KR101247374B1 - Novel peptides with specific binding to Fc domain of IgG and their preparation methods - Google Patents

Abstract

The present invention relates to a novel peptide that selectively binds to the Fc region of immunoglobulin G (Igunoglobulin G; IgG) and a method for preparing the same, and more particularly, to an 11 amino acid sequence that selectively binds to the Fc region of immunoglobulin G. And a method for screening the small molecule peptides using the constructed small molecule peptides, and the Fc region of immunoglobulin G. The novel peptides of the present invention can be usefully used in antibody fixation, antibody purification, immunobiosensor fabrication and drug delivery system (DDS) as fusion proteins or chemical derivatives.

Immunoglobulin G (IgG), Fc, Peptides, Molecular Evolution

Description

Novel peptides with specific binding to Fc domain of IgG and their preparation methods for selectively binding to Fc regions of immunoglobulin VII

The present invention relates to a novel peptide that selectively binds to the Fc region of immunoglobulin G (Igunoglobulin G; IgG) and a method for preparing the same, and more particularly, to an 11 amino acid sequence that selectively binds to the Fc region of immunoglobulin G. Configured small molecule peptides, and methods for screening the small molecule peptides.

Various solid materials (adsorbents, nanoparticles, microparticles, glass, and others) for applications using immune responses, such as immunosensors using antigen-antibody reactions, targeted drug delivery systems, protein chips, diagnostic kits, etc. Immobilizing antibodies on the surface of gold, etc.) or other biomolecules (peptides, proteins, nucleic acids and lipids, etc.) is an indispensable process. Therefore, the technique of fixing the antibody with high reproducibility while maintaining its inherent binding characteristics by adjusting the orientation of the antibody so that the antigen-binding site of the antibody is well exposed to the surface is directly related to the detection sensitivity of the chip or sensor. In addition to being linked, they are indispensable for the bioapplication of drug carriers.

To date, the method of immobilizing the antibody on the surface of the solid substrate mainly depends on the covalent formation reaction lacking physical adsorption or selectivity. However, immobilization of the antibody in this manner results in a decrease in the inherent activity of the antibody due to steric hindrance or random orientation. As an alternative, antibody-binding proteins derived from microorganisms such as Protein A and Protein G may be used. The proteins facilitate binding of the antigen by strongly binding to the Fc region of the antibody that does not participate in antigen-antibody reactions. In addition, the binding of the proteins with the antibody does not require a separate chemical modification has the advantage that does not harm the inherent function of the antibody. Therefore, recent studies have focused on overcoming this problem by modifying antibody-binding proteins by genetic engineering and chemical methods. However, these proteins have disadvantages such as high cost, low stability, contaminant incorporation during purification, reduced antibody activity and lack of selectivity. As a method of overcoming the above problems, research on low molecular weight materials has been made. Until now, methods using dendrimer, iron ions, or calixcrown derivatives have been developed, but these methods show similar binding to all kinds of proteins without orientation control and selectivity to antibodies and are not strong. Has its drawbacks.

Accordingly, the present inventors have been developing a new type of antibody-fixing low-molecular peptide that can overcome the disadvantages of existing antibody-binding proteins or antibody-fixing low-molecular substances, while selectively selecting the Fc region of immunoglobulin G (Immunoglobulin G; IgG). The present invention was completed by identifying novel peptides that strongly bind to each other.

An object of the present invention is a polypeptide that specifically binds to an Fc fragment of immunoglobulin G (Igunoglobulin G; IgG), a method for preparing the polypeptide, and a method for purifying and immobilizing IgG using the polypeptide. To provide.

In order to achieve the above object, the present invention is an amino acid sequence of any one of SEQ ID NOs: 1 to 10, or some amino acid is inserted, deficient or substituted in the amino acid sequence, Fc fragment of immunoglobulin G (Immunoglobulin G; IgG) Provided is a polypeptide that specifically binds to an Fc fragment of an IgG comprising an amino acid sequence whose binding to fragments is maintained.

