CN113999294A

CN113999294A - Immunoglobulin binding proteins and uses thereof

Info

Publication number: CN113999294A
Application number: CN202111640631.6A
Authority: CN
Inventors: 程雷; 江必旺; 刘劲松
Original assignee: Changshu Nanomicro Biological Technology Co ltd; Suzhou Nanomicro Technology Co ltd
Current assignee: Changshu Nanomicro Biological Technology Co ltd; Suzhou Nanomicro Technology Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-02-01
Anticipated expiration: 2041-12-30
Also published as: WO2023124207A1; CN113999294B

Abstract

The invention relates to the technical field of immunoglobulin separation and purification, and particularly relates to an immunoglobulin binding protein and application thereof. The immunoglobulin binding protein is obtained by splicing D, C of staphylococcal protein A and Z-domain partial fragments, and binding protein with improved alkali resistance is unexpectedly obtained, so that the immunoglobulin binding protein can be used for affinity chromatography of immunoglobulin.

Description

Immunoglobulin binding proteins and uses thereof

Technical Field

The invention relates to the technical field of immunoglobulin separation and purification, and particularly relates to an immunoglobulin binding protein and application thereof.

Background

With the advent and development of biotechnology, the prevention and treatment of diseases has revolutionized. Today's biotechnology penetrates almost every corner of our lives, where research directed at antibodies has also performed prominently. From polyclonal antibodies, monoclonal antibodies to recombinant antibodies, every technological leap will give people unlimited surprise. 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 becomes the key, and the good and bad of the purification technology and the large and small scale often determine the vitality of the antibody drug production.

The affinity molecule with special structure is made into solid phase adsorbent and placed in chromatographic column, and when the protein mixture liquid to be separated passes through the chromatographic column, the protein with affinity to the adsorbent is adsorbed and retained in the chromatographic column. The protein without affinity is separated from the separated protein by directly flowing out without being adsorbed, and then the bound protein is eluted by changing the binding condition by using a proper eluent, and the method for separating and purifying the protein is called affinity chromatography. Certain structural sites on some of the biomolecules recognize and bind to other molecules, such as enzyme-substrate recognition binding, receptor-ligand recognition binding, and antibody-antigen recognition binding, which is both specific and reversible, and which can be released by changing conditions. This binding capacity between biomolecules is called affinity. Affinity chromatography is a protein separation and purification method designed according to the principle, and is a chromatographic technique for separating target proteins or other molecules capable of specifically binding to ligands in a protein mixture by using a chromatographic medium covalently linked with the specific ligands.

Affinity chromatography is an adsorption chromatography in which an antigen (or antibody) and a corresponding antibody (or antigen) are specifically bound, and the binding is reversible under certain conditions. Therefore, after the antigen (or antibody) is solid-phased, the corresponding antibody (or antigen) in the liquid phase can be selectively bound on the solid phase carrier, thereby separating from other proteins in the liquid phase and achieving the purpose of separation and purification. The method has the advantages of high efficiency, rapidness, simplicity and the like.

Staphylococcal protein A (staphylococ)The cus protein A, SPA) contains 5 domains capable of specifically binding to Fc segment of antibody IgG molecule, and is staphylococcus aureus (A. aureus)Staphylococcus aureus) A constituent protein in the cell wall. Staphylococcus aureus is a gram-positive bacterium, a common pyogenic infectious bacterium in cross infection in hospitals, has a thallus diameter of about 0.8 mu m, is in a small ball shape, and is named after a bunch of grapes. SPA is capable of binding to many mammalian IgG antibodies (often the Fc fragment) and is therefore one of the earliest proteins used for affinity chromatography and antibody purification.

However, since SPA is also a protein, it requires high physicochemical properties for affinity adsorption and elution, and is particularly resistant to alkaline reagents, and the amount of antibody bound is often not optimal.

Disclosure of Invention

Based on this, there is a need for a strong alkali-resistant immunoglobulin-binding protein and its application.

The invention relates to an immunoglobulin binding protein, which has an amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2.

According to still another aspect of the present invention, the present invention also relates to a protein multimer comprising two or more repeating units selected from either or both of the amino acid sequence represented by SEQ ID NO:1 and the amino acid sequence represented by SEQ ID NO: 2.

