Preparation and application of hexahistidine-tagged protein immunoaffinity purification and enrichment column

A technology of hexahistidine and tagged proteins, which is applied in purification and enrichment devices and in the field of biological separation, can solve the problems of limiting target protein application, detection, non-specific adsorption, and severe elution conditions, and achieve accurate separation and elution The effect of mild conditions and high coupling density

Inactive Publication Date: 2013-05-22
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 and enrichment column
  • Preparation and application of hexahistidine-tagged protein immunoaffinity purification and enrichment column
  • Preparation and application of hexahistidine-tagged protein immunoaffinity purification and enrichment column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of immunoaffinity-purified His-specific polyclonal antibody

[0046] 1.1 His short peptide hapten design

[0047] 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.

[0048] 1.2 His short peptide immunogen preparation

[0049] Take 5 mg / mL KLH 10 mL, add 100 μL saturated Na 2 CO 3 , then add 1 mL 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 G25 Sephadex, add 55 mg of His short peptide hapten, Adjust the pH to 8.5 and react at room temperature for 60 minutes to obtain the His-KLH covalent bond complex, which is the immune antigen after desalting with G25 Sephadex. Using ovalbumin OVA as the carrier protein, the coating antigen His-OVA for detection was prepared by the same method.

[0050] 1.3 Prepara...

Embodiment 2

[0063] Example 2 Preparation of enrichment column for immunoaffinity purification of high-density His-tagged recombinant protein.

[0064] 2.1 Transfer 3 mL of Agarose activated by sodium iodate to the column, wash with 100 mL of distilled water, drip dry, and pass through 0.1 mol / L Na 2 CO 3 , drip dry and set aside;

[0065] 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 20 mg / mL, centrifuge to get the supernatant after complete dissolution;

[0066] 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;

[0067] 2.4 Wash out the unbound antibodies with 20 mL of distilled water and collect them together;

[0068] 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 minutes;

[0069] 2.6 W...

Embodiment 3

[0072] Example 3 His-specific antibody-labeled horseradish peroxidase

[0073] 3.1 Weigh 5 mg immunoaffinity purified His-specific polyclonal antibody, dissolve in 100 μL 0.1 mol / 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;

[0074] 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;

[0075] 3.3 Desalted by G25 Sephadex desalting column;

[0076] 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 present invention involves a kind of biological separation and purification enrichment device, especially a preparation method and application of efficient separation and purification, enriched hexal picinine HIS label restructuring protein. Background technique [0002] Molecular biology and protein groups are popular disciplines in the field of life science research. The use of recombinant protein has increased significantly in recent years.In order to facilitate the labeling, tracking, identification and purification of genetic restructuring proteins, a reorganized protein will be artificially introduced in target protein expression.Therefore, the technology of separation and purification, enrichment of reorganization is increasingly displaying its importance.The separation and purification of the complicated expression system and the rich target protein are a difficult and heavy task, and the affinity layer method is the most widely widely separated...

Claims

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Application Information

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Patent Type & Authority Applications(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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