KR20170098169A - Antibody binding affinity ligand with advanced alkali resistance - Google Patents
Antibody binding affinity ligand with advanced alkali resistance Download PDFInfo
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- KR20170098169A KR20170098169A KR1020170019861A KR20170019861A KR20170098169A KR 20170098169 A KR20170098169 A KR 20170098169A KR 1020170019861 A KR1020170019861 A KR 1020170019861A KR 20170019861 A KR20170019861 A KR 20170019861A KR 20170098169 A KR20170098169 A KR 20170098169A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- C07K1/16—Extraction; Separation; Purification by chromatography
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
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Abstract
The present invention relates to an antibody-affinity ligand for use in the purification of an antibody, wherein in the A, B, C or D domain of protein A, the amino acid first in front of the N-terminus of the 'FNK consecutive sequence' It was found that the amino acids present were vulnerable to the alkali, and it was found that substitution of these amino acids can acquire resistance to alkali without loss of IgG binding ability.
Description
The present invention relates to an antibody affinity ligand used for purification of an antibody, and more particularly, to an antibody-affinity ligand which is more suitable for a column process to be carried out in an alkali condition, To provide an economical antibody affinity ligand.
Most of the currently available antibody therapeutics are purified using an antibody-affinity ligand made of Protein A, the cell membrane protein of Staphylococcus aureus .
Protein A consists of five domains (E, D, A, B and C) and a C-terminal cell-wall-binding region (FEMS Immunol) with high affinity for a specific part of the antibody, Med Microbiol. 1998 Jan; 20 (1): 69-78; J Bacteriol. 1984 Aug; 159 (2): 713719).
In 1966, Forsgren, Sjoquist et al. Reported that the Fc region of human IgG subclasses 1 and 2 was replaced by Protein A (SEQ ID NO: 2). The amino acid sequence of protein A was found in 1984 by Uhlen et al. (The Journal of Biological Chemistry, 259, 1695-1702) (J Immunol 1966 97: 822-827). In 1986, Moks et al. Reported that the five domains of protein A are composed of 56 to 61 amino acids, each of which binds to the Fc region of IgG (Eur J Biochem 156 (3): 637-643).
On the other hand, the monoclonal antibody produced by animal cells, etc., is adsorbed on the protein A ligand bound to the chromatography resin and then separated under low pH conditions. Again, the impurities (nucleic acid, fat, Proteins, etc.) are removed, and the CIP process is performed to regenerate the column.
However, in such a repeated CIP process under alkaline conditions, all or a part of the antibody affinity ligand is separated from the resin, resulting in a fatal result in contamination of the product, or a problem that the ligand is denatured and the recovery rate of the desired antibody is lowered do. The productivity is lowered, and the resin having the expensive antibody affinity ligand must be replaced. In addition, there is a problem that protein A, which may be contained in the final product, must be detected using a separate protein A detection kit in order to detect the leakage of the ligand.
Therefore, it is necessary to have resistance to alkaline conditions of protein A, and various methods have been devised to overcome the vulnerability of alkaline conditions. One of the most popular products on the market today is GE Healthcare's MabSelect SuRe.
This product is based on the Z domain in which two amino acids of the B domain are substituted, some amino acids of which are substituted (FEMS Immunology and Medical Microbiology Volume 20,
However, even if the previously developed products are somewhat resistant to alkali, it is necessary to develop a ligand that maintains high antibody affinity with higher alkali resistance for cost reduction and productivity improvement.
The present invention aims to develop and provide a technique capable of further increasing alkali resistance in various ligands developed based on Protein A or five domains (E, D, A, B and C) therein.
The present invention relates to an Fc region binding ligand of an antibody in which one or more domains of the A, B, C and D domains of protein A are linked and formed, (F), asparagine (N), and lysine (K), in which the first amino acid existing in front of the N-terminal of the 'FNK consecutive sequence' Lt; RTI ID = 0.0 > Fc < / RTI > region binding ligand.
In the Fc region-binding ligand of the antibody of the present invention, the Fc region-binding ligand of the antibody has, for example, one of the A, B, C, and D domains, And the asparagine (N), which is an amino acid present in front, may be substituted with alanine (A).
In the Fc region binding ligand of the antibody of the present invention, the Fc region binding ligand of the antibody is preferably formed by connecting a plurality of A domains. At this time, it is more preferable that four or five A domains are connected.
