CN106188251A - A kind of immunoglobulin-binding proteins mutant and application thereof - Google Patents
A kind of immunoglobulin-binding proteins mutant and application thereof Download PDFInfo
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- CN106188251A CN106188251A CN201510225194.XA CN201510225194A CN106188251A CN 106188251 A CN106188251 A CN 106188251A CN 201510225194 A CN201510225194 A CN 201510225194A CN 106188251 A CN106188251 A CN 106188251A
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Abstract
The present invention relates to a kind of immunoglobulin-binding proteins mutant, by to natural grape pneumococcal proteins A(SpA) E domain amino acid carry out rite-directed mutagenesis and obtain, be combined with other regions in addition to complementary determining region (CDR) of immunoglobulin molecules, and it is prepared into the chromatography media for immunoglobulin affinity chromatograph, for separation, the purification of immunoglobulin.
Description
Technical field
The present invention relates to biological technical field, more specifically the present invention relates to a kind of immunoglobulin-binding proteins mutant and application thereof.A kind of immunoglobulin-binding proteins mutant that the present invention relates to, by to natural grape pneumococcal proteins A(SpA) E domain amino acid carry out rite-directed mutagenesis and obtain, be combined with other regions in addition to complementary determining region (CDR) of immunoglobulin molecules, and it is prepared into the chromatography media for immunoglobulin affinity chromatograph, for separation, the purification of immunoglobulin.
Background technology
SP (Staphylococal
Protein A, SpA) it is a kind of protein separated from aureus cell wall.Nineteen forty-seven, Vevwey finds in some staphylococcus aureus, containing a kind of material, in double diffusion test, can form precipitation with normal human serum.Jensen(1959) have also discovered similar phenomenon, by its named Staphylococal Protein A.Lofkvist in 1963 etc. have separated Staphylococal Protein A, and prove that it is a kind of protein, and have any different with sugar.Grov (1960), by its named SP, is called for short SpA protein A (ProteinA).The gene of coding SpA was cloned and at expression in escherichia coli in nineteen eighty-three.Research to SPA 26S Proteasome Structure and Function finds, SpA molecule comprises A, five homeodomains of B, C, D, E, and each domain has the ability being independently combined with IgG.Under conditions of neutral ph, protein A capture immunoglobulin molecules, discharged at low ph conditions.Therefore, one of separation, purification therapy monoclonal antibody important means at present are had become as using SpA as the affinity chromatograph of part.
Protein A affinity chromatography is the first step of therapeutic monoclonal antibodies downstream purification, containing large number of biological macromole impurity in culture supernatant to be purified, every time or for several times after purification cycle, it is necessary to through incumbent firms, otherwise the performance of chromatography media will be remarkably decreased.In order to extend the life-span of chromatographic column and guarantee purification quality, it is necessary to chromatographic column periodically carries out incumbent firms (CIP), optimal CIP condition is to use the sodium hydroxide solution of 0.5M, cleans 15-20 minute with relatively low flow velocity.But natural or restructuring SpA aglucon is very sensitive to NaOH, changeableness or come off under strong alkaline condition, if selecting the guanidine hydrochloride of high concentration or carbamide to be carried out, it is good that the effect cleaned is nothing like NaOH cleaning performance, and it is the highest to clean cost, the industrialization not being suitable for monoclonal antibody produces.
Protein A degeneration and coming off in chromatography media, is due to protein A in the basic conditions, and agedoite (Asn) and glutamine (Gln) occur desamination reaction, cause Fragmentation.Easily occurring in the aminoacid of deaminizating, agedoite (Asn) is more more sensitive to alkalescence condition than Gln.What desamination reaction speed was the fastest is when the sequence combined with glycine (Gly) containing agedoite (Asn) in peptide chain or agedoite (Asn) and serine (Ser) combine.Owing to " Asparagine-Glycine " sequence is most sensitive for alkalescence, in SpA domain, E contains " Asparagine-Glycine " sequence (N28-G29), therefore speculate that its alkali resistance is worst.The sudden change of current alkali resistance SpA sequence is concentrated mainly on B, C-structure territory, as applied wide alkali resistance SpA affinity media Mabselect SuRe at present, use SpA domain B and carry out the rite-directed mutagenesis of agedoite, be threonine (N23T) by the asparagine mutation of the 23rd.