The present invention also provides a gene comprising any one of SEQ ID NOs: 11 to 20 encoding the polypeptide.

The present invention also provides an expression vector comprising the gene.

The present invention also provides a transformant in which the expression vector is introduced into a host cell.

In addition,

1) culturing the transformant in medium; And

2) provides a method for producing a polypeptide that specifically binds to the Fc region of IgG comprising the step of separating and purifying the polypeptide from the culture.

In addition,

1) preparing a DNA library;

2) preparing a fusion gene capable of expressing the fusion protein by fusing the fluorescent protein gene to the gene of the DNA library of step 1);

3) preparing an expression vector into which the fusion gene of step 2) is inserted;

4) preparing a transformant by introducing the expression vector of step 3) into a host cell and culturing the transformant;

5) treating and washing the culture medium or the lysate of the transformant of step 4) on a solid substrate coated with a polypeptide comprising an Fc fragment of IgG;

6) measuring the fluorescence of the solid substrate of step 5); And

7) It provides a polypeptide selection method that specifically binds to the Fc region of IgG comprising the step of selecting a transformant is detected the fluorescence signal measured.

In addition,

1) activating the carboxyl group of the solid substrate comprising a carboxyl group;

2) a surface treatment step of attaching the polypeptide of claim 1 to the solid substrate of step 1); And

3) It provides a method for producing an IgG monomolecular film consisting of binding IgG to the surface-treated solid substrate of step 2).

In addition,

1) treating the composition containing the antibody to a column to which the polypeptide is adsorbed; And

2) provides a method for purifying an IgG antibody comprising eluting the antibody attached to the column of step 1).

In addition,

1) surface-treating the polypeptide to adhere to the solid substrate; And

2) it provides a method for immobilizing IgG comprising the step of treating IgG to the solid substrate of step 1).

In addition, the present invention provides an immunosensor in which IgG is immobilized through the polypeptide on a solid substrate surface-treated with the polypeptide.

The novel peptides of the present invention can be immobilized by immobilizing IgG on various kinds of solid phase surfaces, and can also be immobilized with different binding affinity according to the origin or subtype of IgG. The surface treatment technology using may be used in the manufacture of various immune sensor / immune chip and the adsorption column material for antibody purification and the targeted DDS transporter for drug delivery and biological medicine (protein, peptide, nucleic acid, etc.).

Hereinafter, the present invention will be described in detail.

In the present invention, the amino acid sequence of any one of SEQ ID NOs: 1 to 10, or some amino acids are inserted, deficient or substituted in the amino acid sequence, and the binding ability of the immunoglobulin G (Immunoglobulin G; IgG) to the Fc fragment is reduced. Provided is a polypeptide that specifically binds to an Fc fragment of an IgG comprising an amino acid sequence to be maintained.

Preferably, the polypeptide is prepared by a manufacturing method including the following steps.

1) preparing a DNA library;

2) fusing a fluorescent protein gene to the carboxy terminus of the DNA base sequence of the prepared DNA library, thereby preparing a fusion gene expressing the fluorescent protein without a stop sequence in the library;

3) inserting the fusion gene into an expression vector using a restriction enzyme and transforming the host cell;

4) culturing the transformant to form colonies, and then selecting and culturing only double fluorescent colonies to secure cell pellets in which the fusion protein is expressed;

5) preparing E. coli cell lysate using the cell pellet lysozyme digestion method; And

6) Screening peptides by dot blotting assay with a library containing the fusion protein constructed from the cell lysate.

In the method, the DNA library of step 1) is phage display method, E. coli surface display) or by using PCR and cloning (Kawasaki M. et al., Biochemical and Biophysical Research Communications, Volume 280, Number 3, 26 January 2001, pp. 842-844 (3)). It is preferable to use one, and a method using PCR and cloning is more preferable, but is not limited thereto.

