN UNIV
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