Additionally, patent of invention (application number CN03806793.5) discloses, the domain B sequence to SpA is utilized to use neighbouring mutating technology (" by pass ") to carry out rite-directed mutagenesis (N23T, N43E), to improve its alkali resistance.Patent of invention (application number 201310097553.9) discloses, and utilizes SpA domain B sequence to carry out rite-directed mutagenesis (N23T, F30A) to improve its alkali resistance.Additionally, patent of invention (application number CN200780036281.4) discloses, and domain C sequence wild type (Cwt) and disappearance " Asn-Lys-Phe-Asn " mutant (Cdel) are obtained the alkali resistance suitable with MabselectSure.
During monoclonal antibody is isolated and purified, the problem that currently used ProteinA medium also exists alkali resistance difference.Protein
Medium A is after repeatedly alkalescence is cleaned, and dynamic adsorption carrying capacity declines, purification efficiency reduces.The present invention is by natural Protein
The E domain of A carries out rite-directed mutagenesis E(N23T, N28D, G29A), compared with prior art, its alkali resistance, dynamic adsorption carrying capacity and purification effect etc. are significantly increased this mutant.
Summary of the invention
A kind of immunoglobulin-binding proteins mutant that the present invention relates to, its aminoacid sequence derives from natural grape pneumococcal proteins A(SpA) E domain.By the agedoite of " alkali is sensitive " in its aminoacid sequence is carried out rite-directed mutagenesis so that it is mutant is compared compared with parent molecules has higher alkali resistance, and the affinity of immunoglobulin is significantly improved by this mutant.
A kind of immunoglobulin-binding proteins mutant of the present invention, utilizes the E domain of natural grape pneumococcal proteins A, suddenlys change, partial amino-acid in its E domain such as N11S, N21A, N23T, N28D, N28A, G29A, N43E, N52A；And the combinatorial mutagenesis of catastrophe point, such as E(N23T, N28D), E(N23T, N28A), E(N23T, N43E) and, E(N28D, N43E), E(N32T, N28D, G29A) etc..Through lot of experiment validation such as active testing, alkali resistance test and affinity tests, wherein preferred embodiment is for being threonine by its 23rd asparagine mutation, and the 28th asparagine mutation is aspartic acid, and the 29th glycine mutation is alanine (N23T, N28D, G29A).
Preferred immunoglobulin-binding proteins mutant is utilized technique for gene engineering fermentation culture, isolated and purified, obtain and have compared with high immunoglobulinlg affinity, immunoglobulin-binding proteins molecule that alkali resistance is strong, affinity chromatography medium preparation method is utilized to be prepared as the chromatography media for immunoglobulin affinity chromatograph this associated proteins molecule, this chromatography media is compared with the chromatography media of existing market, and its alkali resistance, dynamic adsorption carrying capacity machine purification effect are significantly increased.
Accompanying drawing explanation
Fig. 1, BL21/pET32a supernatant SDS-PAGE.
Fig. 2, i Protein A and MabslectSure Performance comparision figure.
Detailed description of the invention
Embodiment
1
, natural
SpA
Alkali resistance catastrophe point select
Natural SpAD alkali resistance depends on its aminoacid sequence, it is considered that, in the basic conditions, it is the major reason causing Fragmentation that the desamination reaction at agedoite (Asn) and glutamine (Gln) occurs.And desamination reaction speed the fastest be when the sequence combined containing agedoite (Asn) and glycine (Gly) in peptide chain or agedoite (Asn) and serine (Ser) combine.And the affinity of SpA Yu IgG depends on the conformation of peptide and protein.
The amino acid sequence analysis in SpA different structure territory see table 1.
Table 1, the amino acid sequence analysis in SpA different structure territory
Choosing containing asparagine residue less E domain as mutagenesis template, " by pass " strategy conventional from conventional SpA sudden change is different, and E domain directly carries out rite-directed mutagenesis.The natural amino acid (such as Ser, Glu) in the first-selected corresponding site of other domains of mutant target aminoacid, if this site is without natural amino acid, selects alanine (Ala).Report before this is not suitable for the N28 of rite-directed mutagenesis, uses the aspartic acid that structure is similar therewith to replace.Rite-directed mutagenesis for SpA domain E see table 2.
Table 2, the rite-directed mutagenesis of SpA domain E
Embodiment
2
, containing domain
E
The preparation of mutant
(1) vector construction containing domain E mutant
Various mutant gene sequences in synthetic table 1.Being connected with the carrier pET32a through NcoI and BamHI enzyme action, convert escherichia coli (BL21/DE3), screening has the transformant of amicillin resistance, and through plasmid extraction, after enzyme action is identified, certification structure territory E mutant gene is cloned in pET32a.
