KR20190114550A - Peptides for forming protein-protein conjugate and the method for forming protein-protein conjugate using the same - Google Patents

Abstract

The present invention relates to a protein comprising the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 10, in which, specifically, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 10, which is a human-derived protein, is used to form a complex of a first target protein and a second target protein, and a bond using the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 10 is specifically bonded with an antigen-antibody reaction and includes a disulfide bond to form a strong bond, thereby being useful for forming the complex of the first target protein and the second target protein.

Description

Peptides for forming protein-protein conjugate and the method for forming protein-protein conjugate using the same}

The present invention relates to a protein-protein conjugate forming mediated peptide and a method for forming a protein-protein conjugate using the same.

Bioconjugation is a technology that combines two molecules containing at least one biomaterial and plays an important role in the development of effective vaccines, affinity substrates, drug delivery systems, and the like.

The most representative bioconjugates are antibody-drug conjugates (ADCs), which are used as therapeutic agents. ADC is a technology that focuses on targeting a specific cell to a drug by taking full advantage of the antibody. It consists of three components: the drug, a monoclonal antibody, and a linker that links the antibody to the drug. ADCs are most commonly used to deliver anticancer agents to tumor cells using antibodies that specifically bind to specific antigens expressed on the surface of cancer cells.

In addition, as a method for discovering a target protein for drug development, protein-protein conjugation is used. In vivo, proteins function through binding to other proteins. Changes in protein expression, changes in cell position, and structural changes through post-translational modification lead to changes in protein binding. Since the abnormalities in protein interactions caused by these changes are associated with diseases, identification of protein interactions using protein-protein binding and discovery of target proteins related to diseases based on these are important processes for drug development.

As methods for conjugating protein components, Mao et al. (Mao, H., et al., J. Am. Chem. Soc. 126 (2004) 2670-2671) describe C- A sortase mediated protein ligation method for terminal modification was developed, and Sato (Sato, H., Advanced Drug Delivery Reviews 54 (2002) 487-504), et al. In order to introduce site-specific polyethylene glycol into proteins, a system using transglutaminase (TGase) was established. Transglutaminase is an enzyme that catalyzes an acyl transfer reaction between the gamma-carboxamide group of glutamine residues and the primary epsilon amino group of lysine. Split inteins also perform protein trans-splicing, a naturally occurring process in living organisms, which is a multistage biochemical reaction consisting of cleavage and formation of peptide bonds. Specifically, two protein fragments including the split intein are combined to form an enzyme, followed by a process of removing the intein itself and catalyzing the linking of the flanking sequence. Intein is functional in exogenous environments and can be used to chemically manipulate virtually any polypeptide backbone, which has been applied to basic biological research as well as in therapeutic development (Shah, NH & Muir, TW, Chem. Sci). 5, 446-461 (2014). Bonds formed through such a reaction are known to be fairly stable bonds, such as being resistant to proteases.

However, since methods for binding such protein components are applied by proteins of non-human origin, there is a possibility of causing an immune response, so the application to therapeutic agents is limited.

Therefore, the present inventors specifically bind to a variant of human-derived 3LRH intrabody and a variant of 3LRH intrabody to remove a factor that can induce an immune response in forming bioconjugation. The present invention was completed by making peptides that form a protein and confirming that they can mediate specific and firm binding of two or more proteins.

It is an object of the present invention to provide a protein comprising an amino acid sequence as set out in SEQ ID NO: 6 or SEQ ID NO: 10, a polynucleotide encoding the protein, a first using a peptide consisting of the amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 10 A method of forming a conjugate of a target protein and a second target protein, and a combination of the first target protein and the second target protein formed by the forming method.

In order to achieve the above object, the present invention provides a protein comprising an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 10.

The present invention also provides a polynucleotide encoding the protein.

In addition, the present invention includes 1) a first fusion protein comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and a first target protein, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and a second target protein. Preparing a second fusion protein; And 2) mixing the first fusion protein and the second fusion protein to form a bond between the first fusion protein and the second fusion protein. ) Provides a formation method.

The present invention also provides a conjugate of the first target protein and the second target protein formed by the method for forming a conjugate of the first target protein and the second target protein.

The present invention can form a conjugate of a first target protein and a second target protein using a peptide consisting of an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 10, which is a protein derived from human, Binding using a peptide consisting of the amino acid sequence set forth in No. 10 is specific for binding in an antigen-antibody reaction and can form a strong bond including disulfide bonds, thereby forming a conjugate of the first target protein and the second target protein. It can be usefully used.

