Immunoglobulin binding protein and its application

A protein and multimer technology, applied in the field of immunoglobulin-binding proteins, can solve the problems of high requirements on physical and chemical properties, unsatisfactory binding antibody loading, and poor resistance to alkaline reagents.

Active Publication Date: 2022-03-29
SUZHOU NANOMICRO TECH CO LTD +1
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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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of protein multimer

[0053] Expression of engineering bacteria:

[0054] All the gene synthesis work in this experiment was entrusted to Nanjing GenScript to complete the expression strain E.coli BL (DE3).

[0055] The protein multimer prepared in this example is a six-segment repeat, and its monomers are obtained by splicing partial fragments of D-domain, C-domain and Z-domain:

[0056] E-domain

[0057] AQHDEAQQNA FYQVLNMPNL NADQRNGFIQ SLKDDPSQSA NVLGEAQKLN DSQAPK

[0058] D-domain

[0059] ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK

[0060] A-domain

[0061] ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK

[0062] B-domain

[0063] ADNKFNKEQQ NAFYEILHLP NLNEEQRNGF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0064] C-domain

[0065] ADNKFNKEQQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKEILAEAKK LNDAQAPK

[0066] Z-domain

[0067] VDNKFNKEQQ NAFYEILHLP NLNEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

[0068] The sequence of the r...

Embodiment 2

[0117] Embodiment 2 Preparation of Protein A affinity filler

[0118] activation

[0119] 1. Accurately measure 300ml of 2mol / L NaOH solution, fully mix the solution with 200g of PMMA microspheres after draining and pour it into the reaction kettle, turn on the stirring motor and set the speed to 200rpm, set the temperature of the water bath to 40°C, and seal the reaction 30min.

[0120] 2. Accurately measure 125ml of dimethylformamide and 75ml of 1,4-butanediol glycidyl ether into the reaction kettle, seal and react for 2.5h.

[0121] 3. Take down the reactor, pour the microsphere mixture into a suction filter funnel to drain, and wash with 2CV deionized water. Then wash with 2CV alcohol and deionized water respectively, and drain until no water drips.

[0122] coupling

[0123] 1. Accurately weigh 10 g of the activated microspheres and place them in a cleaned and dried Erlenmeyer flask.

[0124] 2. Weigh 0.3g of the ligand and dissolve it fully with 12.8ml of 50mM PB bu...

Embodiment 3

[0129] 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 spliced ​​from partial fragments of D, C and Z-domain of staphylococcal protein A, and unexpectedly obtains a binding protein with improved alkali resistance, which can then be used for affinity chromatography of immunoglobulins.

Description

technical field [0001] The 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, the prevention and treatment of diseases have undergone revolutionary changes. Today's biotechnology has penetrated into almost every corner of our lives, and the research on antibodies is also outstanding. From polyclonal antibodies, monoclonal antibodies to recombinant antibodies, every technological leap will give people unlimited surprises. However, like all other protein drugs, antibody production technology, production scale and purification technology are important technical links that restrict antibody production. The antibody purification technology has become the key. The quality of the purification technology and the size of the scale often determine the vitality of an antibody drug production. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C07K16/00C07K1/22C12N15/31B01J20/286B01J20/30
CPCC07K14/31C07K16/00C07K1/22B01J20/286
Inventor 程雷江必旺刘劲松
Owner SUZHOU NANOMICRO TECH CO LTD
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