(2) strain containing structure E mutant builds and fermentation
Use CaCl2PET32a-P is converted e. coli bl21/DE3 by method, screens transformant on the LB flat board containing ampicillin, obtains recombinant conversion (BL21/pET32a) containing pET32a through plasmids detection and restriction analysis.Picking colibacillus engineering BL21
/ pET32a, is inoculated in LB culture medium, inoculum concentration 1 ~ 2%V/V, in 30 DEG C of overnight incubation, above-mentioned cultured seed culture medium aseptically presses l next day:
10-1:5 is inoculated on fermentation medium, and 30 DEG C ferment to OD600Reach 0.4 ~ 0.8, be warming up to 42 DEG C of inductions, after 5 hours, receive centrifugal receipts bacterium.
Take a small amount of cell and add 2X sample-loading buffer, PAGE gel electrophoresis is done by standard, Fig. 1 shows, there is a new protein band (the second from left) in the position of 20kD in supernatant after the broken bacterium of the BL21/pET32a of induction, convert in BL21/DE3 bacterium of PET32 empty carrier and the precipitation after the broken bacterium of BL21/pET32a of induction and occur without sub-band, it was demonstrated that recombiant protein ProteinA abduction delivering in BL21/pET32a.(each swimming lane of Fig. 1 is respectively as follows: marker, supernatant after induction, supernatant before induction).
(3) domain E mutant purification
By above-mentioned collection thalline NaCl ten phosphate buffer (pH7.
0 ~ 8. 0) suspending, ultrasonication, 4 DEG C are centrifuged, and collect supernatant and obtain crude extract.Through Ni
Sepharose 6FF purification is with the mutant protein of his label.Re-use EK enzyme enzyme action and remove the His purification tag of mutant protein.
Experimental example
1
, domain
E
The combination activity of mutant
Still have and human IgG binding ability for the SpA domain E mutant after checking rite-directed mutagenesis.Surface plasma technical measurement is used to use the combination activity of said mutation body and human IgG antibody.Utilize Biacore 2000(Uppsala,
Sweden), by polyclone IgG and the HAS(feminine gender reference of people) it is coupled at CMS sensor chip (Biacore).With 10 kinds of different concentration (100-550nM) HBS (10 mM HEPES, 0. 15M NaCl, 3. 4
MM EDTA, 0. 005% surfactant P20, pH 7. 4) in prepare recombinant protein A mutant solution.Sample is expelled to surface as copy using the flow velocity of random order and 30 ul/min, uses the HCl regenerating surface of 10 mM.Utilize BIA 3. 0. 2 b software analysis data.Utilize Lange wrong that model and calculate apparent kinetic constant and affinity constant (as shown in table 3).Compared with other mutants, E(N23T, N28D, G29A) have higher affinity, E(N23T, N28D, G29A with IgG) there is the aminoacid sequence shown in SEQ ID NO:1, the nucleotide sequence shown in SEQ ID NO:2.
Table 3, utilize Biacore that SpA domain E mutant is combined determination of activity
Experimental example
2
, domain
E
Mutant STUDY ON ALKALI RESISTANCE
In order to investigate the alkali resistance of different E structure domain mutant, according to the following step, the different E mutant agarose gel of preparation, and fill to affinity column, investigate the change of its dynamic carrying capacity according to the IgG purifying procedure of standard.Result (table 4) shows, E(N23T, N28D, G29A) after 7 CIP (7hour), its dynamic carrying capacity is still maintained at 95%, is much better than the mutant of other E domains.
1, the preparation of E structure domain mutant agarose gel:
1) reacting in aqueous medium with agarose gel with epoxychloropropane, sodium hydroxide, react 2-3 hour at a temperature of 30-60 DEG C, reaction drains solvent with distilled water cleaning after terminating to neutrality
2) recombinant protein A of above-mentioned product Yu the present invention is reacted 15-20 hour at a temperature of 5-25 DEG C, clean after reaction alkane and drain solvent again, i.e. obtain recombinant protein A agarose gel
3) it is filled to 20mL(XK/16/20 by above-mentioned) 10cm Gao Zhuzhong, for subsequent experimental.