Figure 1 shows the docking of the huntingtin peptide-3LRH antibody structure and protein A using a molecular modeling program.
Figure 2 shows the amino acid sequence of the overlapping regions of the huntingtin peptide and protein A and the amino acid sequence of the engineered epitope of # 8188 protein A.
Figure 3 shows the alignment of the helix (helice) of the protein A and huntingtin peptide (red color is the huntingtin amino acid, gray is the amino acid of the protein A domain).
FIG. 4 shows the amino acid sequence in which cysteine is introduced into 3LRH ^cys antibody and # 8188 protein A for disulfide bridge formation between 3LRH ^cys antibody and # 8188 ^cys protein A. FIG.
Figure 5 shows the crystal structure of 3LRH antibody and # 8188 Protein A.
FIG. 6 is an enlarged view of the engineered epitope portion of # 8188 Protein A of FIG. 5 (amino acid residues of # 8188 Protein A and huntingtin peptide interacting with 3LRH antibody in gray and red, respectively).
7 compares the structure of native protein A and engineered protein A, # 8188 protein A. FIG.
8 shows the results of SDS-PAGE analysis for confirming disulfide bridge formation of 3LRH ^cys antibody and # 8188 ^cys protein A. FIG.
Figure 9 shows the crystal structure of # 8188 ^cys protein A bound to the 3LRH ^cys antibody (disulfide bridge is indicated by a yellow line, electron density map around the disulfide bridge is represented by a gray mesh).

Hereinafter, the present invention will be described in detail.

The present invention provides a protein comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10.

The protein preferably comprises an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 10 at the N-terminus, internal or C-terminus, but is not limited thereto.

The protein may be a fusion protein of the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10 with any desired protein.

The peptide consisting of the amino acid sequence described in SEQ ID NO: 6 is a 3LRH ^cys antibody that is a variant of the 3LRH antibody, in the present invention, the 3LRH antibody binds to a short alpha helix peptide present in the human huntingtin protein, SEQ ID NO: An immunoglobulin light chain composed of the amino acid sequence set forth in 1. The 3LRH ^cys antibody is a mutated tyrosine (Y), the 53rd amino acid of the 3LRH antibody, to cysteine (cystein, C).

In the present invention, # 8188 protein A refers to a peptide consisting of the amino acid sequence of SEQ ID NO: 4 having a helical epitope to which the 3LRH antibody can bind, # 8188 ^cys protein A is a variant of the # 8188 protein A By peptide means the peptide consisting of the amino acid sequence shown in SEQ ID NO: 8. Specifically, the # 8188 ^cys protein A may be a mutated alanine (alanine, A) that is the 51st amino acid of the # 8188 protein A with cysteine (cystein, C).

The peptide consisting of the amino acid sequence of SEQ ID NO: 6 and the peptide consisting of the amino acid sequence of SEQ ID NO: 8 may form a bond between the peptides. Specifically, among the amino acid sequences set forth in SEQ ID NO: 8, the region of SEQ ID NO: 46 to amino acids 59 (SEQ ID NO: 9) is a region which forms a bond with the 3LRH ^cys antibody, and more specifically, described in SEQ ID NO: 9 Among the amino acid sequences, the 2nd (K, lysine), 6th (C, cysteine), 9th (S, serine), 10th (L, leucine) and 13th (F, phenylalanine) amino acids are combined with 3LRH ^cys antibody. Is an important amino acid in the formation of bonds. Among the amino acid sequences set forth in SEQ ID NO: 9, 2nd (K, lysine), 6th (C, cysteine), 9th (S, serine), 10th (L, leucine) and 13th (F, phenylalanine) amino acids Peptides consisting of the amino acid sequence set forth in SEQ ID NO: 10, conserved only, may form a bond with the 3LRH ^cys antibody. If the amino acid sequence of the region of the protein that binds the antibody in the protein is preserved, the specific amino acids directly involved in the binding of the antibody toward the antibody, and if the remaining amino acid sequence is replaced with another amino acid, the binding force similar to the original antigen-antibody binding Can be combined.

The amino acid sequence described in SEQ ID NO: 10 may be an amino acid sequence described in SEQ ID NO: 9.

The present invention also provides a polynucleotide encoding the protein. In the case of the peptide consisting of the amino acid sequence described in SEQ ID NO: 6, the polynucleotide encoding the peptide may be the nucleotide sequence of SEQ ID NO: 7. In addition, in the case of a peptide consisting of the amino acid sequence of SEQ ID NO: 8 comprising the amino acid sequence of SEQ ID NO: 10, the polynucleotide encoding the peptide may be the nucleotide sequence of SEQ ID NO: 11, but is not limited thereto. no.

According to a specific example of the present invention, the present inventors prepared # 8188 protein A into which a spiral epitope into which protein 3A binds 3LRH antibody was introduced. Specifically, the alignment of the helices of protein A and huntingtin peptides is directed to amino acids (K47 (not shown), E50, S51, P54 (not shown) and K55 (not shown) directed toward 3LRH antibodies in protein A and important for binding to the antibody). 3), and the amino acids of protein A were replaced with the corresponding amino acids of the huntingtin peptide to prepare # 8188 protein A with engineered epitopes (FIG. 2 and table). 2).

In addition, the inventors confirmed that # 8188 protein A and the 3LRH antibody form a complex, and the complex is substantially identical to the structure of the complex of the original huntingtin peptide-3LRH antibody that binds by antigen-antibody reaction ( 5 to 7).

