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Preparation and Application of Hexahistidine-tagged Protein Immunoaffinity Purification Enrichment Column

A technology of hexahistidine and tagged proteins, applied in the field of biological separation, purification and enrichment devices, can solve the problems of limited application of target proteins, poor detection and purification effects, severe elution conditions, etc., to achieve accurate separation, even The effect of high coupling rate and high coupling density

Inactive Publication Date: 2015-11-18
NANNING LANGUANG BLUE LIGHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the affinity chromatography columns currently used for separation, purification and enrichment of His recombinant proteins mainly include metal chelate affinity columns and affinity columns using enzyme substrates and reactive fuels as ligands, these affinity columns have non-specific Significant shortcomings such as heterosexual adsorption, severe elution conditions, and poor purification effects, and the metal chelate affinity column also has the defect that metal ions fall off into the eluent, which will limit the subsequent application of the separated and purified target protein. detection

Method used

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  • Preparation and Application of Hexahistidine-tagged Protein Immunoaffinity Purification Enrichment Column
  • Preparation and Application of Hexahistidine-tagged Protein Immunoaffinity Purification Enrichment Column
  • Preparation and Application of Hexahistidine-tagged Protein Immunoaffinity Purification Enrichment Column

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of the His-specific polyclonal antibody of embodiment 1 immunoaffinity purification

[0045] 1.1 His short peptide hapten design

[0046]Using 4 groups of amino acid residues in His hexahistidine, divide them into 2 groups and introduce cysteine ​​sulfhydryl SH groups in opposite directions to prepare thiolated His short peptides.

[0047] 1.2 His short peptide immunogen preparation

[0048] Take 5mg / mLKLH10mL, add 100μL saturated Na 2 CO 3 , then add 1mL of 10% SDS, mix well, boil for 5 minutes to denature, then react with iodoacetyl-NHS to generate iodoacetylated KLH, remove excess iodoacetic acid by G25Sephadex, add 55mg of His short peptide hapten, adjust pH=8.5 After reacting at room temperature for 60 minutes, the His-KLH covalent bond complex was obtained, which was obtained after desalting with G25Sephadex as the immune antigen. Using ovalbumin OVA as the carrier protein, the coating antigen His-OVA for detection was prepared by the same method. ...

Embodiment 2

[0061] Example 2 Preparation of high-density His-tagged recombinant protein immunoaffinity purification enrichment column.

[0062] 2.1 Transfer 3 mL of Agarose activated by sodium iodate into the column, wash with 100 mL of distilled water, drip dry, and pass through 0.1 mol / L Na of 2 times the volume of Agarose 2 CO 3 , drip dry and set aside;

[0063] 2.2 Add 300 mg of immunoaffinity-purified His-specific polyclonal antibody with 0.1 mol / L Na 2 CO 3 Dissolve to make the concentration reach 20mg / mL, centrifuge to take the supernatant after complete dissolution;

[0064] 2.3 Add the supernatant to the affinity column in step 2.1, collect the effluent and pass through the column again, repeat 3-5 times;

[0065] 2.4 Wash out the unbound antibodies with 20mL distilled water and collect them together;

[0066] 2.5 Prepare 1.2 mL of sodium borohydride solution with a concentration of 10 mg / mL with distilled water, add it to the column, shake and mix, and react at 4 °C for 30...

Embodiment 3H

[0070] Embodiment 3His-specific antibody-labeled horseradish peroxidase

[0071] 3.1 Weigh 5 mg immunoaffinity purified His-specific polyclonal antibody, dissolve in 100 μL 0.1mol / L Na 2 CO 3 , react for 10 minutes, according to the amount of antibody: horseradish peroxidase = 1:1, add 5 mg of activated horseradish peroxidase, mix well;

[0072] 3.2 Add 1 mg of sodium borohydride, mix well and exhaust, place at -20°C for 1 minute, then move to 4°C for 2 hours;

[0073] 3.3 Desalted by G25Sephadex desalting column;

[0074] 3.4 Add BSA so that the final concentration of BSA is 1 mg / mL, add 50% glycerol, and detect the titer of the labeled antibody by ELISA.

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Abstract

The invention provides an His-tagged recombinant protein immunoaffinity purification and enrichment column capable of efficiently and specifically enriching hexahistidine-tagged protein and a preparation method and application thereof. The provided immunoaffinity purification and enrichment column comprises an activated agarose filler coupled with an immunoaffinity-purified His-specific polyclonal antibody and a plastic column loaded with the immunoaffinity filler, wherein the His-specific polyclonal antibody coupled with the immunoaffinity filler is extracted by an immunoaffinity method. The immunoaffinity purification and enrichment column is prepared by loading the immunoaffinity filler into the special plastic column; and the immunoaffinity purification and enrichment column is high in efficiency and strong in specificity while enriching His-tagged recombinant protein, is mild in diluting conditions, can be repeatedly utilized and is an ideal material for separating, purifying and enriching the His-tagged recombinant protein.

Description

technical field [0001] The invention relates to a biological separation, purification and enrichment device, in particular to a preparation method and application of an immunoaffinity purification and enrichment column for efficient separation and purification and enrichment of hexahistidine His-tagged recombinant protein. Background technique [0002] Molecular biology and proteomics are popular subjects in the field of life science research, and the use of recombinant proteins has greatly increased in recent years. In order to facilitate the labeling, tracking, identification and purification of genetically recombinant proteins, a tag polypeptide is artificially introduced during the expression of the target protein to form a recombinant protein. Therefore, the technology of separation, purification and enrichment of recombinant proteins is increasingly showing its importance. Separating, purifying, and enriching target proteins from complex expression systems is a arduou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/38C07K1/22G01N1/34
Inventor 谢体三谢芝勋庞耀珊潘丽金宁欢欢张芬萧浩
Owner NANNING LANGUANG BLUE LIGHT BIOTECH
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