2, standardized IgG purification chromatography eluant program:
1) combine liquid and clean balance affinity chromatography chromatographic column
2) 0.2ml/min loading, sample is 10mg/ml people polyclone IgG, and applied sample amount is 20CV(column volume)
3) combining the unconjugated immunoglobulin of buffer solution for cleaning, consumption is 6CV
4) elution buffer 1.0ml/min carries out eluting, and consumption is 6 CV
5) combining buffer to be balanced chromatographic column, consumption is 6 CV
6) CIP alkali liquor is used to carry out incumbent firms, base for post matter and 0.5M
Between NaOH, time of contact is 30 minutes.
3, the buffer used:
In conjunction with buffer formulation: 20mMPBS adds 150mM
NaCl(0.92g NaH2PO4·2H2O, 5.04g
Na2HPO4·12H2O, 8.77g NaCl, SuperQ are water-soluble is adjusted to 7.0 to 1L, pH)；
Eluent formula: citric acid 3.8g, disodium citrate 0.82g, SuperQ are water-soluble is adjusted to 3.0 to 1L, pH.
CIP solution: 0.5M NaOH.
Table 4, the alkaline stability research of different structure territory E mutant
Experimental example
3
, development of new type alkali-resistant fibre affinity media (
i
Protein A
) performance study
(1) E(N23T, N28D, G29A) preparation of tetramer agarose gel (i Protein A)
By the method for chemosynthesis, design synthesis E(N23T, N28D, G29A) sequence of tetramer repeated fragment of gene, method as described in embodiment 1, express, the above-mentioned E(N23T of purification, N28D, G29A) tetramer albumen.
E mutant (N23T, N28D, G29A) tetrameric agarose gel is prepared according to the preparation method of above-described embodiment.
(2) i Protein A affinity media compares with the alkali resistance of MabselcetSure affinity media
For investigating E(N23T, N28D, G29A further) tetrameric alkali resistance, use MabselectSure prepacked column (5ml) and iProteinA post (XK165, loadings 5ml), carry out IgG purification as follows, measure the dynamic carrying capacity of chromatographic column through different CIP circulations.Result (table 5) shows, i Protein A affinity media, compared with commercially available alkali resistance affinity media (MabselctSure), has more preferable alkali stability.The results are shown in Table 5 and Fig. 2.
Standardized IgG purification chromatography chromatography eluant program:
1) buffer balance chromatography chromatographic column is combined
2) 0.2ml/min loading, sample is 10mg/ml people polyclone IgG, and applied sample amount is 20CV(column volume)
3) combining buffer solution for cleaning and be not associated with albumen, consumption is 6 CV
4) elution buffer 1.0ml/min carries out eluting, and consumption is 6 CV
5) combining buffer to be balanced chromatographic column, consumption is 6 CV
6) CIP alkali liquor is used to carry out incumbent firms, base for post matter and 0.5M
Between NaOH, time of contact is 60min.
Table 5, i Protein A affinity media compare with the alkali resistance of MabselctSure affinity media
(3) i
Protein A affinity media carrying capacity dynamic with MabselcetSure and purification effect compare
According in embodiment 4.1, utilize the IgG of the 40th CIP recycling elution to receive liquid and calculate the dynamic carrying capacity of iProtein A and MabselctSure.Meanwhile, use HPLC method (TSKgel G3000SW chromatographic column) to be analyzed IgG purity in two media eluent and aggressiveness content, calculate IgG monomer purity and aggressiveness content.Result (subordinate list 6) shows, compared with iProteinA with MabselctSure, has the most dynamically carrying capacity and purification effect.
In addition, patent CN10152278 (A) is recorded, domain C sequence wild type (Cwt) and disappearance " Asn-Lys-Phe-Asn " mutant (Cdel), only obtain the alkali resistance suitable with MabselectSure, and repeatedly its dynamic carrying capacity is less than MabselectSure after CIP.Therefore, i Protein A alkali resistance disclosed by the invention and purification effect are better than Cwt and Cdel equally.