In addition, the inventors of the present invention, the 3LRH ^cys antibody mutated tyrosine (Y), the 53rd amino acid tyrosine (Y) of the 3LRH antibody to the short alpha helix peptide present in the human huntingtin protein (cystein, C) # 8188 ^cys protein A was prepared by mutating alanine (A), the 51st amino acid of # 8188 protein A having a helical epitope to which 3LRH antibodies can bind (cystein, C) (Table 1). 3 and Table 4).

In addition, the inventors prepared a complex of 3LRH ^cys antibody- # 8188 ^cys antigen protein using 3LRH ^cys antibody and # 8188 ^cys protein A (see FIG. 4).

In addition, the inventors of the SDS-PAGE analysis confirmed that the disulfide bridge between the 3LRH ^cys antibody and # 8188 ^cys protein A was formed in the non-reducible gel to shift the band. As a result of analyzing the crystallization structure, the electron density map It was confirmed that the disulfide bridge is formed through (see FIGS. 8 and 9).

Therefore, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 8 is derived from human beings and does not have side effects such as inducing an immune response, and the binding between the peptides can be specifically bound because they bind by antigen-antibody reactions. In addition, since a disulfide bond can form a strong bond, it can be used as a useful system for bioconjugation between fusion proteins containing each of these peptides.

In addition, the present invention includes 1) a first fusion protein comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 6 and a first target protein, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10 and a second target protein. Preparing a second fusion protein; And 2) mixing the first fusion protein and the second fusion protein to form a bond between the first fusion protein and the second fusion protein. ) Provides a formation method.

In the present invention, "target protein" means a protein or peptide to be conjugated, and may be included without limitation in kind or size as long as it is a protein or peptide.

In the present invention, "fusion protein" is an artificially synthesized protein in which two or more proteins or peptides are linked to each other, and specifically, a peptide composed of the target protein and the amino acid sequence described in SEQ ID NO: 6 or SEQ ID NO: 10 of the present invention. It may include.

As used herein, the term “first fusion protein” refers to a fusion protein comprising a peptide (3LRH ^cys antibody) consisting of an amino acid sequence represented by SEQ ID NO: 6 and a first target protein, and “second fusion protein” refers to SEQ ID NO: 10. It means a fusion protein comprising a peptide consisting of the amino acid sequence described as and the second target protein.

The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 may be present at the N-terminus, internal or C-terminus of the first target protein sequence. In addition, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 may be present at the N-terminus, internal or C-terminus of the sequence of the second target protein.

In the present invention, a "conjugate of a first target protein and a second target protein" is used to prepare the first fusion protein and the second fusion protein in order to bind any first target protein and the second target protein. , Refers to a conjugate obtained by binding the prepared first fusion protein and the second fusion protein. Specifically, the conjugate is between a peptide consisting of the amino acid sequence of SEQ ID NO: 6 contained in the first fusion protein (3LRH ^cys antibody) and a peptide consisting of the amino acid sequence of SEQ ID NO: 10 contained in the second fusion protein. It is formed by a bond.

In the step of preparing the fusion protein of step 1), the fusion protein may be prepared by a chemical synthesis method known in the art, or by cloning and expressing a polynucleotide encoding the fusion protein in an expression vector.

The binding of step 2) may be a bond between the peptide consisting of the amino acid sequence of SEQ ID NO: 6 and the peptide consisting of the amino acid sequence of SEQ ID NO: 10.

The binding between the peptides may be bound by an antigen-antibody reaction. In addition, the binding between the peptides may further include a disulfide bond to the one bound by the antigen-antibody reaction.

The amino acid sequence described in SEQ ID NO: 10 may be an amino acid sequence described in SEQ ID NO: 9.

The present invention also provides a conjugate of the first target protein and the second target protein formed by the method of forming a conjugate of the first target protein and the second target protein.

According to a specific embodiment of the present invention, the present inventors cloned a polynucleotide encoding 3LRH ^cys antibody and # 8188 ^cys protein A into a vector and transformed the vector into E. coli to transform the 3LRH ^cys antibody and # 8188 ^cys. Protein A was prepared and a complex of 3LRH ^cys antibody- # 8188 ^cys antigen protein was prepared (see Table 3, Table 4 and FIG. 4). Therefore, when a fusion protein of 3LRH ^cys antibody and a first target protein and # 8188 ^cys protein A and a fusion protein of a second target protein are mixed, a conjugate between the target protein is formed by binding between 3LRH ^cys antibody and # 8188 ^cys protein A. It can manufacture.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> 3LRH  Construction of Antibodies

Polynucleotides encoding 3LRH antibodies were synthesized based on amino acid sequence information stored in a PDB database.