The invention also relates to nucleic acids, vectors, host cells and methods of production relating to the immunoglobulin-binding proteins and protein multimers.

According to a further aspect of the invention, the invention also relates to an affinity chromatography medium and an affinity chromatography separation device containing the affinity chromatography medium, wherein the chromatography medium comprises a solid phase carrier and a ligand grafted on the solid phase carrier;

the ligand is an immunoglobulin-binding protein as described above or a protein multimer as described above.

The invention also relates to the use of the above product for the isolation or enrichment of immunoglobulins from a liquid medium.

The immunoglobulin binding protein is obtained by splicing D, C of staphylococcal protein A and a partial fragment of Z-domain, and is transformed to unexpectedly obtain the immunoglobulin binding protein with high alkali resistance, so that the immunoglobulin binding protein can be used for affinity chromatography of immunoglobulin.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

SEQ ID NO. 2 is a substitution of amino acid Q at position 12 of SEQ ID NO. 1 with A. The immunoglobulin-binding protein may bind to the Fc region of an immunoglobulin.

In the present invention, the term "immunoglobulin" is a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments comprising complementarity determining regions (CDR regions), particularly full-length antibodies comprising an Fc fragment or variants thereof. The term "full length antibody" includes polyclonal antibodies and monoclonal antibodies. Variants comprising an Fc segment are well known in the art, e.g., scFv-Fc and the like. The type of antibody can be selected from IgG, IgA, IgM, IgE, IgD. Preferably, the antibody is at least one of IgG (IgG 1, IgG2, IgG3, or IgG 4), IgA, and IgM. Furthermore, the term "immunoglobulin" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional), humanized (humanized) antibodies and human antibodies, as well as related synthetic isomeric forms (isofoms).

The Fc portion of an "immunoglobulin" may be from animals including humans and all animal husbandry (e.g., livestock and pets) and wild, preferably mammals, more preferably: pig, dog, rabbit, human, monkey, mouse, and cow.

In some embodiments, an "immunoglobulin" may also be a fusion protein.

The "immunoglobulin" is preferably an antibody drug, in particular a monoclonal antibody drug, examples of which may be selected from the group consisting of antibodies as shown below:

