Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction

A protein and specific technology, applied in the field of protein immobilization, can solve the problems of long period and low specificity, and achieve the effect of solving the long period of protein immobilization

Pending Publication Date: 2020-11-24
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] The purpose of the present invention is to provide a one-step directional immobilization method for proteins based on Halo-tag specific dehalogenation reaction, which can effectively solve the problems of low specificity and long period in current protein immobilization methods

Method used

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  • Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction
  • Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction
  • Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction

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Embodiment 1

[0034] refer to figure 1 , the one-step directional immobilization method of protein based on Halo-tag specific dehalogenation reaction provided by the present invention, specifically comprises the following steps:

[0035] Step1: Haloalkanoic acid modified separation medium

[0036] Haloalkanoic acids can be selected from: 6-chlorohexanoic acid, 6-bromohexanoic acid, 6-fluorohexanoic acid, 6-iodohexanoic acid and all saturated acids of the above-mentioned halogens.

[0037] The separation medium can be selected: resin microspheres, porous microspheres, etc. modified by functional groups such as hydroxylation, amination, mercaptolation, and carboxylation.

[0038] In this embodiment, 6-chlorohexanoic acid is selected as the halogenated alkanoic acid, and aminated porous microspheres are selected as the separation medium.

[0039] The process of modifying aminated porous microspheres with 6-chlorohexanoic acid is as follows:

[0040] Dissolve 6-chlorohexanoic acid (1.2eq), a...

Embodiment 2

[0047] refer to figure 1, the one-step directional immobilization method of protein based on Halo-tag specific dehalogenation reaction provided by the present invention, specifically comprises the following steps:

[0048] Step1: Haloalkanoic acid modified separation medium

[0049] In this embodiment, 6-chlorohexanoic acid is selected as the halogenated alkanoic acid, and aminated porous microspheres are selected as the separation medium.

[0050] The process of modifying aminated porous microspheres with 6-chlorohexanoic acid is as follows:

[0051] Dissolve 6-chlorohexanoic acid (1.2eq), aminated porous microspheres (1.0eq) and diisopropylethylamine (3.0eq) in DMF (5mL), then add HATU (1.2eq), and stir at room temperature for 2 The 6-chlorohexanoic acid can be modified to the surface of the aminated porous microspheres within hours to obtain the 6-chlorohexanoic acid-modified microspheres.

[0052] Step2: One-step directional immobilization of Halo-tag fusion protein

...

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Abstract

The invention discloses a protein one-step directional immobilization method based on a Halo-tag specific dehalogenation reaction. According to the method, a Halo-tag gene is recombined to an inactiveC terminal of target receptor protein, the surface of a separation medium is modified with halogenated alkanoic acid through an acylation reaction, induced expression is performed, and then by utilizing a specific dehalogenation reaction between C-terminal dehalogenase of a recombinant receptor and haloalkane on the surface of the separation medium, one-step directional covalent immobilization ofthe Halo-tag recombinant receptor in a cell lysis solution is realized. The novel protein one-step directional immobilization method is established. Due to the fact that one-step high-specificity immobilization of the protein can be achieved through the cell lysis solution, the problems that the protein immobilization period is long and the specificity of a protein immobilization method is low are effectively solved.

Description

technical field [0001] The invention relates to a protein immobilization method, in particular to a one-step directional immobilization method for protein based on the specific dehalogenation reaction of halogenated alkane dehalogenase (Halo-tag), and belongs to the technical field of protein immobilization. Background technique [0002] At present, the protein immobilization methods reported in the literature can be divided into: physical adsorption method, chemical bonding method and immobilization method based on specific tag affinity. Among the above three methods: [0003] (1) The physical adsorption method is to immobilize proteins on the surface of solid substrates through non-specific adsorption. Although this method is the easiest to operate in the process of protein immobilization, since this method immobilizes proteins on the surface of solid materials through non-specific adsorption, the selectivity for the target protein is low. The target protein is purified,...

Claims

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Application Information

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IPC IPC(8): C07K17/04
CPCC07K17/04
Inventor 赵新锋李倩王静曾凯竹
Owner NORTHWEST UNIV
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