SEQ ID NO: Antibodies
name order
Kinds order One 3LRH Amino acid sequence MGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQ
QLPGRAPELLMYDDDLLAPGVSDRFSGSRSGTSASLTISGLQSE
DEADYYAATWDDSLNGWVFGGGTKVTVLSA 2 3LRH Sequence
(5 '→ 3') ATGGGTAGTCAACCAGTACTTACACAAAGCCCGTCTGTGTCTGCCGCTCCACG
TCAGAGAGTCACCATCTCAGTTAGTGGTTCTAATTCAAATATAGGATCGAACA
CAGTGAACTGGATTCAGCAGTTGCCTGGCCGTGCCCCGGAGTTATTAATGTAT
GATGATGATTTATTAGCACCCGGAGTATCAGATCGCTTTTCAGGAAGCCGTAG
TGGCACTAGTGCGTCCTTAACCATTAGCGGGTTACAGTCTGAAGACGAAGCGG
ACTATTACGCAGCTACGTGGGATGATAGTTTAAATGGCTGGGTTTTTGGTGGT
GGTACTAAAGTTACGGTGCTGTCCGCA

Polynucleotides encoding the 3LRH antibody (SEQ ID NO: 2) were cloned into the pET28a vector and then transformed into E. coli strain BL21 (DE3) for protein production. Transformed E. coli BL21 (DE3) was inoculated in LB medium added with 50 μg / mL kanamycin and incubated at 37 ° C. When the value of OD ₆₀₀ reached 0.7, 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to induce protein production and incubated overnight at 21 ° C. After obtaining E. coli , the harvested cell pellets are resuspended in lysis buffer containing 20 mM Tris pH 8.0, 200 mM NaCl, and 0.5 mM PMSF, and then resuspended with a microfluidizer (Microfluidics). Homogenized. After centrifugation, the supernatant was loaded onto a Ni-NTA metal affinity column, and the buffer containing 20 mM Tris pH 8.0, 200 mM NaCl and 500 mM imidazole pH 8.0 was removed. 6XHis-tagged protein was eluted.

To remove the 6XHis tag, the eluted antibody protein was treated with thrombin (Sigma, Cat #. T4648) overnight. The cleaved 3LRH antibody was purified by loading it on a Superdex 200 size-exclusion column (GE Healthcare) equilibrated with 20 mM Tris pH 8.0, 200 mM NaCl.

< Example  2> Epitope  Construction of Introduced # 8188 Protein A

An epitope capable of binding 3LRH antibody to protein A was attempted. 3LRH antibodies bind to an alpha helical stretch of 14 amino acids in human huntingtin protein.

The huntingtin peptide structure bound to the 3LRH antibody was overlapped with the C terminal helix of protein A (see FIG. 1). FIG. 2 shows the first 10 amino acids of the huntingtin peptide overlapped with the molecular modeling program and the last 10 amino acids of the C-terminal helix of Protein A (see FIG. 2).

To design the epitope in protein A, the structural alignment of the helix of protein A with the huntingtin protein resulted in lysine (K47) (not shown), which is the 47th amino acid in protein A, and glutamine (E50). ) And the 51st amino acid serine (S51) was directed towards the antibody and was found to be important for binding (see Figure 3). In addition, proline (P54) (not shown), the 54th amino acid in protein A, and lysine (K55) (not shown), the 55th amino acid, were also directed toward the antibody and important for binding.

Thus, the amino acids of protein A except lysine were replaced with the corresponding amino acids of the huntingtin peptide to prepare # 8188 protein A having an engineered epitope (see FIG. 2 and Table 2). Since lysine (K47) was found at both the huntingtin peptide and the corresponding position in the protein A sequence, no change was necessary, and the 54th and 55th amino acids, the N-terminal regions of Protein A, were 13, the N-terminal region of the huntingtin peptide. To the 18th amino acid sequence.

SEQ ID NO: protein
name order
Kinds order 3 Protein A Amino acid sequence MFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEA
KKLNESQAPK 4 # 8188
Protein A Amino acid sequence MFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEA
KKLNKAQASLKSFQ 5 # 8188
Protein A Sequence
(5 '→ 3') ATGTTCAACAAGGATCAGCAGAGCGCCTTCTACGAGATCCTGAACATGCCGA
ACCTGAACGAGGCCCAGCGCAACGGCTTCATCCAGAGCCTGAAGGATGATCC
AGCCAGAGCACGAACGTGCTGGGTGAAGCTAAGAAACTGAACAAAGCTCAGG
CTTCTCTGAAATCTTTCCAG

Specific manufacturing process of # 8188 protein A is as follows. Polynucleotide encoding SEQ ID NO: # 8188 (SEQ ID NO: 5) was obtained by PCR using synthetic oligonucleotides. Specific PCR procedure is as follows. 20 seconds in the denaturation step (95 ° C.) using a gene encoding protein A as a template and using a forward primer (SEQ ID NO: 12) and a reverse primer (SEQ ID NO: 13); 20 seconds in the annealing step (56 ° C.); PCR (30 cycles in total) was performed according to the 30 second condition in the extension step (72 ° C.). (Forward primer (SEQ ID NO: 12): ACTACCATATGTTCAACAAGGATCAGCAGAGCGCC; Reverse primer (SEQ ID NO: 13): ACTACGCGGCCGCTCACTGGAAAGATTTCAGAGAAGCCTGAGCTTTGTTCAGTTTCTTAGCTTCACCCAGCACGTTC)