anti-GD 2 antibody 3F8, abamectin (Abagonomab), Abciximab (Abciximab), ACZ885 (canakinumab), Adalimumab (Adalilimumab), Addenamumab (Adalelimumab), Addenamumab (Adecatuzumab), Aframomumab (Afelimomab), Atubumab (Afutuzumab), Pezidomab (Alizezumab pegol), Alemtuzumab (Allituzumab), pentoxydum-tuzumab (Altuzumab pentate), Maumomab (Anatomab mafenox), Anluzumab (Anlunuzumab) (IMA-638), Adinizumab (Apotuzumab), Aselimumab (Artumomab), Atenizumab (Atolizumab), Abelizumab (Abelizumab), Abelizumab (Bezizumab), Abelizumab (Abelizumab), Abelimumab (Abelizumab), Abelizumab (Abelimex), Abelimexib), Abelizumab (Abelizumab), Abelix (Abelimex), Abelix (Abelix), Abelimex (Abelix), Abelix (Abelix), Abelimex (Abelix), Abelix (Abelix) and Abelix) and Abelix (Abelix) Abelix (Abelix) in Abelix), Abelix) in (Abelix) in, Bixizumab (Biciromab), bivatuzumab-DMl (Bivatuzumab mertansine), Lantuzumab (Blinatumomab), Brentuximab vedotin, Briakiumab, Carnazumab (Canakinmumab), Memantimab (Canuzumab mertansine), Carluumab pentapeptide (Capromab pendend), Cartuzumab (Catuzaxomaxomab), Celizumab (Celizumab), Petzuzumab (Certifizumab), Cetuzumab (Cetuximab), Cetuzumab (Cituzumab), Cetuzumab (Cixtuzumab), Clulizumab (Cleniumumab), Cetuzumab tetatan, CNTO148 (Cetuzumab), Cetuzumab (Cixutuzumab), Cetuzumab (Cleniumumab), Cetuzumab (Clenimexituzumab), Cetuzumab (Cetuzumab), Cetuvulizumab), Cetuzumab (Cetuzumab), Cetuvu (Cetuzumab), Cetuvu), Cetuzumab (Cetuvu), Cetuzumab (Cetuvu), Cetuzumab (Cetuvu, Cetuvu (Cetuvu), Cetuzumab), Cetuvu (Cetuvu), Cetuvu (Cetuvu), Cetuvu (Cetuvu ), Cetuvu (Ctu), Cetuvu (Cetuvu) and E (Cetuvu), Cetuvu (Cetuvu) and E (Cetuvu) and Cetuvu (Cetuzumab (Cetuvu), Cetuvu (Cetuzumab), Cetuvu (Ctu), Cetuvu (Cetuvu) and E (Cetuvu), Cetuvu) and E (Cetuvu, Epilozumab (Edbeclomab), efamozumab (Efalizumab), efuzumab (Efantumumab), efuzumab (Efangumtumab), eimitumumab (Elsiiumumab), pegolimumab (Enlimomab pegol), Ceipilimumab (Epitmometatuxen), Epratuzumab (Epratuzumab), Erlizumab (Erlizumab), Erimazemazumab (Ertumaxomab), Edazazumab (Etarazezumab), Evomab (Exbiviruzumab), Fanolisomab (Fanolisomab), Faralmomab (Faralimomab), Faralmomab (Faralimoab), Novizumab (Felvizumab), Nozazumab (Fezakinumab), Figituzumab (Figituzumab), Argituzumab (Familizumab), Fraveluzumab (Ferzivumgiumtuzumab), Gezakizumab (Gezakinumab), Gajirimuzumab (Gejirimuzumab), Gajirimuzumab), Ejingumazumab (Gejivu (Gojivu), Gajivumab (Gojivu, Gajivu-Ejintezumab), Ejintezomib (Gejivub), Egovizumab), Ejintezomib (Gejimazokumazokumazumab), Ejin, Infliximab (Infliximab), Infliximab (Intetumumab), itumumab (inolomab), itumumab (inolimab), itumumab ozolomicin (Inotuzumab ozogamicin), Ipilimumab (Ipilimumab), itumumab (Iratumumab), Keliximab (Keliximab), labezumab (Labetuzumab), lekulizumab (Lebrikizumab), lemuzumab (lemasizumab), lemuzumab (lemalesamumab), lemadumab (ledeluzumab), lexamumab (lexatuzumab), livir (libirumamab), toluzumab (Matuzumab), lucamuzumab (Lucatumumab), luciximab (lumimab), macumumab (Mapatumumab), macumumab (macumumab), macumumab (Matuzumab), macumumab (macranthumumab), macumumab (macumumab 0), macumumab (macumumab-CD), macumumab (miumumab), macumumab (miumumab 0), macumumab (macumumab), macumumab (miumumab), macumumab (r-CD 0), macumumab (r), macumumab (r-c), macumumab (r-CD) and the like, Tanaclizumab (Nacolomab tafentox), tanamezumab (Naptomumab estafenatox), Natalizumab (Natalizumab), nembacuzumab (Nebacumab), Netuzumab (Netuzumab), Neruimumab (Nerelimomab), Nimotuzumab (Nimotuzumab), Nivolumab (Nivolumab), Munavolumab (Nofetumumab), Oruizumab (Ocriluzumab), Otuzumab (Odulimob), Oxamunmumab (Ofatumumab), Oxamunmumab (Omalizumab), Moorezumab (Oportuzumab), Oxogluzumab (Oregovizumab), Oxalizumab (Oxalizumab), Pacifumab (Pacifumab), Jujulizumab (Primulizumab), Rituzumab (Primulizumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuzumab (Pirimuzumab), Pirimuz, Ranibizumab (Rafiviruzumab), Ramucirumab (Ramucirumab), Ranibizumab (Ranibizumab), Raxikumab (Raxibacumab), Regawimab (Regavirumab), Raylelizumab (Reslizumab), Rituximab (Rituzumab), Rituximab (Rituximab), Rotatuzumab (Robartuzumab), rolizumab (Rontalizumab), Roveluzumab (Rovelizumab), rolizumab (Ruplizumab), Satuzumab (Satumomab), Sevivimab (Seviruzumab), Sibrolizumab (Sibrotuzumab), Sibrolizumab (Sifamozumab), Sefatuzumab (Sifalizumab), Sifatuzumab (Sifalizumab), Supiazumab (Siflulizumab), Supiazumab (Supializumab), Sufanuzumab (Sufanuzumab), Sufanuzumab (Sufantaslizumab), Sufanuzumab (Tafantaslizumab), Sufanuzumab (Tafanuzumab), Sufanuzumab (Tafantasumab), Tafanuzumab), Sufanuzumab (Tafantamuzumab), Tafantamutab (Tafanuzumab), Tafantamuzumab (Tafantamuzumab), Tafantamova (Tafantamova-Tafantame (Tafantam), Tafantame (Tafantamovab), Tafantamovab (Tafantamovab), Tafantamova-e (Tafantamovab), Tafantamovab (Tafantamovab), Tafantamova (Tafantamova-e (Tafantamovab), Tafantamovab (Tafantamovab), Tafantamb), Tafantamova-e (Tafantamb (Tafantamovab), Tafantamb), Tafantamovab), Tafantame (Tafantamb), Tafantamuzumab), Tafantamb (Tafantamb), Tafantame (Tafantamb), Tafantamuzumab), Tafantame (Tafantamb), Tafantame (Tafantamuzumab), Tafantamova-e (Tafantame (Tafantamuzumab), Tafantame (Tafantamb), Tafantamuzumab), Tafantamb (Tafantamb), Tafantame-e (Tafantamb), Tafantamb (Tafantamb), Tafantame-e (Tafantamb), Tafantamb (Tafantame, Attentiomab (Telimomab aritox), temitumomab (tentumomab), teneriximab (Teneliximab), tilizumab (Teplizumab), TGN1412, tiximumab (ticimumab), Tremelimumab (Tremelimumab), tegafur-lizumab (tigitumumab), TNX-355 (ibalizumab), TNX-650, TNX-901 (talizumab), tollizumab (Tocilizumab), tollizumab (Toralizumab), tollizumab (Tositumomab), Trastuzumab (Trastuzumab), Tremelimumab (tremulumab), simukumab (tuzumab), tuveluzumab), ulizumab (tuviruzumab), uralizumab (Trastuzumab), Trastuzumab (Trastuzumab), vemuralizumab), vemuratuzumab (vetuzumab), vimuzumab), vemuralizumab (tuzumab), vemuralizumab (vemuralizumab), vemuralizumab (vemuralizumab), tuzumab (tuzumab), vemuralizumab (vemuralizumab), taclizumab (vemuralizumab), taclizumab (taclizumab), taclizumab (taclizumab), taclizumab (taclizumab), taclizumab (taclizumab), taclizumab (taclizumab), taclizumab (taclizumab), taclizumab (taclizumab), taclizumab (tacrolimus (, Zaolimumab (Zanolimumab), ziralumab (Ziralimumab), and azimomab (zoimob aritox).