In the method, the fluorescent protein of step 2) is preferably EGFP (enhanced green fluorescent protein), but is not limited thereto. The inventors fused the fluorescent protein (EGFP) gene to the carboxy terminus of the DNA nucleotide sequence of the DNA library prepared above, thereby producing a fluorescent protein by expressing the fluorescent protein without the stop sequence in the library.

In the above method, the expression vector of step 3) is always an expression vector, more preferably a pHCE IIB vector, but is not limited thereto. The host cell may be a prokaryote such as E. coli or Bacillus subtilis , and may also be derived from yeast, insect cells, plant cells, animal cells, such as Saccharomyces cerevisiae . It may be one eukaryotic cell. The expression vector introduction method into the host cell may be any method known to those skilled in the art.

In the above method, the screening of step 6) is preferably a dot blotting assay method, but is not limited thereto.

The present inventors analyzed using a dot blot, and the fluorescence rate of the whole colonies was about 50 to 60%, and the final 10 fusion proteins were selected by screening 2000 fluorescent colonies (see FIG. 2). Gene sequences of the colonies were analyzed (see Table 1) to obtain nucleotide sequences of SEQ ID NOs: 11 to 20, and the amino acid sequences of SEQ ID NOs: 1 to 10 were identified through the nucleotide sequences (see Table 2).

In addition, the inventors measured by using a dot blot analysis method to determine the binding capacity of the obtained peptide to IgG. Specifically, the FcBP-EGFP candidate peptide was applied to the surface of the nitrocellulose membrane treated with human, rabbit, rat, mouse and goat immunoglobulin G, followed by washing and remaining. The binding force and the binding site were measured by measuring the fluorescence intensity. As a result, the peptide comprising the amino acid sequence of SEQ ID NOS: 1 to 10 of the present invention showed a high binding capacity to IgG (see Fig. 3).

The present invention also provides a gene comprising any one of SEQ ID NOs: 11 to 20 encoding the polypeptide.

The present invention also provides an expression vector comprising the gene.

The expression vector is always preferably an expression vector, but is not limited thereto.

In addition, the present invention provides a transformant in which the expression vector is introduced into a host cell.

The host cell may be a prokaryote such as E. coli or Bacillus subtilis . It may also be eukaryotic cells derived from yeast, insect cells, plant cells, animal cells such as Saccharomyces cerevisiae . The expression vector introduction method into the host cell may be any method known to those skilled in the art.

In addition,

1) culturing a transformant incorporating a vector containing a vector comprising a gene comprising a nucleotide sequence of any one of SEQ ID NOs: 11 to 20 encoding the polypeptide in a medium; And

2) provides a method for producing a polypeptide that specifically binds to the Fc region of IgG comprising the step of separating and purifying the polypeptide from the culture.

As the culture medium, it is preferable to select a medium suitable for the transformant from the culture medium known to those skilled in the art. The purification can use any purification method known to those skilled in the art.

The host cell may be a prokaryote such as E. coli or Bacillus subtilis . In addition, Saccharomyces cerevisiae ) and eukaryotic cells derived from yeast, insect cells, plant cells, animal cells. The expression vector introduction method into the host cell may be any method known to those skilled in the art.

In addition,

1) preparing a DNA library;

2) preparing a fusion gene capable of expressing the fusion protein by fusing the fluorescent protein gene to the gene of the DNA library of step 1);

3) preparing an expression vector into which the fusion gene of step 2) is inserted;

4) preparing a transformant by introducing the expression vector of step 3) into a host cell and culturing the transformant;

5) treating and washing the culture medium or the lysate of the transformant of step 4) on a solid substrate coated with a polypeptide comprising an Fc fragment of IgG;

6) measuring the fluorescence of the solid substrate of step 5); And

7) It provides a polypeptide selection method that specifically binds to the Fc region of IgG comprising the step of selecting a transformant is detected the fluorescence signal measured.

