Immunoglobulin binding protein and its application

An immunoglobulin and binding protein technology, applied in the direction of immunoglobulin, application, separation method, etc., can solve the problems of high requirements on physical and chemical properties, unsatisfactory binding antibody loading, poor resistance to alkaline reagents, etc.

Active Publication Date: 2020-12-04
SUZHOU NANOMICRO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, since SPA itself is also a protein, it requires high physical and chemical properties during affinity adsorption and elution, especially poor tolerance to alkaline reagents, and its binding antibody loading is often not ideal.

Method used

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  • Immunoglobulin binding protein and its application
  • Immunoglobulin binding protein and its application
  • Immunoglobulin binding protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of protein multimers

[0060] Engineering bacteria expression:

[0061] All the gene synthesis work in this experiment was entrusted to Nanjing GenScript to express the strain E.coli BL (DE3).

[0062] The protein multimer prepared in this example is a six-segment repeat, and its monomer is obtained by splicing partial fragments of E-domain, C-domain and Z-domain:

[0063] E-domain

[0064] AQHDEAQQNA FYQVLNMPNL NADQRNGFIQ SLKDDP SQSA NVLGEAQKLN DSQAPK

[0065] D-domain

[0066] ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK

[0067] A-domain

[0068] ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK

[0069] B-domain

[0070] ADNKFNKEQQ NAFYEILHLP NLNEEQRNGF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0071] C-domain

[0072] ADNKFNKEQQ NAFYEILHLP NLT EEQRNGF IQSLKDDPSV SKEILAEAKK LNDAQAPK

[0073] Z-domain

[0074] VDNKFNKEQQ NAFYEILHLP NLN EEQRNAF IQSLKDDP SQ SANLLAEAKK LNDAQAPK

[0075] The sequence of the resulti...

Embodiment 2

[0140] Example 2 Preparation of Protein A Affinity Filler

[0141] activation

[0142] 1. Accurately measure 300ml of 2mol / L NaOH solution, fully mix the solution with 200g of dried PMMA microspheres and pour it into the reaction kettle, turn on the stirring motor, set the rotation speed to 200rpm, set the temperature of the water bath to 40°C, and seal the reaction. 30min.

[0143] 2. Accurately measure 125ml of dimethylformamide and 75ml of 1,4-butanediol glycidyl ether into the reaction kettle, and seal the reaction for 2.5h.

[0144] 3. Remove the reactor, pour the microsphere mixture into a suction filter funnel, drain and rinse with 2 CV of deionized water. Then wash with 2CV of alcohol and deionized water respectively, and drain until no water drips.

[0145] coupling

[0146] 1. Accurately weigh 10 g of the activated microspheres and place them in a washed and dried conical flask.

[0147]2. Weigh 0.3g of the ligand and fully dissolve it with 12.8ml of buffer. Ad...

Embodiment 3

[0152] Example 3 Preparation of Protein A affinity filler

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PUM

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Abstract

The invention relates to the technical field of immunoglobulin separation and purification, in particular to an immunoglobulin binding protein and its application. The immunoglobulin binding protein is obtained by splicing the E, C and Z-domains of staphylococcal protein A, and unexpectedly obtains an increase in antibody loading and alkali resistance properties, and can be used for affinity chromatography of immunoglobulins.

Description

technical field [0001] The present invention relates to the technical field of immunoglobulin separation and purification, in particular to an immunoglobulin binding protein and its application. Background technique [0002] With the emergence and development of biotechnology, revolutionary changes have taken place in the prevention and treatment of diseases. Today's biotechnology has penetrated into almost every corner of our lives, and the research on antibodies has also made outstanding achievements. From polyclonal antibodies, monoclonal antibodies to recombinant antibodies, every technological leap will give people infinite surprises. However, like all other protein drugs, antibody production technology, production scale and purification technology are important technical links that restrict antibody production. And antibody purification technology has become the key. The quality of purification technology and the size of the scale often determine the vitality of an a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C07K16/00C07K1/22C12N15/31C12P21/02B01D15/38
CPCC07K14/31C07K16/00B01D15/3809
Inventor 江必旺程雷
Owner SUZHOU NANOMICRO TECH CO LTD
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