According to still another aspect of the present invention, the present invention also relates to a protein multimer comprising two or more repeating units selected from either or both of the amino acid sequence represented by SEQ ID NO:1 and the amino acid sequence represented by SEQ ID NO: 2. When the sequence contains the repeating units of the two sequences, the connection arrangement sequence of SEQ ID NO 1 and SEQ ID NO 2 is disordered, and SEQ ID NO 1 or SEQ ID NO 2 can be connected with the sequence of the sequence or can be connected with each other.

In some embodiments, the protein multimer contains 4, 5, 6, 7, or 8 of the repeating units.

In some embodiments, the protein multimer also has a terminal coupling group at the N-terminus.

In some embodiments, the terminal coupling group comprises arginine and/or cysteine.

It will be readily understood that the invention also claims proteins substantially identical to said immunoglobulin-binding protein or said protein multimer, such proteins being substantially identical to the immunoglobulin-binding protein of SEQ ID NO:1 or SEQ ID NO:2, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity, and retains the function of specifically binding to an immunoglobulin; furthermore, such proteins are still capable of specifically binding immunoglobulins and subsequently eluting them after soaking in 0.5mol/L NaOH for 24h at 25 ℃ and can maintain a loading of about 56 mg/ml.

The invention also relates to nucleic acids encoding the immunoglobulin-binding proteins as described above or the protein multimers as described above.