In addition,

1) treating the composition containing the antibody to a column to which the polypeptide is adsorbed; And

2) provides a method for purifying an IgG antibody comprising eluting the antibody attached to the column of step 1).

In addition,

1) activating the carboxyl group of the solid substrate comprising a carboxyl group;

2) a surface treatment step of attaching the polypeptide of claim 1 to the solid substrate of step 1); And

3) It provides a method for producing an IgG monomolecular film consisting of binding IgG to the surface-treated solid substrate of step 2).

In the above method, the solid substrate of step 1) is biodegradable organic polymer nanoparticles such as CM-5 Au sensor chip, magnetic microparticles, glass plate, gold nanoparticles, PLGA or various (micro) well plate [(micro) well plate It may be any one selected from the group consisting of]. The solid substrate is characterized in that it has a carboxyl group, when the carboxyl group is activated it can be fixed by reaction with the terminal amine group of the peptide of the present invention.

In the above method, the IgG of step 3) is preferably human, rabbit, rat or goat derived IgG, but is not limited thereto.

By the surface treatment of the present invention, the solid substrate may bind proteins to adjust the orientation, and increase the uniformity of the solid substrate surface. The method for preparing a monomolecular film using the IgG-fixing peptide of the present invention is physically and chemically stable compared to the method using an antibody-binding protein (protein A, protein G, protein A / G or protein L), and various chemical formulas It is possible to use a very wide range of uses, and also characterized by showing a high selectivity and binding capacity for IgG compared to the method using a conventional low molecular weight compound.

In addition,

1) surface-treating the polypeptide to adhere to the solid substrate; And

2) it provides a method for immobilizing IgG comprising the step of treating IgG to the solid substrate of step 1).

In addition, the present invention provides an immunosensor in which IgG is immobilized through the polypeptide on a solid substrate surface-treated with the polypeptide.

The immunosensor can be obtained by surface treatment with a peptide specifically binding to the Fc region of IgG on a carboxyl group-activated solid substrate and immobilizing the IgG to be detected.

Detection of the antigen-antibody reaction using the immunosensor may vary depending on the type of solid substrate. That is, when the solid substrate is a CM-5 Au sensor chip, the antigen-antibody binding degree can be measured by surface plasmon resonance while injecting the antigen into the immune sensor at a constant rate. In addition, in the case where the solid substrate is magnetic microparticles, the magnetic microparticles themselves may be boiled in a buffer solution and then measured by PAGE method. In addition, when the solid substrate is a glass plate can be measured by measuring the fluorescence by treating the fluorescent labeled IgG to the peptide.

Hereinafter, the present invention will be described in more detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example １> FcBP - EGFP  Positive control ( Positive Control ) Construction of Expression Vector

The inventors of the present invention provide an antibody-binding peptide (CysAlaTrpHisLeuGlyGluLeuValTrpCys) at the 5 'end of a nucleotide sequence of a fluorescent gene in order to fuse an antibody-binding peptide (FcBP) and an enhanced green fluorescent protein (EGFP) (SEQ ID NO: A fusion vector was constructed by inserting DNA containing the information of 21). The base sequence corresponding to the antibody binding peptide was inserted when designing the primer. The primers are as follows: Forward primer sequence: 5'-GAA TTC CAT ATG GAC TGT GCA TGG CAC CTG GGA GAA CTG GTC TGG TGC ACC GTG AGC AAG GGC GAG GAG CTG TTC-3 '(NdeI) (SEQ ID NO: 22), and reverse primer sequences: 5′-CCG CTC GAG CTT GTA CAG CTC GTC CAT GCC GAG CTC GAG-3 ′ (Xho I) (SEQ ID NO: 23).