Amplified # 8188 polynucleotide (SEQ ID NO: 5) was cloned into the pET28a vector and then transformed into E. coli strain BL21 (DE3) for protein production. Transformed E. coli BL21 (DE3) was inoculated in LB medium added with 50 μg / mL kanamycin and incubated at 37 ° C. When the value of OD ₆₀₀ reached 0.6, it was further incubated for 4 hours at 37 ℃ after adding 0.5 mM IPTG. E. coli was obtained, then resuspended in lysis buffer containing 20 mM Tris pH 8.0, 200 mM NaCl and 0.5 mM PMSF and homogenized with a microfluidizer (Microfluidics). Cell suspension was removed by centrifugation and the supernatant was loaded onto a Ni-NTA chromatography column. 6XHis-tagged proteins are eluted using a buffer comprising 20 mM Tris pH 8.0, 200 mM NaCl and 500 mM imidazole pH 8.0, and the imidazole is a PD-10 desalting column (GE Healthcare). Removal was performed to obtain purified # 8188 Protein A.

< Example  3> 3LRH ^cys  Antibodies and # 8188 ^cys  Construction of Protein A

In order to apply an antigen-antibody binding reaction between 3LRH antibody and # 8188 protein A to prepare a nanostructure, etc., the two polypeptide molecules were linked by a disulfide bridge. Design 2.0 program was used to determine the position to introduce cysteine (Cys) for the 3rd amino acid tyrosine, 53rd amino acid and # 8188 protein A, alanine, the 51st amino acid.

< Example  3-1> 3LRH ^cys  Construction of Antibodies

In order to prepare 3LRH ^cys antibody (Table 3) mutated tyrosine (Y), the 53rd amino acid of 3LRH with cysteine (C), overlap PCR was performed.

Using the gene encoding the 3LRH antibody as a template, PCR was performed using forward primer 1 (SEQ ID NO: 14) and reverse primer 1 (SEQ ID NO: 15) to obtain template 1. Then, template 2 was obtained by PCR using forward primer 2 (SEQ ID NO: 16) and reverse primer 2 (SEQ ID NO: 17). Template 1 and template 2 were simultaneously PCR with forward primer 1 and reverse primer 2 to complete overlap PCR.

(Forward primer 1 (SEQ ID NO: 14): ACTACCATATGGGTAGTCAACCAGTACTTACACAA;

Reverse primer 1 (SEQ ID NO: 15): ATCATCATCACACATTAATAACTCCGGGGCACGGC;

Forward primer 2 (SEQ ID NO: 16): TTATTAATGTGTGATGATGATTTATTAGCACCCG;

Reverse primer 2 (SEQ ID NO: 17): ACTACGCGGCCGCTCATGCGGACAGCACCGTAACT)

SEQ ID NO: Antibodies
name order
Kinds order One 3LRH Amino acid sequence MGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQ
QLPGRAPELLM Y DDDLLAPGVSDRFSGSRSGTSASLTISGLQSE
DEADYYAATWDDSLNGWVFGGGTKVTVLSA 6 3LRH ^cys Amino acid sequence MGSQPVLTQSPSVSAAPRQRVTISVSGSNSNIGSNTVNWIQ
QLPGRAPELLM C DDDLLAPGVSDRFSGSRSGTSASLTISGLQSE
DEADYYAATWDDSLNGWVFGGGTKVTVLSA 7 3LRH ^cys Sequence
(5 '→ 3') ATGGGTAGTCAACCAGTACTTACACAAAGCCCGTCTGTGTCTGCCGCTCCACG
TCAGAGAGTCACCATCTCAGTTAGTGGTTCTAATTCAAATATAGGATCGAACA
CAGTGAACTGGATTCAGCAGTTGCCTGGCCGTGCCCCGGAGTTATTAATGTGT
GATGATGATTTATTAGCACCCGGAGTATCAGATCGCTTTTCAGGAAGCCGTAG
TGGCACTAGTGCGTCCTTAACCATTAGCGGGTTACAGTCTGAAGACGAAGCGG
ACTATTACGCAGCTACGTGGGATGATAGTTTAAATGGCTGGGTTTTTGGTGGT
GGTACTAAAGTTACGGTGCTGTCCGCA

The polynucleotide encoding the amplified 3LRH ^cys antibody (SEQ ID NO: 7) was cloned into the pET28a vector, and then 3LRH ^cys antibody was obtained by the method described in Example 1.

< Example  3-2> # 8188 ^cys  Construction of Protein A

To mutate alanine (A), the 51st amino acid of protein A # 8188, to cysteine (C), PCR was performed with a polynucleotide encoding protein # 8188 A as a template to encode # 8188 ^cys protein A. A polynucleotide was prepared.