The invention also relates to a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.

In some embodiments, the vector of the present invention may further comprise a gene used for screening (e.g., an antibiotic resistance gene), a nucleic acid for producing a fluorescent protein, or the like. The fluorescent protein can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.

The invention also relates to a host cell comprising a nucleic acid as described above or a vector as described above.

The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. The host cell is preferably a prokaryotic cell, more preferably E.coli, e.g.E.coli BL (DE 3).

The present invention also relates to a method of producing an immunoglobulin-binding protein as described above or a protein multimer as described above, comprising:

culturing a host cell as described above under suitable culture conditions; and

recovering the immunoglobulin-binding protein or protein multimer thus produced from the culture medium or from the cultured cells.

The invention also relates to an affinity chromatography medium, which comprises a solid phase carrier and a ligand grafted on the solid phase carrier;

the ligand is the immunoglobulin binding protein or the protein multimer.

In some embodiments, the solid support is selected from any one of bentonite, glass microspheres, quartz microspheres, magnetic beads, calcium hydroxy phosphate, alumina, polyacrylamide gel, starch gel, dextran gel, cellulose, agarose, silicon, ceramic, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters, gelatin, polyurethane, polystyrene divinyl benzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinyl pyrrolidone.

The solid phase carrier may be selected from suitable shapes, forms, materials and modifications depending on the application. The solid support surface can be substantially flat or planar. Or may be circular or the like. The shape of the surface of the solid support includes, but is not limited to, pores, depressions, pillars, ridges, channels, membranes, or the like. The solid support is preferably a particulate material having a substantially planar surface. The shape of the particulate matter is preferably spherical or substantially spherical.

Further, the solid support may be non-porous, but preferably comprises one or more pores, more preferably a porous network, allowing free passage of macromolecules. In some embodiments, the pore diameter is 300-5000A; in the present invention, "non-porous" means that the matrix support has no pores that are appreciably measurable, for example, a pore diameter ≦ 20A.

In some embodiments, the solid support is activated by covering certain groups (e.g., hydroxyl groups, etc.) on the surface of the solid support with an intermediate compound (e.g., CNBr) and by providing new chemical groups (e.g., -CN) on the surface of the solid support, such that the new groups can covalently react with the affinity ligand to achieve covalent attachment.

Thus, it will be readily appreciated that the surface of the solid support may also be modified with groups for activation, such as at least one of epoxy, amino, aldehyde, hydroxyl, carboxyl, and thiol groups. The modification sites typically also comprise derivatizing groups with these corresponding chemical functionalities.

The invention also relates to an affinity chromatography separation device containing the affinity chromatography medium.

The affinity chromatography separation device can be SPE solid phase extraction column, centrifuge tube with separation membrane, separation membrane (membranes), fast detection biochip (bio-chips), fiber bundle column, monolithic column and conventional analytical grade or preparative chromatographic column, etc.

The invention also relates to the use of an affinity chromatography medium as described above, or an affinity chromatography separation device as described above, for separating or enriching immunoglobulins from a liquid medium.

In some embodiments, the use comprises at least one step of treating the affinity chromatography media or the affinity chromatography separation device under basic conditions.

In some embodiments, the alkaline conditions should be in the vicinity of the pH provided by 0.5mol/L NaOH, such as 7 to14, or 10 to14, or 12 to14, or 13 to14, or 13.5 to 14. The alkaline treatment time may be up to 24 hours, for example 8 hours, 16 hours or 20 hours.

In some embodiments, the liquid medium is a sample comprising immunoglobulins or a solution thereof after dilution. Any sample comprising immunoglobulins may be used in the present invention. The immunoglobulin-containing sample may comprise, for example, a cell culture (particularly a cell culture supernatant), a blood extract (particularly a serum extract), and ascites (e.g., ascites of an animal such as a rat, a mouse, a rabbit, an sheep, a horse, a cow, a camel, etc.). As an example, antibodies can be expressed in Chinese Hamster Ovary (CHO) cells in a stirred tank bioreactor.

Embodiments of the present invention will be described in detail with reference to examples.

EXAMPLE 1 preparation of protein multimers

Expressing engineering bacteria:

all the gene synthesis work in the experiment is finished by entrusting Nanjing Kinsley to express the strain E.coli BL (DE 3).