Using the EGFP gene as a template, 1 μl of DNA, 20 μΜ forward primer and 1 μl of reverse gene, 2.5 μl of polymerase activity buffer, 2 μl of dNTP mixture, 1 μl of Taq polymerase, and 16.5 μl of sterile distilled water were added. After mixing to μl, PCR reaction was performed under the following conditions: initial denaturation at 95 ° C. for 5 minutes, denaturation at 95 ° C. for 30 seconds, annealing 55 ° C. for 1 minute, elongation at 72 ° C. for 2 minutes, 25 repetitions, and final elongation 72 10 min, reaction termination and storage (4 ° C).

The PCR reaction product was identified by DNA fragmentation of about 800 bp through 1% agarose gel electrophoresis. The final PCR reaction product and the pET 28a vector (Novagen, Cat. No. 69864-3) were used at 37 ° C. with restriction enzymes Nde I (TaKaRa, Cat. No. 1161A) and Xho I (TaKaRa, Cat. No. 1094A). After the reaction for 2 hours, the plasmid DNA was prepared by ligation using a DNA Ligation kit (TaKaRa, Cat. No. 6022). The prepared plasmid DNA was confirmed to be a fusion vector (28a-FcBP-EGFP) containing an antibody-binding peptide (FcBP) and a fluorescent gene (EGFP) by amplifying in E. coli. The fusion protein expressed from the produced fusion vector was used as a control of this experiment.

< Example  2> Peptides  For creating libraries DNA  library

To prepare the peptide library, the inventors first produced a single-stranded synthetic DNA library (Bioneer, Korea) containing 27 random DNA sequences (A, T, G, C) capable of generating 9 peptide libraries. Kawasaki M. et al., Biochemical and Biophysical Research Communications, Volume 280, Number 3, 26 January 2001, pp. 842-844 (3). DNA library prepared above (base sequence: 5′-GCC GCA GCC ATA TGA AAG ATG AAT GTN NNN NNN NNN NNN NNN NNN NNN NNN NNT GCG AAT TCG AGC TCC GTC GA-3 ′; N: A, T, G or C (SEQ ID NO .: 24) as a template, and a forward primer [base sequence; 5'-GC CGC AGC CAT ATG AAA GAT GAA TGT-3 '( Nde I) (SEQ ID NO: 25)] and reverse primer [SEQ ID NO: 5'-CTC GCC CTT GCT CAC TCG ACG GAG CTC GAA-3' ( Megaprimer 1 was prepared by performing a PCR reaction (SEQ ID NO: 26)] (PCR conditions: initial denatured 95 ° C. 5 minutes, denatured 95 ° C. 30 seconds, annealing 55 ° C. 1 minute, elongation 72 ° C. 1 minute, 25 Repeat, final elongation 72 ° C. 10 min, reaction termination and storage 4 ° C.).

The PCR reaction product was electrophoresed on a 1% agarose gel, and a DNA fragment of about 800 bp was identified. Manufactured Mega primer 1 and the reverse primer (base sequence: a 27) for each mold the first ㎕, EGFP gene: 5'-ATT CGC GGA TCC CTT GTA CAG CTC GTC CAT GCC GAG-3 '(HⅠ Bam) (SEQ ID NO: 1 μl of DNA, 2.5 μl of polymerase activity buffer, 2 μl of dNTP mixture, 1 μl of Taq polymerase, and 16.5 μl of sterile distilled water were mixed to make the final 25 μl, followed by PCR reaction under the following conditions (PCR). Conditions: initial denaturation 95 ° C. 5 min, denaturation 95 ° C. 30 sec, annealing 55 ° C. 1 min, elongation 72 ° C. 2 min, 25 repetitions, final elongation 72 ° C. 10 min, reaction termination and storage 4 ° C.) Has a Nde I restriction enzyme site, a library sequence capable of encoding nine peptides, a sequence encoding an EGFP gene, and a Bam HI restriction enzyme site in order from the 5′-end.

