Preparation and application of flag peptide tagged recombinant protein immunoaffinity purification and enrichment column

Abstract

The invention provides a D-tag tagged recombinant protein immunoaffinity purification and enrichment column capable of efficiently and specifically enriching flag peptide 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 D-tag specific polyclonal antibody and a plastic column loaded with the immunoaffinity filler, wherein the D-tag 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 D-tag tagged recombinant protein, is mild in diluting conditions, can be repeatedly utilized and is an ideal material for separating, purifying and enriching the D-tag 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 highly efficient separation and purification and enrichment of flag peptide D-tag tag 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 a...

Examples

Embodiment 1

[0045] Example 1 Preparation of Immunoaffinity Purified D-tag-specific Polyclonal Antibody

[0046] 1.1 Design of D-tag short peptide hapten

[0047] Using a short peptide composed of 8 amino acid residues, namely aspartyl-tyrosyl-lysyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine, using These 8 amino acid residues are divided into two kinds and the cysteine ​​sulfhydryl SH group is introduced in opposite directions to prepare sulfhydrylated D-tag short peptides.

[0048] 1.2 Preparation of D-tag short peptide immunogen

[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 D-tag short peptide half For the antigen, adjust the pH to 8.5, react at room temperature for 60 minutes, and obtain the D-tag-KLH covalent bond complex, which is the immune antigen after desalting with G25...

Embodiment 2

[0063] Example 2 Preparation of enrichment column for immunoaffinity purification of recombinant protein with high-density D-tag tag.

[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 Dilute 300 mg of immunoaffinity-purified D-tag-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;

[00...

Embodiment 3

[0072] Example 3 D-tag-specific antibody-labeled horseradish peroxidase

[0073] 3.1 Weigh 5 mg immunoaffinity purified D-tag 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.

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 highly efficient separation and purification and enrichment of flag peptide D-tag tag 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 a...

Examples

Embodiment 1

[0045] Example 1 Preparation of Immunoaffinity Purified D-tag-specific Polyclonal Antibody

[0046] 1.1 Design of D-tag short peptide hapten

[0047] Using a short peptide composed of 8 amino acid residues, namely aspartyl-tyrosyl-lysyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine, using These 8 amino acid residues are divided into two kinds and the cysteine ​​sulfhydryl SH group is introduced in opposite directions to prepare sulfhydrylated D-tag short peptides.

[0048] 1.2 Preparation of D-tag short peptide immunogen

[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 D-tag short peptide half For the antigen, adjust the pH to 8.5, react at room temperature for 60 minutes, and obtain the D-tag-KLH covalent bond complex, which is the immune antigen after desalting with G25...

Embodiment 2

[0063] Example 2 Preparation of enrichment column for immunoaffinity purification of recombinant protein with high-density D-tag tag.

[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 Dilute 300 mg of immunoaffinity-purified D-tag-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;

[00...

Embodiment 3

[0072] Example 3 D-tag-specific antibody-labeled horseradish peroxidase

[0073] 3.1 Weigh 5 mg immunoaffinity purified D-tag 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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