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A method for site-directed covalent immobilization of histidine-tagged proteins based on vinyl sulfone surface reaction

A technology of histidine tag and vinyl sulfone, which is applied in the field of fixed-point covalent immobilization of histidine-tagged proteins, can solve the problems of inability to achieve fixed-point fixation, and achieve the effect of low cost, high stability and low cost

Active Publication Date: 2021-01-15
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fixed-site immobilization cannot be achieved due to the random distribution of amino groups on the protein surface

Method used

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  • A method for site-directed covalent immobilization of histidine-tagged proteins based on vinyl sulfone surface reaction
  • A method for site-directed covalent immobilization of histidine-tagged proteins based on vinyl sulfone surface reaction
  • A method for site-directed covalent immobilization of histidine-tagged proteins based on vinyl sulfone surface reaction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Methyl Tripolyethylene Glycol (OH-EG 3 -OMe, Tsi Ai Chemical Industry Development Co., Ltd.) and divinyl sulfone (DVS) are miscible at a volume ratio of 1:5, and 20mg / ml of dimethylaminopyridine (DMAP, sigma-aldrich) is added to react at 25°C 2h, purified by column chromatography to obtain methyl tripolyethylene glycol vinyl sulfone derivatives (VS-EG 3 -OMe).

[0035] 1 equivalent of VS-EG 3-OMe and 2 equivalents of N-Boc-L-histidine (Acros Organics) and 2 equivalents of N-α-Boc-L-lysine (Acros Organics) were dissolved in PB buffer (20 mM Sodium dihydrogen) at 25°C for 48h. The reaction solution was lyophilized and redissolved in D 2 O, characterized by carbon NMR spectroscopy, and VS-EG 3 -OMe, N-Boc-L-histidine and N-α-Boc-L-lysine were characterized by carbon nuclear magnetic resonance. Such as figure 1 , the results showed that the characteristic peaks of vinyl groups at 131.5ppm and 135.7ppm basically disappeared, and the peaks at 137.8ppm and 40ppm could b...

Embodiment 2

[0037] Place the gold-plated silicon wafer in 0.2mg / mL Hydroxy-EG 6 -Undecanethiol ((11-mercaptoundecyl)hexa(ethylene glycol), Dongren Chemical Technology Co., Ltd.) was reacted in ethanol solution at 25°C for 24h. It was then placed in a solution containing 10% (v / v) DVS and 1 mM triphenylphosphine (PPh 3 , Tianjin Damao Chemical Reagent Factory) in acetonitrile, 25 ℃ for 6h. Rinse with acetonitrile and blow dry with nitrogen to obtain a vinyl sulfone-terminated polyethylene glycol surface (VS-EG 6 ) of gold-plated silicon wafers. VS-EG 6 The gold-plated silicon wafers on the surface were reacted with N-Boc-L-histidine and N-α-Boc-L-lysine in PB buffer solution of pH 6.5, pH 7.0 and pH 8.0 respectively at 25°C for 12h . X-ray photoelectron spectroscopy was used to characterize the elements on the surface of the gold-plated silicon wafer after reaction and calculate the ratio of N1s to S2p on the surface. The larger the ratio, the more amino acids reacted on the surface. ...

Embodiment 3

[0039] Primers were designed according to the nucleotide sequence of the target gene (primer sequence: 5'-CATATGGCTGAAGCTGGTAT-3', 5'-CCGCTGCTTCCTAATAAAGCTT-3'). Use the HaloTag-6His gene sequence in the commercially purchased pH6HTC plasmid (Promega Biotechnology Co., Ltd., item number: G803A) as a template for PCR amplification, and PCR uses Tks Gflex DNA Polymerase (Bao Biological Engineering Co., Ltd., item number: R060) Amplify. The PCR reaction conditions were denaturation at 98°C for 10s, annealing at 60°C for 15s, extension at 68°C for 30s, and 30 cycles. The PCR product was detected by agarose electrophoresis, and the target gene fragment was recovered according to Takara MiniBESTAgarose Gel Extraction Kit Ver 4.0 (Bao Biological Engineering Co., Ltd., catalog number: 9762), and the target gene fragment was connected to the PET21a plasmid (Beijing Tianenze Gene Technology Co., Ltd. company), the HaloTag-6His-PET21a plasmid was obtained. The HaloTag-6His-PET21a plasm...

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Abstract

The invention discloses a new method for fixed point covalent fixing of a histidine tag protein based on vinyl sulfone surface reaction. The method for fixed point covalent fixing of the histidine tagprotein based on vinyl sulfone surface reaction comprises the following steps: taking a substrate material containing a vinyl sulfone group on the surface as a protein fixing carrier, and realizing the reaction of the histidine tag and the surface vinyl sulfone under room temperature in a protein solution under a near neutral condition, thereby realizing the fixed point covalent fixing of the histidine tag protein on the vinyl sulfone surface. The new method for fixed point covalent fixing provided by the invention has high directionality and specificity; the fixed protein is high in stability; the reaction condition is mild and the experimental method is simple; reagents used in the method are all conventional reagents and are low-cost; the new method for fixed point covalent fixing of the histidine tag protein provided by the invention has an extensive application prospect in the fields of enzyme immobilization, biochip preparation, micro-array preparation and targeting drug-delivery system construction.

Description

technical field [0001] The invention belongs to the field of protein immobilization, and relates to a method for fixed-point covalent immobilization of histidine tagged protein based on vinyl sulfone surface reaction. [0002] technical background [0003] Protein immobilization has important applications in the fields of biosensing, enzyme immobilization, and targeted drug delivery systems. Traditional random immobilization methods (such as NHS-activated carboxyl method) will cause the active site of the protein to be masked, thereby affecting the biological activity of the immobilized protein. The fixed-site immobilization method can fully expose the active site by controlling the orientation of the protein on the surface, thereby effectively improving the biological activity of the immobilized protein. Finding the specific reaction between the target protein and the surface functional groups is the basis for the realization of site-specific immobilization. Commonly used ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K17/02C12N11/02C12N11/14C12N11/089A61K47/42
CPCA61K47/42C07K17/02C12N11/02
Inventor 程昉李明洋董继程汪晴
Owner DALIAN UNIV OF TECH
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