The final PCR reaction product was treated with Nde I and Bam HI (TaKaRa, Cat. No. 1010A) restriction enzymes (37 ° C., 2 hours), and DNA was purified by agarose gel electrophoresis. Alkaline Phosphatase, Calf Intestinal, NEB, Cat.No.M0290S) was treated with pHCE IIB vector (TaKaRa, Cat.No. BL009) treated with the same restriction enzyme to selectively remove and purify the phosphate group at the end of DNA. Used. Each restriction gene treated with the restriction enzyme was inserted into the pHCE IIB expression vector by ligation by T4 DNA Ligase, thereby completing the preparation of a plasmid DNA library for peptide library production. The prepared plasmid library was directly transformed into E. coli Rosetta cells (Novagen) and then proceeded.

< Example  3> using E. coli Peptides  Library expression

The present inventors spread the E. coli transformed in Example 2 onto an LB plate (Luria-Bertani plate) containing an ampicillin antibiotic and incubated at 37 ° C. for 18 hours to colonize colonies. Formed (FIG. 2). Among the colonies, only the fluorescent colonies were selected and inoculated in 4 mL of LB medium containing ampicillin and incubated at 25 ° C. for 20 to 24 hours to express the FcBP candidate peptide-EGFP fusion protein. Cell pellets recovered by centrifugation (16600 g , 4 ° C., 5 minutes) were treated with 450 μl lysis buffer (10 mM BME, 4.5 mM PMSF, 1 mM EDTA, 0.5% Tween-). 20, 10% glycerol). Then, lysozyme was added to a final concentration of 1 mg / ml and the cells were lysed by slow stirring at room temperature for 2 hours. The cell lysate was centrifuged (16600 g , 4 ° C., 30 minutes), and then the supernatant was taken and all the library members were diluted to adjust the concentration to a similar fluorescence intensity and used for dot blotting assay.

< Example  4> Dot blot analysis  Used FcBP - EGFP Fusion protein  Library Screening

Dot blotting assay was performed on a nitrocellulose membrane (BioTrace NT membrane, Pall, Cat. No. 66485) using an Easy-Titer ELIFA System (Pierce pierce, Cat. No. 77000). . The system is used between a 96-well plate and a vacuum chamber to insert five types of immunoglobulin G (Ig, human, rat, rabbit, mouse and goat) through a well. G) [human IgG (Sigma, Cat. No. I8640), mouse IgG (Sigma, Cat. No. I4131), rabbit IgG (Sigma, Cat. No. I5006), mouse IgG (Sigma, Cat. No. I8765) , Goat IgG (Sigma, Cat. No. I9140)] were added to each of 10 μg (0.1 mg / ml 100 μl) and physically adsorbed onto the membrane. Then add 3% BSA solution (150 μl) (Sigma, Cat. No. BSAS-100) dissolved in PBS buffer to block the voids on the membrane and remove the remaining BSA solution. To wash with PBS buffer (150 μl).

100 μl of the cell lysis solution prepared as described above was added thereto, and allowed to stand for 5 minutes, followed by washing three times with a washing buffer (PBS buffer, pH 7.4, 0.05% Tween-20, 150 μl). Then, the membrane was separated from the apparatus and soaked in the wash buffer, and then washed by stirring for 30 minutes. The extent to which the candidate peptide binds to the immunoglobulin constant region (Fc region) was confirmed by photographing fluorescence derived from green fluorescent protein (EGFP) linked to the peptide with LAS-3000 (FUJIFILM) (FIG. 3).

As a result, the fluorescence rate of the whole colonies was about 50 to 60%, and the final 10 fusion proteins were selected by screening 2000 fluorescent colonies. The plasmids of these candidates were then purified using a Plasmid Mini Extraction Kit (Bioneer, Cat. No. K-3111) and sequenced to sequence as shown in Table 1 and Table 2 below. It was confirmed.