The Fc region binding ligand of the antibody in which a plurality of A domains are linked in the Fc region binding ligand of the antibody of the present invention is further characterized in that asparagine (N) which is the 23rd amino acid from the N-terminal is substituted with another amino acid, It is preferable that glycine (G) which is the 29th amino acid from the N-terminal is replaced with another amino acid and asparagine (N) which is the 43th amino acid from the N-terminal is replaced with another amino acid. Asparagine (N), which is the 23rd amino acid from the N-terminal, is substituted with threonine (T), glycine (G), which is the 29th amino acid from the N-terminal, is substituted with alanine The asparagine (N), which is the 43rd amino acid, is substituted with glutamic acid (E).
In the Fc region-binding ligand of the antibody of the present invention, the Fc region binding ligand of the antibody preferably has the entire X domain or a part thereof bound to the C-terminal.
In the present invention, in the A, B, C or D domain of protein A, it is presumed that the position between the first amino acid existing before the N-terminal of the 'FNK consecutive sequence' And it is possible to obtain resistance to an alkali without loss of antibody (IgG) binding ability by substituting these first anterior amino acid or second anterior amino acid with another amino acid.
It has also been found that productivity can be greatly improved if the C domain of the A, B, C, or D domain is appended to the X domain or a subsequence thereof.
Fig. 1 shows the decomposition pattern and the N-terminal sequence analysis of the degraded band in accordance with the 0.8M NaOH treatment of Protein A. Fig.
FIG. 2 is a block diagram showing the structure of a protein A (denoted by 'PA' in FIG. 2), an A4 domain (denoted by 'A4' in FIG. 2), an A5 domain ') Was treated with 0.5 M NaOH for 5.1 hr, and SDS-PAGE was developed.
Figure 3 shows the expression levels of 'A5-X ligand' with X domains linked to the A4, A5 and A5 domains.
Fig. 4 shows the result of measurement of IgG binding ability with time after confirmation of alkaline decomposition pattern of A (N4K) 5 and A (N3A, N4K) 5 and alkali treatment.
FIG. 5 shows SDS PAGE photographs of the results of a comparative experiment (0.8M NaOH treatment for 1 hour) of the alkali resistance of three kinds of ligands and the control group PA (protein A).
The present invention relates to an Fc region binding ligand of an antibody in which one or more domains of the A, B, C and D domains of protein A are linked and formed, (F), asparagine (N), and lysine (K), in which the first amino acid existing in front of the N-terminal of the 'FNK consecutive sequence' Lt; RTI ID = 0.0 > Fc < / RTI > region binding ligand. At this time, the Fc region-binding ligand of the antibody may comprise, for example, asparagine (N), which is an amino acid secondarily to the N-terminal of the FNK consecutive sequence in any one of the domains A, B, C and D May be substituted with alanine (A).
In the present invention, through the experiment of the present invention as described below, the amino acid existing first in the N-terminal side of the 'FNK consecutive sequence' existing on the A, B, C and D domains of protein A and the amino acid It was confirmed that the resistance to alkaline can be increased by substituting the amino acid existing in the first or the amino acid existing in the second before with the other amino acid in the wild type amino acid.
On the other hand, in the Fc region binding ligand of the antibody of the present invention, the Fc region binding ligand of the antibody may be formed by connecting a plurality of A domains, for example. At this time, the Fc region binding ligand of the antibody is preferably formed by connecting four or five A domains.
Many of the existing Fc region binding ligands have been developed based on the B-domain and have not been developed based on the A-domain. However, in the present invention, it has been confirmed that Fc region binding ligands having increased resistance to alkaline can be produced without loss of antibody binding ability even on the basis of A-domain.
Meanwhile, in the Fc region binding ligand of the antibody of the present invention, the Fc region binding ligand of the antibody in which one or more of the A-domains are linked is further comprised of asparagine (N ) Is further substituted with another amino acid, glycine (G), which is the 29th amino acid from the N-terminus, is substituted with another amino acid, and asparagine (N), which is the 43th amino acid from the N-terminus, is substituted with another amino acid. In the Fc region binding ligand of the antibody of the present invention, asparagine (N), which is the 23rd amino acid from the N-terminal, is substituted with threonine, and glycine (G) which is the 29th amino acid from the N- Alanine, and asparagine (N), which is the 43rd amino acid from the N-terminus, may be replaced with glutamic acid.