Table 6, i Protein A affinity media compare with MabselectSure purification effect
SEQUENCE
LISTING
<110>
Shanghai Zhangjiang Biological Technology Co
<120>
A kind of immunoglobulin-binding proteins mutant and application thereof
<130>
20150506
<160>
2
<170>
PatentIn version 3.5
<210>
1
<211>
58
<212>
PRT
<213>
Artificial sequence
<400>
1
Asn Ala Ala
Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val
1
5
10
15
Leu Asn Met
Pro Asn Leu Thr Ala Asp Gln Arg Asp Ala Phe Ile Gln
20
25
30
Ser Leu Lys
Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala
35
40
45
Gln Lys Leu
Asn Asp Ser Gln Ala Pro Lys
50
55
<210>
2
<211>
174
<212>
DNA
<213>
Artificial sequence
<400>
2
aacgcggcgc
agcatgatga agcgcagcag aacgcgtttt atcaggtgct
gaacatgccg 60
aacctgaccg
cggatcagcg cgatgccttt attcagagcc tgaaagatga
tccgagccag 120
agcgcgaacg
tgctgggcga agcgcagaaa ctgaacgata gccaggcgcc
gaaa 174
Claims (7)
1. the associated proteins mutant of a binding domain-immunoglobulin molecule, it is characterised in that by natural grape pneumococcal proteins A(SpA) E domain in partial amino-acid suddenly change, described sudden change selected from N23T and/or N28D and/or G29A sudden change.
2. the associated proteins mutant of a kind of binding domain-immunoglobulin molecule described in claim 1, it is characterised in that described immunoglobulin molecules associated proteins mutant has aminoacid sequence shown in SEQ ID NO:1, has SEQ
Nucleotide sequence shown in ID NO:2.
3. a mutain tetramer, it is characterised in that be made up of as unit associated proteins molecule described in claim 1,2.
4. the albumen encoding any one in claim 1,2,3 or polymeric nucleic acid.
5. an expression vector, it is characterised in that express nucleic acid described in claim 4.
6. the application of the associated proteins mutant of a kind of binding domain-immunoglobulin molecule described in claim 1,2,3, it is characterized in that, associated proteins molecule described in claim 1,2,3 is coupled on solid phase carrier agarose gel, prepares a kind of chromatography media for affinity chromatograph.
7. the application of the associated proteins mutant of a kind of binding domain-immunoglobulin molecule described in claim 1,2,3,6, it is characterised in that combine immunoglobulin molecules in liquid, separates one or more immunoglobulin molecules in liquid.
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| CN106188251B CN106188251B (en) | 2020-07-31 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109721645A (en) * | 2017-12-29 | 2019-05-07 | 兆生生物科技南京有限公司 | A kind of albumin A of gene mutation and its application |
| CN114315996A (en) * | 2021-12-31 | 2022-04-12 | 博格隆(浙江)生物技术有限公司 | Preparation method of recombinant Protein A and affinity chromatography medium |
| CN114605508A (en) * | 2022-05-11 | 2022-06-10 | 北京达成生物科技有限公司 | Antibody binding proteins capable of binding to the Fc region of an antibody molecule and uses thereof |
| CN116217680A (en) * | 2023-01-10 | 2023-06-06 | 珠海冀百康生物科技有限公司 | Immunoglobulin binding proteins with high alkali stability and uses thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011005341A2 (en) * | 2009-04-03 | 2011-01-13 | University Of Chicago | Compositions and methods related to protein a (spa) variants |
| CN102516372A (en) * | 2002-03-25 | 2012-06-27 | 通用电气健康护理生物科学股份公司 | A mutated immunoglobulin-binding protein |
-
2015
- 2015-05-06 CN CN201510225194.XA patent/CN106188251B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516372A (en) * | 2002-03-25 | 2012-06-27 | 通用电气健康护理生物科学股份公司 | A mutated immunoglobulin-binding protein |
| WO2011005341A2 (en) * | 2009-04-03 | 2011-01-13 | University Of Chicago | Compositions and methods related to protein a (spa) variants |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109721645A (en) * | 2017-12-29 | 2019-05-07 | 兆生生物科技南京有限公司 | A kind of albumin A of gene mutation and its application |
| CN114315996A (en) * | 2021-12-31 | 2022-04-12 | 博格隆(浙江)生物技术有限公司 | Preparation method of recombinant Protein A and affinity chromatography medium |
| CN114605508A (en) * | 2022-05-11 | 2022-06-10 | 北京达成生物科技有限公司 | Antibody binding proteins capable of binding to the Fc region of an antibody molecule and uses thereof |
| CN114605508B (en) * | 2022-05-11 | 2022-07-29 | 北京达成生物科技有限公司 | Antibody binding proteins capable of binding to the Fc region of an antibody molecule and uses thereof |
| CN116217680A (en) * | 2023-01-10 | 2023-06-06 | 珠海冀百康生物科技有限公司 | Immunoglobulin binding proteins with high alkali stability and uses thereof |
| CN116217680B (en) * | 2023-01-10 | 2024-02-02 | 珠海冀百康生物科技有限公司 | Immunoglobulin binding proteins with high alkali stability and uses thereof |
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