DNA base sequence of antibody binding peptide Kinds DNA sequence SEQ ID NO: EGFP 1 DNA TGT CAT AAG AGG AGT TTT TGG GCG GAT AAT TGC SEQ ID NO: 11 EGFP 2 DNA TGT CGT ACG CAG TTT CGG CCT AAT CAG ACT TGC SEQ ID NO: 12 EGFP 3 DNA TGT CAG CTG TGC GAC TTC TGG CGC ACT CGA TGC SEQ ID NO: 13 EGFP 4 DNA TGT TTT GAG GAT TTT AAT GAG CAG AGG ACT TGC SEQ ID NO: 14 EGFP 5 DNA TGT CTT GCG AAG TTT TTG AAG GGG AAG GAT TGC SEQ ID NO: 15 EGFP 6 DNA TGT TGG CAT AGA CGA ACT CAT AAG ACC TTC TGC SEQ ID NO: 16 EGFP 7 DNA TGT CGG ACG ATT CAG ACG AGG TCT CAT TGG TGC SEQ ID NO: 17 EGFP 8 DNA TGT ATT AAG TTG GCT CAG CTT CAT TCT GTT TGC SEQ ID NO: 18 EGFP 9 DNA TGT TGG AGG CAT CGT AAT GCT ACT GAG TGG TGC SEQ ID NO: 19 EGFP 10 DNA TGT CAG AAT TGG ATT AAG GAT GTG CAT AAG TGC SEQ ID NO: 20

Amino acid sequence of an antibody binding peptide Kinds Amino acid sequence SEQ ID NO: EGFP 1 C H K R S F W A D N C SEQ ID NO: 1 EGFP 2 C R T Q F R P N Q T C SEQ ID NO: 2 EGFP 3 C Q L C D F W R T R C SEQ ID NO: 3 EGFP 4 C F E D F N E Q R T C SEQ ID NO: 4 EGFP 5 C L A K F L K G K D C SEQ ID NO: 5 EGFP 6 C W H R R T H K T F C SEQ ID NO: 6 EGFP 7 C R T I Q T R S H W C SEQ ID NO: 7 EGFP 8 C I K L A Q L H S V C SEQ ID NO: 8 EGFP 9 C W R H R N A T E W C SEQ ID NO: 9 EGFP 10 C Q N W I K D V H K C SEQ ID NO: 10

1 is a diagram showing a process of screening a peptide specifically binding to the Fc region of IgG.

FIG. 2 is a diagram showing fluorescent colonies expressing FcBP-EGFP fusion proteins on LB plates inoculated with E. coli transformed with an expression vector library.