It has been reported in the prior art that when the 23rd amino acid, the 29th amino acid and the 43rd amino acid of the N-terminal are substituted with other amino acids, the resistance to the alkali can be increased. However, it has been confirmed that when the present invention is further applied to the present invention, it is possible to produce a ligand having increased resistance to an alkali while increasing binding ability to an antibody.
On the other hand, in the Fc region binding ligand of the antibody of the present invention, the Fc region binding ligand of the antibody may preferably have the entire X domain or a part thereof bound to the C-terminal. When the entire X domain or a part thereof is connected to the C-terminal of the A, B, C, and D domains, the productivity of the corresponding domain is improved.
Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.
[ Experimental Example 1: by alkali Protein Confirmation of decomposition pattern of A]
Experiments were conducted on the alkali resistance of Protein A (manufacturer: Repligen, catalog No.: 10-2001-1M).
Protein A (PA) was reacted with 0.8 M NaOH for 60 minutes and developed by SDS-PAGE. D, A, B and C domains of protein A were analyzed by SDS-PAGE, and N-terminal amino acids of the degraded bands (1, 2, .
Figure 1 shows the N-terminal sequence analysis results of the 0.8 M NaOH degradation pattern of protein A and the degraded band. As shown in FIG. 1, the N-terminal amino acid sequence of the N-terminal region in the 1, 2, and 3 bands on the SDS-PAGE was the sequence of the FNK .
This result was compared with the domain sequence of A (SEQ ID NO: 1), B (SEQ ID NO: 2), C (SEQ ID NO: 3), D (SEQ ID NO: 4) and E A specific sequence ' FNK ', common at the N-terminal side of the domain, the C domain and the D domain was identified.
Therefore, the inventors infer that the presence of this particular continuous sequence 'FNK' may be related to the sensitivity to the alkali, and infer that the alkali resistance can be increased through modification of the N-terminal anterior amino acid of this continuous sequence The following experiments were carried out.
[ Experimental Example 2: by alkali Protein A domain decomposition pattern of A]
In this experiment, we tried to confirm the alkali decomposition pattern of 'A domain' of protein A based on the above experimental results. For this purpose, a ligand consisting of four repeating sequences of the A domain and a repeating sequence of five repeating sequences of the A domain were prepared, respectively, and their alkali resistance was tested. As a comparative experimental group, a ligand composed of 4 Z domains prepared by the patent EP 2,202,310 A2 was prepared and used.
The A-domain ligand thus prepared was examined for the alkali degradation pattern in the same manner as in Experimental Example 1, and the binding activity of IgG was also measured.
As a result, it was confirmed that the degree of decomposition and decomposition pattern of Protein A were similar in case of A4 domain and A5 domain. FIG. 2 is a block diagram showing the structure of a protein A (denoted by 'PA' in FIG. 2), an A4 domain (denoted by 'A4' in FIG. 2), an A5 domain ') Was treated with 0.5 M NaOH for 5.1 hr, and SDS-PAGE was developed. In the case of protein A, A4, and A5 domains, decomposition bands were observed after 5.1 hr treatment. However, it was confirmed that the decomposition degree of Z4 domain was low.
On the other hand, when treated at a concentration of 0.5M NaOH for various times, denaturation or the like occurred in a high concentration alkaline state, and thus the antibody adsorbing ability as a final role could be lost. Further, IgG binding ability was further measured to confirm whether the binding ability was changed.
As a result of the experiment, as shown in Table 1 below, the IgG binding ability of the protein A, A4, and Z4 domains was similar in the comparatively low alkaline conditions (0.1M NaOH, 0.3M NaOH). However, the adsorption capacity of the protein A, A4, and A5 was lowered in the 0.5M NaOH / 24hr treatment condition, while the Z4 domain showed relatively high antibody Respectively.
0.1 M NaOH
0.5 M NaOH
[ Experimental Example 3: Improvement of purification yield of A4, A5 domain]
5 < / RTI > batch cultures were performed to produce the A4 domain and the A5 domain. As a result, overall expression rate and purification yield were lower than that of protein A. Accordingly, the present inventors deduce that the X domain in protein A has a certain influence on the expression rate, and the X domain (SEQ ID NO: 6) is added to the C-terminus of the A5 domain.
The ligand of the A5-X domain was constructed, fermented and purified to confirm the degree of alkali resistance by SDS-PAGE. Figure 3 shows the expression levels of A4, A5 and A5-X ligands.