The protein polymer prepared in the embodiment is a six-segment repeat, and the monomer of the protein polymer is obtained by splicing partial fragments of D-domain, C-domain and Z-domain:

E-domain

AQHDEAQQNA FYQVLNMPNL NADQRNGFIQ SLKDDPSQSA NVLGEAQKLN DSQAPK

D-domain

ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK

A-domain

ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK

B-domain

ADNKFNKEQQ NAFYEILHLP NLNEEQRNGF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

C-domain

ADNKFNKEQQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV SKEILAEAKK LNDAQAPK

Z-domain

VDNKFNKEQQ NAFYEILHLP NLNEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQAPK

the sequence of the resulting monomer was:

VDAQQAKFDK DQQSAFYEIL HLPNLTEEQR NAFIQSLKDDPSV SKEILAEAKK LNDAQAPK

（SEQ ID NO:1）

the protein multimer (DC-1) sequence was:

VDAQQAKFDK DQQSAFYEIL HLPNLTEEQR NAFIQSLKDDPSV SKEILAEAKK LNDAQAPK

VDAQQAKFDK DQQSAFYEIL HLPNLTEEQR NAFIQSLKDDPSV SKEILAEAKK LNDAQAPKC

simultaneously designing six sections of repetitive sequences of DC-2, wherein the sequence of the obtained monomer is as follows:

VDAQQAKFDK DAQSAFYEIL HLPNLTEEQR NAFIQSLKDDPSV SKEILAEAKK LNDAQAPK(SEQ ID NO:2)

the protein multimer (DC-2) sequence was:

VDAQQAKFDK DAQSAFYEIL HLPNLTEEQR NAFIQSLKDDPSV SKEILAEAKK LNDAQAPK

VDAQQAKFDK DAQSAFYEIL HLPNLTEEQR NAFIQSLKDDPSV SKEILAEAKK LNDAQAPKC

1. transformation of the plasmid:

1) 0.03ml of competent cells and 4ng of plasmid DNA were mixed and placed in ice bath for 30 min.

2) The Ep tube was placed in a 42 ℃ water bath for 2 minutes of thermal shock and immediately placed on ice for 1 minute.

3) 70u1 LB medium was added to Ep tube and mixed well, and cultured at 37 ℃ for 30 min.

4) Spread on LB plates containing appropriate concentrations of antibiotic.

5) After being cultured overnight at 37 ℃, the bacterial plaque is taken out and is a positive clone.

2. And (3) bacterial fermentation:

1) two 50ml small shake flasks of LB medium were prepared and placed in a autoclave and sterilized at 121 ℃ for 30 min. And after the sterilization is finished and the cooling is finished, adding antibiotic which is subjected to sterilization and filtration on a super clean bench. Selecting plate cultured monoclonal bacteria, inoculating into small shake flask, and culturing in shaking table at 37 deg.C and 250 rpm. After about 8 hours of culture, it is ready for use.

2) After the culture medium of the 7L fermentation tank is configured and the pH electrode is calibrated, the pH electrode and the dissolved oxygen electrode are installed on the fermentation tank and are placed into a sterilization pot together with the tank body for sterilization. And (5) placing the prepared supplementary materials into a sterilization pot for sterilization.

3) After the sterilized fermentation tank is connected with a stirring motor, 100g of supplementary material is added by a peristaltic pump, the pH is adjusted to 7.0 by ammonia water, and then initial aeration and stirring are set.

4) Taking 100 mu L of the bacteria cultured in the small shake flask, inoculating the bacteria into the fermentation tank from the inoculation port by using a sterile injector, automatically controlling the dissolved oxygen to be 40%, and starting culturing by opening stirring and the dissolved oxygen linkage. Feeding materials when the dissolved oxygen starts to rapidly rise after automatic culture, starting to introduce oxygen, closing stirring and dissolved oxygen linkage at the moment, opening oxygen and dissolved oxygen linkage, adding IPTG (isopropyl-beta-thiogalactoside) for induction after 5 hours of feeding materials, and inducing until the feeding materials are completely consumed.

5) And centrifuging the fermentation liquor to obtain bacterial sludge, and freezing and storing.