Figure 3 is a diagram showing the results of analyzing the attachment of the Fc region of the FcBP-EGFP fusion protein using the dot blotting assay (Dot Blotting Assay) method.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel peptides with specific binding to Fc domain of IgG and          their preparation methods <130> 8p-06-42 <160> 27 <170> Kopatentin 1.71 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 1 <400> 1 Cys His Lys Arg Ser Phe Trp Ala Asp Asn Cys   1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 2 <400> 2 Cys Arg Thr Gln Phe Arg Pro Asn Gln Thr Cys   1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 3 <400> 3 Cys Gln Leu Cys Asp Phe Trp Arg Thr Arg Cys   1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 4 <400> 4 Cys Phe Glu Asp Phe Asn Glu Gln Arg Thr Cys   1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 5 <400> 5 Cys Leu Ala Lys Phe Leu Lys Gly Lys Asp Cys   1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 6 <400> 6 Cys Trp His Arg Arg Thr His Lys Thr Phe Cys   1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 7 <400> 7 Cys Arg Thr Ile Gln Thr Arg Ser His Trp Cys   1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 8 <400> 8 Cys Ile Lys Leu Ala Gln Leu His Ser Val Cys   1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 9 <400> 9 Cys Trp Arg His Arg Asn Ala Thr Glu Trp Cys   1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> EGFP 10 <400> 10 Cys Gln Asn Trp Ile Lys Asp Val His Lys Cys   1 5 10 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> EGFP 1 DNA <400> 11 tgtcataaga ggagtttttg ggcggataat tgc 33 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 2 DNA <400> 12 tgtcgtacgc agtttcggcc taatcagact tgc 33 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 3 DNA <400> 13 tgtcagctgt gcgacttctg gcgcactcga tgc 33 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 4 DNA <400> 14 tgttttgagg attttaatga gcagaggact tgc 33 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 5 DNA <400> 15 tgtcttgcga agtttttgaa ggggaaggat tgc 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 6 DNA <400> 16 tgttggcata gacgaactca taagaccttc tgc 33 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 7 DNA <400> 17 tgtcggacga ttcagacgag gtctcattgg tgc 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> 223 EGFP 8 DNA <400> 18 tgtattaagt tggctcagct tcattctgtt tgc 33 <210> 19 <211> 33 <212> PRT <213> Artificial Sequence <220> 223 EGFP 9 DNA <400> 19 Thr Gly Thr Thr Gly Gly Ala Gly Gly Cys Ala Thr Cys Gly Thr Ala   1 5 10 15 Ala Thr Gly Cys Thr Ala Cys Thr Gly Ala Gly Thr Gly Gly Thr Gly              20 25 30 Cys     <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> EGFP 10 DNA <400> 20 tgtcagaatt ggattaagga tgtgcataag tgc 33 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Fc binding protein <400> 21 Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys   1 5 10 <210> 22 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 22 gaattccata tggactgtgc atggcacctg ggagaactgg tctggtgcac cgtgagcaag 60 ggcgaggagc tgttc 75 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 23 ccgctcgagc ttgtacagct cgtccatgcc gagctcgag 39 <210> 24 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> DNA library <220> <221> variation (222) (27) .. (53) <223> n is A, T, G or C <400> 24 gccgcagcca tatgaaagat gaatgtnnnn nnnnnnnnnn nnnnnnnnnn nnntgcgaat 60 tcgagctccg tcga 74 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 25 gccgcagcca tatgaaagat gaatgt 26 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 26 ctcgcccttg ctcactcgac ggagctcgaa 30 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 27 attcgcggat cccttgtaca gctcgtccat gccgag 36  

Claims (17)

A polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 10 that specifically binds to an Fc fragment of immunoglobulin G (Igunoglobulin G; IgG). Gene encoding the polypeptide of claim 1. The gene according to claim 2, comprising a nucleotide sequence of any one of SEQ ID NOs: 11-20. An expression vector comprising the gene of claim 2. The expression vector according to claim 4, wherein the expression vector is always an expression vector. A transformant incorporating the expression vector of claim 4 into a host cell. The transformant of claim 6, wherein the host cell is E. coli . delete delete delete delete delete 1) activating the carboxyl group of the solid substrate comprising a carboxyl group; 2) Surface treatment for attaching a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 10 that specifically binds to an Fc fragment of immunoglobulin G (Igunoglobulin G; IgG) to the solid substrate of step 1) step; And 3) A method for preparing an IgG monolayer consisting of binding IgG to the surface-treated solid substrate of step 2). The method of claim 13, wherein the solid substrate is composed of biodegradable organic polymer nanoparticles such as CM-5 Au sensor chip, magnetic microparticles, glass plate, gold nanoparticles, PLGA, or various (micro) well plates. It is any one selected from the group. 1) treating a composition comprising an antibody in a column adsorbed a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 10 that specifically binds to an Fc fragment of immunoglobulin G; step; And 2) Purifying IgG antibody comprising the step of eluting the antibody attached to the column of step 1). 1) surface-treating a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 10 that specifically binds to an Fc fragment of immunoglobulin G; And 2) Method of immobilizing IgG comprising the step of treating IgG to the solid substrate of step 1). IgG is immobilized via the polypeptide on a solid substrate surface-treated with a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 10 that specifically binds to an Fc fragment of immunoglobulin G (Igunoglobulin G; IgG) Immune sensor.

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