As shown in FIG. 3, by attaching the X domain, the expression level was improved and the purification yield was also improved.
[ Example 1: Experiment to increase the resistance of the A domain to alkali]
In the case of the A domain, the B domain has the sequence of NNFNKE at the N-terminal NKFNKE position of the C domain. When the asparagine (N), which is the fourth amino acid of the A-domain, is replaced by lysine (K), the amino acid sequence is the same as the B domain and the C domain.
Once the A (N4K) 5 domain (-> NKFNKE) was constructed, it was purified and tested for alkali resistance. As expected, treatment with 0.5 M NaOH for at least 10.2 hrs resulted in complete degradation. This shows that between the first amino acid (K in this experiment) and the second amino acid (N in this experiment) toward the N-terminus of the 'FNK consecutive sequence' Location.
Therefore, in the present embodiment, the amino acid existing second to the N-terminal side of the 'FNK continuous sequence' and the first amino acid existing in front of the 'FNK continuous sequence' are replaced with alanine (A) and lysine (K) A (N3A, N4K) 5 ligand.
The ligand thus produced was examined for the alkali degradation pattern in the same manner as in Experimental Example 1, and IgG binding activity (binding activity) was also measured.
As a result of the experiment, it was confirmed that the alkali resistance was remarkably increased, and the IgG binding ability was also remarkably decreased as compared with the control group (FIG. 4). Fig. 4 shows the result of measurement of IgG binding ability with time after confirmation of alkaline decomposition pattern of A (N4K) 5 and A (N3A, N4K) 5 and alkali treatment.
From the above experiment, it was found that any one or more of the amino acid existing second to the N-terminal side of the 'FNK consecutive sequence' on the A, B, C, We could be confident that we could increase the resistance to alkaline without loss of IgG binding ability.
[ Example 2: Experiment to further increase the resistance of the A domain to alkali]
In various documents, it has been reported that when N23, G29, and N43, which are alkali fragile portions of the D domain, the B domain, and the C domain, are substituted with other amino acids, the resistance to alkali can be increased.
In the present invention, a ligand is newly designed to increase the alkali resistance to the ligand constructed in Example 1 by utilizing the results of the preceding studies.
N3A, N4K, N23T, G29A, and N29E were added to the A (N3A, N4K) 5-X ligand (A''5-X) constructed in Example 1 by further adding N23T, G29A, N43E) 5-X ligand (A '"' '5-X). At this time, a substituted ligand (A '' '5-X) having only three portions of N23T, G29A and N43E except for N3A and N4K was separately constructed and used as a comparative group to confirm the importance of alkali resistance of N3 and N4 .
The experimental results are shown in Fig. FIG. 5 shows SDS PAGE photographs of the results of a comparative experiment (0.8M NaOH treatment for 1 hour) of the three kinds of ligands prepared above and the control group of PA (protein A). As shown in FIG. 5, it was found that the degree of decomposition of A''5-X, A'''5-X, and A'''''5-X samples was lower than that of PA, which is a control group, (The lower the resolution, the darker the band below the thick band). In addition, A''5-X showed lower degree of decomposition than A'''5-X, showed higher alkali resistance, showed the lowest degree of degradation of A''''5-X, showed alkali resistance Was the highest.
On the other hand, the antibody binding ability to the ligands used above was measured, and the results were as shown in Table 2 below. As the control group, PA (protein A), A5-X (the X domain was bound to the five A domains), A'5-X ligand (the fourth amino acid N was replaced with K (N4K) A domain And the X domain was combined with the five combined). The experimental group consisted of 5 A-5 ligands (the third amino acid, N, replaced by A (N3A), and the fourth amino acid, N, replaced by K (N4K) 5 'X ligand (N3A, G29A and N43E), A' '' '5-X ligand (N3A, N4K, N23T, G29A, and N43E were combined, and the X domains were combined with five A domains).
0.5M
As shown in Table 2, when 0.5 M NaOH was treated with various samples (ligands), A''5-X, A'''5-X and A'''''5-X samples were treated with a control A5-X and A'5-X samples, respectively, over time.
From the above-mentioned experimental results, it can be seen that substitution of previously known amino acid moieties can show considerably high alkali resistance and antibody affinity. However, as in the present invention, the amino acid residues in the N- It was concluded that substitution of the amino acid present in front of the first one resulted in higher alkali resistance and IgG binding ability.