3. Ligand purification:

1) and (3) dissolving thallus: according to thalli (wet weight): cell disruption solution =1:10 (kg: L) cells were dissolved in cell disruption solution until no visible clumpy cells were observed

2) Breaking the bacteria: crushing the dissolved thallus with a high-pressure homogenizer at 800 + -100 bar for 2 times

3) First removal of hetero-proteins: adjusting pH of the crushed thallus to 2.0 + -0.2, centrifuging, collecting supernatant

4) And (3) removing the foreign protein for the second time: adjusting the pH of the supernatant of the first centrifugation to 4.2 +/-0.1, and collecting the supernatant after centrifugation

5) UNiGEl 80SP chromatography:

and (3) treatment of the sample liquid: the supernatant from the second centrifugation was adjusted to pH 4.1. + -. 0.05 and filtered through a 0.45 μm filter

Loading: the filtered sample liquid is loaded, and the sample volume flow rate (20 ml/min)

And (3) balancing after sample loading: washing with solution A for more than 3CV (column volume) until UV280 value is leveled off

Impurity washing: washing with impurity washing solution 2CV

And (3) elution: washing the eluent, and collecting the peak: UV280 value of 50 +/-10 mAU-50 +/-10 mAU

Regeneration: the flushing of the solution B is more than 2CV until the UV280 value is leveled off

CIP: 1M NaOH for 10 min

Washing with water: 2CV of purified Water washing

And (3) storage: 10mM NaOH wash at least 1CV

The buffer formulations are shown in table 1:

TABLE 1

EXAMPLE 2 preparation of Protein A affinity Filler

Activation of

1. Accurately measuring 300ml of 2mol/L NaOH solution, fully mixing the solution with 200g of PMMA microspheres after being dried, pouring the mixture into a reaction kettle, turning on a stirring motor, setting the rotating speed to be 200rpm, setting the temperature of a water bath kettle to be 40 ℃, and sealing and reacting for 30 min.

2. Accurately measuring 125ml of dimethylformamide and 75ml of 1, 4-butanediol glycidyl ether into a reaction kettle, and sealing for reaction for 2.5 h.

3. The reaction kettle was taken down, the microsphere mixture was poured into a suction filtration funnel and drained and washed with 2CV deionized water. Then washing with 2CV alcohol and deionized water respectively, and pumping to dry until no water drops.

Coupling of

1. 10g of the activated microspheres are accurately weighed and placed in a washed and dried conical flask.

2. 0.3g of ligand was weighed and dissolved sufficiently in 12.8ml of 50mM PB buffer. Adding a certain amount of sodium sulfate solid into the microspheres to enable the final concentration to be 1-1.5 mol/L, adding the protein solution and the sodium sulfate into a conical flask filled with 10g of microspheres, and fully and uniformly mixing.

3. Adjusting the pH value of the microsphere mixture to 8.5 (error is about 0.1) by using 5mol/L NaOH and 50% phosphoric acid, introducing excessive nitrogen into the conical flask, sealing the conical flask by using a sealing film, putting the conical flask into a shaking table, setting the temperature of the shaking table to be 37 ℃, setting the rotation speed to be 180rpm, and reacting for 10-15 h.

End seal

1. Pouring the coupled microspheres into a filter flask, washing with 2CV deionized water, transferring the microspheres into a conical flask, and adding an appropriate amount of 0.2mol/L Na with the same volume as the microspheres₂CO₃Adding thioglycerol with the final concentration of 1-10%, adjusting the pH to 9.0, and reacting for 4-8 h.

2. Washing the reacted microspheres with 2CV deionized water and 2CV 0.1-0.5M NaOH, draining, storing in 20% ethanol, and storing in a refrigerator at 4 deg.C.

EXAMPLE 3 preparation of Protein A affinity Filler

Alkali resistance test

Preparation work is required in the early stage of carrying out load measurement on the bonded microspheres:

1. preparing a buffer solution: the buffer was prepared with 150 mM NaCl and 20 mM PB, pH 7.0. After the buffer solution is prepared, the buffer solution is filtered by a filter membrane and can be used after being filtered.

2. Preparing an antibody: the proportion of normally formulated antibody is antibody (multi-antibody): buffer =1: 24. 10ml of antibody is measured by a measuring cylinder with a proper volume and placed in a beaker, 190ml of filtered buffer solution is measured and added into the beaker, the mixture is mixed uniformly, then the mixture is injected into a loading ring by a syringe, and air bubbles in the loading ring are emptied.