SEQ ID NO: protein
name order
Kinds order 4 # 8188
Protein A Amino acid sequence MFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEA
KKLNK A QASLKSFQ 8 # 8188 ^cys
Protein A Amino acid sequence MFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEA
KKLNK C QASLKSFQ 9 # 8188 ^cys
Binding Peptides in Protein A Amino acid sequence KKLNKCQASLKSFQ 10 # 8188 ^cys
Binding Peptides in Protein A Amino acid sequence xKxxxCxxSLxxFx 11 # 8188 ^cys
Protein A Sequence
(5 '→ 3') ATGTTCAACAAGGATCAGCAGAGCGCCTTCTACGAGATCCTGAACATGCCGA
ACCTGAACGAGGCCCAGCGCAACGGCTTCATCCAGAGCCTGAAGGATGATCC
GAGCCAGAGCACGAACGTGCTGGGTGAAGCTAAGAAACTGAACAAATGTCAG
GCTTCTCTGAAATCTTTCCAG

Specifically, the mutations were obtained by PCR using synthetic oligonucleotides. Specific PCR procedure is as follows. 20 seconds in the denaturation step (95 ° C.) using a gene encoding protein # 8188 as a template and using a forward primer (SEQ ID NO: 18) and a reverse primer (SEQ ID NO: 19); 20 seconds in the annealing step (56 ° C.); PCR (30 cycles in total) was performed according to the 30 second condition in the extension step (72 ° C.). (Forward primer (SEQ ID NO: 18): ACTACCATATGTTCAACAAGGATCAGCAGAGCGCC; Reverse primer (SEQ ID NO: 19): ACTACGCGGCCGCTCACTGGAAAGATTTCAGAGAAGCCTGACATTTGTTCAGTTTCTTAGCTTCACC)

Polynucleotides encoding the amplified # 8188 ^cys protein A (SEQ ID NO: 11) were cloned into the pET28a vector and then transformed into E. coli strain BL21 (DE3) for protein production. Transformed E. coli BL21 (DE3) was inoculated in LB medium added with 50 μg / mL kanamycin and incubated at 37 ° C. When the value of OD ₆₀₀ reached 0.6, it was further incubated for 4 hours at 37 ℃ after adding 0.5 mM IPTG. E. coli was obtained, then resuspended in lysis buffer containing 20 mM Tris pH 8.0, 200 mM NaCl and 0.5 mM PMSF and homogenized with a microfluidizer (Microfluidics). Cell suspension was removed by centrifugation and the supernatant was loaded onto a Ni-NTA chromatography column. 6XHis-tagged proteins are eluted using a buffer comprising 20 mM Tris pH 8.0, 200 mM NaCl and 500 mM imidazole pH 8.0, and the imidazole is a PD-10 desalting column (GE Healthcare). Removal was carried out to obtain purified # 8188 ^cys protein A.

< Example  4> 3LRH  Complex Construction of Antibody- # 8188 Protein A

To prepare a complex of 3LRH antibody- # 8188 protein A, the 3LRH antibody prepared in Example 1 and # 8188 protein A prepared in Example 2 were mixed at a 1: 1 molar ratio at 4 ° C. for 1 hour. It was.

Only the complex of 3LRH antibody- # 8188 protein A was purified using Superdex 200 gel-filtration chromatography.

< Example  5> 3LRH ^cys  Antibody- # 8188 ^cys  Complex production of antigenic proteins

3LRH ^cys antibody - # 8188 ^cys protein to produce the conjugate of A, carried out in the 3LRH ^cys antibody and # 8188 ^cys protein A non-reducing environment (non-reducing conditions) to one prepared in Example 3 1: 1 molar ratio (molar ratio) Mixed at 4 ° C. overnight.

Only complexes of 3LRH ^cys antibody- # 8188 ^cys antigen peptide were purified using Superdex 200 gel-filtration chromatography.

< Experimental Example  1> 3LRH  Crystal structure confirmation of complex of antibody- # 8188 protein A

The complex of 3LRH antibody- # 8188 protein A prepared in Example 4 was crystallized using a crystallization solution containing 35% PEG 4000, 0.1 M MOPS pH 6.5 and 30% glycerol as a cryoprotectant. Crystals were frozen in liquid nitrogen at -170 ° C.

Diffraction data were collected at Pohang Accelerator Laboratory's 7A and 5C beam lines, and the package HKL2000 was used to process the diffraction data (HKL research).

Early stages of structure identification and refinement were calculated by molecular replacement technique using the PHASER program. The atomic model was built by iterative modeling and refinement using the program COOT and PHENIX.

In crystal structure, the modified C-terminal helix of # 8188 protein A interacted with the 3LRH antibody as expected (see FIG. 5). Mutated amino acid residues for epitope production are shown in red.

The structure of the amino acid interacting with the 3LRH antibody is substantially the same as that of the complex of the original huntingtin peptide-3LRH antibody, and the overall structure of # 8188 Protein A and native Protein A can be overlapped with Cα rmsd of 0.408 kPa (FIGS. 6 and 7).

< Experimental Example  2> using SDS-PAGE 3LRH ^cys  Antibody- # 8188 ^cys  Protein A Liver Disulfide  Confirmation of formation of disulfide bridge

As described in Example 5, when 3LRH ^cys antibody and # 8188 ^cys Protein A were mixed, the formation of disulfide bridges was monitored by performing SDS-PAGE under reducing and non-reducing conditions.