<110> AP TECHNOLOGY CO., LTD. <120> Antibody binding affinity ligand with advanced alkali resistance <130> AP-2015-0206 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 1 Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 50 55 <210> 2 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 2 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 3 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 3 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 4 <211> 61 <212> PRT <213> Staphylococcus aureus <400> 4 Ala Asp Ala Gln Gln Asn Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe 1 5 10 15 Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly 20 25 30 Phe Ile Gln Ser Leu Lys Asp Asp Ser Ser Gln Ser Thr Asn Val Leu 35 40 45 Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 50 55 60 <210> 5 <211> 56 <212> PRT <213> Staphylococcus aureus <400> 5 Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30 Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35 40 45 Leu Asn Asp Ser Gln Ala Pro Lys 50 55 <210> 6 <211> 13 <212> PRT <213> Staphylococcus aureus <400> 6 Glu Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Ser Ala 1 5 10
Claims (7)
(F), asparagine (N), and lysine (K), which are present on any one of domains A, B, C and D, The Fc region binding ligand of the antibody is characterized in that the amino acid present or the second preceding amino acid is replaced by another amino acid.
The Fc region binding ligand of the antibody may comprise,
Wherein the asparagine (N), which is the amino acid secondarily to the N-terminal side of the FNK consecutive sequence, is substituted with alanine (A) in any one of the A, B, C and D domains Fc region binding ligand.
The Fc region binding ligand of the antibody may comprise,
A domains are linked to each other to form an Fc region binding ligand of the antibody.
The Fc region binding ligand of the antibody may comprise,
Wherein the Fc region binding ligand of the antibody is formed by connecting four or five A domains.
The Fc region binding ligand of the antibody may further comprise,
Asparagine (N), which is the 23rd amino acid from the N-terminal, is substituted with another amino acid,
Glycine (G), which is the 29th amino acid from the N-terminal, is substituted with another amino acid,
Wherein the 43rd amino acid asparagine (N) from the N-terminus is replaced by another amino acid.
Asparagine (N), which is the 23rd amino acid from the N-terminal, is substituted with threonine (T)
Glycine (G), which is the 29th amino acid from the N-terminal, is substituted with alanine (A)
Wherein the asparagine (N) from the N-terminal is substituted with glutamic acid (E).
The Fc region binding ligand of the antibody,
The Fc region binding ligand of the antibody is characterized in that the C-terminus is entirely or partly bound to the X domain.
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| WO2008127457A2 (en) * | 2006-12-06 | 2008-10-23 | Repligen Corporation | Nucleic acids encoding recombinant protein a |
| KR101067181B1 (en) * | 2002-03-25 | 2011-09-22 | 지이 헬스케어 바이오-사이언시스 에이비 | A Mutated Immunoglobulin-Binding Protein |
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| US9051375B2 (en) * | 2010-12-21 | 2015-06-09 | The University Of Western Ontario | Alkali-resistant variants of protein A and their use in affinity chromatography |
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| KR101067181B1 (en) * | 2002-03-25 | 2011-09-22 | 지이 헬스케어 바이오-사이언시스 에이비 | A Mutated Immunoglobulin-Binding Protein |
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| WO2008127457A2 (en) * | 2006-12-06 | 2008-10-23 | Repligen Corporation | Nucleic acids encoding recombinant protein a |
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Non-Patent Citations (5)
| Title |
|---|
| EP 2,202,310 A2에는,"Caustic stable chromatography ligands"를 제목으로 하는 알칼리 저항성을 갖는 새로운 프로테인 A가 기재되어 있다. |
| PCT/CA2011/001370에는, "Novel alkali-resistant variants of protein a and their use in affinity chromatogram"을 제목으로 하는 알칼리 저항성을 갖는 새로운 프로테인 A가 기재되어 있다. |
| Protein Engineering.,1(2):107-113(1987.)* * |
| 일본특허 공개번호 2006-304633에는,"Immunoglobulin binding protein"을 제목으로 하는 알칼리 저항성을 갖는 새로운 프로테인 A가 기재되어 있다. |
| 일본특허 공개번호 2006-320220에는,"DNA SEQUENCE USED FOR PRODUCTION OFFUSION POLYPEPTIDE, RECOMBINANT EXPRESSION VECTOR, TRANSFORMANT, AND METHOD FOR PRODUCING THE FUSION POLYPEPTIDE"를 제목으로 하는 알칼리 저항성을 갖는 새로운 프로테인 A가 기재되어 있다. |
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