3. Column assembling: the column used in the test is a 1 ml PP column, the microspheres are homogenized firstly, the microspheres are homogenized to a proper water-soluble ratio, the homogenate liquid is not viscous and not diluted, then the microspheres are slowly dripped into a column tube by a dropper, the lower end of the column tube is slowly extracted by an injector, the spheres are slowly deposited, when about 1 ml of the spheres are deposited, the dripping is stopped, and then an upper sieve plate is placed and is plugged by a plunger. The loaded column was then equilibrated with affinity A solution (Ph7.4 in PBS), and the equilibration was complete when the UV light was flat and the value was constant.

4. Measuring the loading capacity: instrument SCG antibody concentration: 2.15mg/ml flow rate: 0.2ml/min

After the measurement, the load graph was stored, then 0.5mol of sodium hydroxide was introduced into the column, the volume of the introduced alkali was about 30ml, after the passage of the alkali, the column was placed in a 25 ℃ incubator, and after 24 hours of storage, the load was measured again with the same antibody, and the pH of the elution was 3.0. The measured values are shown in table 2 below (10% DBC):

TABLE 2 alkali resistance test data

Note: the alkali treatment is the ratio of the result of 10% DBC (dynamic loading) of the multi-resistance of the microspheres after soaking in 0.5mol/L NaOH at 25 ℃ for 24h to the loading of the non-soaked microspheres.

As is clear from the above, the mutated DC-1 and DC-2 have high alkali resistance.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the invention is subject to the appended claims, and the description can be used for explaining the contents of the claims.

Sequence listing

<110> Suzhou nano micro-technology GmbH, ever-mature nano-micro-technology GmbH

<120> immunoglobulin-binding proteins and uses thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 61

<212> PRT

<213> Artificial Sequence

<400> 1

Val Asp Ala Gln Gln Ala Lys Phe Asp Lys Asp Gln Gln Ser Ala Phe

1 5 10 15

Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala

20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu

35 40 45

Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55 60

<210> 2

<211> 61

<212> PRT

<213> Artificial Sequence

<400> 2

Val Asp Ala Gln Gln Ala Lys Phe Asp Lys Asp Ala Gln Ser Ala Phe

1 5 10 15

Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala

20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu

35 40 45

Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55 60

Claims

1. An immunoglobulin binding protein having an amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2.

2. A protein multimer comprising two or more repeating units selected from either one or both of the amino acid sequence represented by SEQ ID NO. 1 and the amino acid sequence represented by SEQ ID NO. 2.

3. The protein multimer of claim 2, containing 4, 5, 6, 7, or 8 of said repeating units.

4. The protein multimer according to claim 2 or 3, further having a terminal coupling group at the N-terminus.

5. The protein multimer of claim 4, the terminal coupling group comprising arginine and/or cysteine.

6. A nucleic acid encoding the immunoglobulin-binding protein of claim 1 or the protein multimer of any one of claims 2-5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. A method of producing the immunoglobulin-binding protein of claim 1 or the protein multimer of any one of claims 2-5, comprising:

culturing the host cell of claim 8; and

10. An affinity chromatography medium comprising a solid support and a ligand grafted to the solid support;

the ligand is an immunoglobulin-binding protein according to claim 1 or a protein multimer according to any one of claims 2 to 5.

11. The affinity chromatography media of claim 10, wherein the solid support is selected from any one of bentonite, glass microspheres, quartz microspheres, magnetic beads, calcium hydroxyapatite, alumina, polyacrylamide gel, starch gel, dextran gel, cellulose, agarose, silicon, ceramics, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters, gelatin, polyurethane, polystyrene divinyl benzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, and polyvinyl pyrrolidone.

12. An affinity chromatography separation apparatus comprising the affinity chromatography medium according to any one of claims 10 to 11.

13. Use of the affinity chromatography medium of any one of claims 10 to 11, or the affinity chromatography separation device of claim 12, for separating or enriching immunoglobulins from a liquid medium.

14. Use according to claim 13, the liquid medium being selected from cell cultures, ascites or blood extracts, or their diluted solutions.