Referring to FIG. 8, in the non-reducing gel, a disulfide bridge was formed between 3LRH ^cys antibody and # 8188 ^cys protein A, and the band was shifted. After overnight culture (16 hr), the disulfide bridge was formed at almost 100% efficiency. It was confirmed that. With only # 8188 ^cys protein A, with no antibody to bind, homodimers were formed under non-reducing conditions.

Cysteine mutations can increase the stability of the complex. In addition, disulfide bridges can be formed only when the two cysteines are close enough to each other, making them a convenient tool for confirming the successful design of epitopes and the formation of complexes. Thus binding of # 8188 ^cys protein A to 3LRH ^cys antibody can be rapidly monitored by non-reducing SDS-PAGE, using only minimal amounts of protein without purification.

< Experimental Example  3> 3LRH ^cys  Antibody- # 8188 ^cys  Confirmation of Crystallization Structure of Complex of Antigen Protein

The complex of 3LRH ^cys antibody- # 8188 ^cys protein A prepared in Example 5 was crystallized using a crystallization solution containing 45% PEG 4000, 0.1 M HEPES pH 7.5 and 30% glycerol as a cryoprotectant. Crystals were frozen in liquid nitrogen at -170 ° C.

Diffraction data were collected at Pohang Accelerator Laboratory's 7A and 5C beam lines, and the package HKL2000 was used to process the diffraction data (HKL research).

Early stages of structure identification and refinement were calculated using the molecular replacement technique using the PHASER program. The atomic model was built by iterative modeling and refinement using programs COOT and PHENIX.

In the structure of the complex of 3LRH ^cys antibody- # 8188 ^cys protein A, the electron density map clearly shows the disulfide bridge (FIG. 9). The disulfide bridge is indicated by a yellow line in FIG. 9, and the electron density map around the disulfide bridge is indicated by a gray mesh. It also shows that the disulfide bridge itself does not interfere with the interaction between 3LRH ^cys antibody- # 8188 ^cys protein A.

The complex system of 3LRH ^cys antibody- # 8188 ^cys protein A stabilized with disulfide bridges can be a useful tool for bioconjugation. Since 3LRH and # 8188 Protein A are derived from human proteins, the limitations of conventional bioconjugation methods that have been previously limited as therapeutic agents due to side effects such as immunological complications can be solved. In addition, since 3LRH ^cys antibody or # 8188 ^cys protein A is small in size, there is an advantage that it can be easily produced in large quantities as a fusion tag in E. coli .

<110> Korea Advanced Institute of Science and Technology (KAIST) <120> Peptides for forming protein-protein conjugate and the method for          forming protein-protein conjugate using the same <130> 2017P-11-031 <160> 19 <170> KoPatentIn 3.0 <210> 1 <211> 115 <212> PRT <213> Artificial Sequence <220> 3223H antibody <400> 1 Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala   1 5 10 15 Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser Asn Ile              20 25 30 Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg Ala Pro          35 40 45 Glu Leu Leu Met Tyr Asp Asp Asp Leu Leu Ala Pro Gly Val Ser Asp      50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser  65 70 75 80 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr Trp Asp                  85 90 95 Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val             100 105 110 Leu ser ala         115 <210> 2 <211> 345 <212> DNA <213> Artificial Sequence <220> 3223H antibody <400> 2 atgggtagtc aaccagtact tacacaaagc ccgtctgtgt ctgccgctcc acgtcagaga 60 gtcaccatct cagttagtgg ttctaattca aatataggat cgaacacagt gaactggatt 120 cagcagttgc ctggccgtgc cccggagtta ttaatgtatg atgatgattt attagcaccc 180 ggagtatcag atcgcttttc aggaagccgt agtggcacta gtgcgtcctt aaccattagc 240 gggttacagt ctgaagacga agcggactat tacgcagcta cgtgggatga tagtttaaat 300 ggctgggttt ttggtggtgg tactaaagtt acggtgctgt ccgca 345 <210> 3 <211> 55 <212> PRT <213> Artificial Sequence <220> <223> Protein A <400> 3 Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met   1 5 10 15 Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys              20 25 30 Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu          35 40 45 Asn Glu Ser Gln Ala Pro Lys      50 55 <210> 4 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> # 8188 Protein A <400> 4 Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met   1 5 10 15 Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys              20 25 30 Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu          35 40 45 Asn Lys Ala Gln Ala Ser Leu Lys Ser Phe Gln      50 55 <210> 5 <211> 176 <212> DNA <213> Artificial Sequence <220> <223> # 8188 Protein A <400> 5 atgttcaaca aggatcagca gagcgccttc tacgagatcc tgaacatgcc gaacctgaac 60 gaggcccagc gcaacggctt catccagagc ctgaaggatg atccagccag agcacgaacg 120 tgctgggtga agctaagaaa ctgaacaaag ctcaggcttc tctgaaatct ttccag 176 <210> 6 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> 3LRHcys antibody <400> 6 Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val Ser Ala Ala   1 5 10 15 Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn Ser Asn Ile              20 25 30 Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly Arg Ala Pro          35 40 45 Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro Gly Val Ser Asp      50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Thr Ile Ser  65 70 75 80 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala Thr Trp Asp                  85 90 95 Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val             100 105 110 Leu ser ala         115 <210> 7 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> 3LRHcys antibody <400> 7 atgggtagtc aaccagtact tacacaaagc ccgtctgtgt ctgccgctcc acgtcagaga 60 gtcaccatct cagttagtgg ttctaattca aatataggat cgaacacagt gaactggatt 120 cagcagttgc ctggccgtgc cccggagtta ttaatgtgtg atgatgattt attagcaccc 180 ggagtatcag atcgcttttc aggaagccgt agtggcacta gtgcgtcctt aaccattagc 240 gggttacagt ctgaagacga agcggactat tacgcagcta cgtgggatga tagtttaaat 300 ggctgggttt ttggtggtgg tactaaagtt acggtgctgt ccgca 345 <210> 8 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> # 8188cys Protein A <400> 8 Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met   1 5 10 15 Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys              20 25 30 Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu          35 40 45 Asn Lys Cys Gln Ala Ser Leu Lys Ser Phe Gln      50 55 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> # 8188cys Protein A-peptide <400> 9 Lys Lys Leu Asn Lys Cys Gln Ala Ser Leu Lys Ser Phe Gln   1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> # 8188cys Protein A-peptide <400> 10 Xaa Lys Xaa Xaa Xaa Cys Xaa Xaa Ser Leu Xaa Xaa Phe Xaa   1 5 10 <210> 11 <211> 177 <212> DNA <213> Artificial Sequence <220> <223> # 8188cys Protein A <400> 11 atgttcaaca aggatcagca gagcgccttc tacgagatcc tgaacatgcc gaacctgaac 60 gaggcccagc gcaacggctt catccagagc ctgaaggatg atccgagcca gagcacgaac 120 gtgctgggtg aagctaagaa actgaacaaa tgtcaggctt ctctgaaatc tttccag 177 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> # 8188 Protein A-Forward primer <400> 12 actaccatat gttcaacaag gatcagcaga gcgcc 35 <210> 13 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> # 8188 Protein A-Reverse primer <400> 13 actacgcggc cgctcactgg aaagatttca gagaagcctg agctttgttc agtttcttag 60 cttcacccag cacgttc 77 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 3LRHcys antibody-Forward primer 1 <400> 14 actaccatat gggtagtcaa ccagtactta cacaa 35 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 3LRHcys antibody-Reverse primer 1 <400> 15 atcatcatca cacattaata actccggggc acggc 35 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 3LRHcys antibody-Forward primer 2 <400> 16 ttattaatgt gtgatgatga tttattagca cccg 34 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 3LRHcys antibody-Reverse primer 2 <400> 17 actacgcggc cgctcatgcg gacagcaccg taact 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> # 8188cys Protein A-Forward primer <400> 18 actaccatat gttcaacaag gatcagcaga gcgcc 35 <210> 19 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> # 8188cys Protein A-Reverse primer <400> 19 actacgcggc cgctcactgg aaagatttca gagaagcctg acatttgttc agtttcttag 60 cttcacc 67

Claims (10)

A protein comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10.
A polynucleotide encoding the protein of claim 1.
The protein of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 10 is an amino acid sequence set forth in SEQ ID NO: 9.
1) a first fusion protein comprising a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 6 and a first target protein, and a second fusion protein comprising a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 10 and a second target protein Preparing a; And
2) a conjugate of the first target protein and the second target protein, comprising mixing the prepared first fusion protein and the second fusion protein to form a bond between the first fusion protein and the second fusion protein. Forming method.
The method according to claim 4,
The peptide consisting of the amino acid sequence represented by SEQ ID NO: 6 is present at the N-terminus, internal or C-terminus of the first target protein sequence,
The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 is present at the N-terminus, internal or C-terminus of the sequence of the second target protein, conjugate of the first target protein and the second target protein Forming method.
The method according to claim 4,
The binding of step 2) is a conjugate of the first target protein and the second target protein, which is a bond between the peptide consisting of the amino acid sequence of SEQ ID NO: 6 and the peptide consisting of the amino acid sequence of SEQ ID NO: 10 Forming method.
The method according to claim 6,
The binding between the peptides is bound by an antigen-antibody reaction, the method of forming a conjugate (conjugate) of the first target protein and the second target protein.
The method according to claim 7,
The bond between the peptide further comprises a disulfide bond (disulfide bond), the method of forming a conjugate (conjugate) of the first target protein and the second target protein.
The method of claim 4, wherein the amino acid sequence of SEQ ID NO: 10 is an amino acid sequence of SEQ ID NO: 9, and a method of forming a conjugate of the first target protein and the second target protein.
A conjugate of a first target protein and a second target protein formed by the method of claim 4.

Images