JP2018080131A - Drd1-derived tag peptide, and antibody to recognize drd1 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody that recognizes DRD1, and a method of purifying protein using a peptide derived from an epitope of the antibody.SOLUTION: Proteoliposome of DRD1 is prepared as an antigen, and on the basis of the antigen, prepared is an antibody. Then, on the basis of the sequence of an epitope of the antibody, determined is a peptide sequence derived from the epitope of the antibody. Thus, established are an antibody that recognizes DRD1, and a method of purifying protein using the peptide derived from the epitope of the antibody.SELECTED DRAWING: None

Description

  The present invention relates to an antibody that recognizes dopamine D1 receptor (dopamine receptor D1, DRD1), a peptide derived from the epitope of the antibody, and a protein purification method using them.

(Anti-membrane protein antibody)
Membrane proteins are important drug discovery targets, but there are few anti-membrane protein antibodies with high specificity and affinity on the market. This is because it is very difficult to construct a membrane protein expression system and to purify the membrane protein.
In recent years, a membrane protein synthesis system using a wheat cell-free protein synthesis system has been developed at the Proteoscience Center of Ehime University. Thereby, a high-quality antibody can be produced by using a cell-free synthesized membrane protein as an antigen (see: Non-Patent Document 1: http://www.pros.ehime-u.ac.jp). /introduce/outline.html).
In addition, ISAAC (Immunospot-array assay on a chip) technology for isolating antibody-producing cells using a microchip and obtaining the antibody gene is known. The ISAAC technology has facilitated the production of rabbit monoclonal antibodies that have been difficult to produce in the past (see Non-patent Document 2 and Patent Document 1).

(Tag for protein purification)
Protein purification tags are used in biochemical and cell biological studies of proteins, and are important for the purification of soluble proteins and membrane proteins. In particular, the epitope tag can be easily added by PCR or the like, and has advantages such as high specificity and affinity. However, existing epitope tags (FLAG, V5, HA, etc.) lack variations and have problems such as low sensitivity and non-specific detection.

(Affinity tag system)
The affinity tag system is generally composed of an epitope tag and an antibody that specifically recognizes the epitope tag, and uses the affinity between the epitope tag and the antibody. The target protein fused with the epitope tag is used for detection by immunoblotting and immunofluorescence staining, purification of the target protein, etc., and is an essential tool in life science.
Therefore, several types of affinity tags are widely used. However, existing tags are not very sensitive and specific, and all contain amino acid residues that undergo post-translational modifications such as phosphorylation and ubiquitination, making them unsuitable for signal transduction studies. There is a point.
Furthermore, for simultaneous detection in immunostaining and immunoprecipitation, there is a high need for high-performance new tags, such as the need for many types of tags.

JP 2014-73100 A

http://www.pros.ehime-u.ac.jp/introduce/outline.html Nature Med, 15: 1088-1092, 2009.

It is an object to provide a tag peptide that is optimal for purification of membrane proteins and the like. The present inventors have stated that “DRD1 has a limited expression site and is a membrane protein and is not detected by a normal cell extraction method. Therefore, an antibody that recognizes DRD1 and a peptide derived from an epitope of the antibody are identified. The idea was that if used, membrane proteins and the like could be purified with high sensitivity and specificity. Furthermore, a protein purification method (affinity tag system) using an antibody that recognizes DRD1 and a peptide derived from an epitope of the antibody has not been reported.
Therefore, an object of the present invention is to provide a protein purification method using an antibody that recognizes DRD1 and a peptide derived from an epitope of the antibody.

  The present inventor prepared DRD1 proteoliposome as an antigen, and prepared an antibody based on the antigen. Next, the peptide sequence derived from the epitope of the antibody was determined based on the sequence of the epitope of the antibody. Then, a protein purification method using an antibody recognizing DRD1 and a peptide derived from the epitope of the antibody was established.

That is, the present invention is as follows.
1. The following formula (I);
Gly-Gln-His-X-Thr (I)
A tag peptide comprising an amino acid sequence represented by (wherein X represents a hydrophobic amino acid), an amino acid sequence represented by Gln-His-Pro-Thr, or an amino acid sequence represented by His-Pro-Thr; Peptide for tag peptide elution.
2. 2. The tag peptide or tag peptide elution peptide according to item 1 above, wherein X is Pro, Val, Trp, Gly, Leu, Phe or Met.
3. 3. The tag peptide or tag peptide elution peptide according to 1 or 2 above, wherein X is Pro or Val.
4). 4. The tag peptide according to any one of the preceding items 1 to 3, wherein the tag peptide eluting peptide has an amino acid sequence of Thr-Ser-Gln-Asn on the N-terminal side of the peptide for eluting tag peptide.
5. A tag peptide fusion protein in which the tag peptide according to any one of items 1 to 4 is fused.
6). A protein purification method comprising the following steps.
(1) A step of contacting a protein containing the tag peptide according to any one of items 1 to 4 or a solution containing the tag peptide fusion protein according to item 5 with an antibody or antibody fragment that recognizes DRD1 (2) 6. Step of adding tag peptide eluent 7. The method for purifying a protein according to item 6, wherein the tag peptide eluent contains the peptide for eluting tag peptide according to any one of items 1 to 4.
8). 8. The method for purifying a protein according to item 7 above, wherein X is Pro, Val, Trp, Gly, Leu, Phe or Met.
9. 9. The method for purifying a protein according to item 7 or 8, wherein the tag peptide elution peptide further has an amino acid sequence of Thr-Ser-Gln-Asn on the N-terminal side.
10. 10. The method for purifying a protein according to any one of items 7 to 9, wherein the tag peptide elution peptide has a higher binding affinity for an antibody or antibody fragment that recognizes DRD1 than the tag peptide.
11. 11. The method for purifying a protein according to any one of 7 to 10 above, wherein a combination of the tag peptide and the peptide for eluting the tag peptide is as follows.
(1) Tag peptide is GQHVT-COOH, tag peptide elution peptide is GQHPT-COOH (2) Tag peptide is GQHVT-COOH, tag peptide elution peptide is TSQNGQHPT-COOH (3) Tag The peptide is GQHWT-COOH and the peptide for eluting the tag peptide is GQHPT-COOH (4) The tag peptide is GQHWT-COOH and the peptide for eluting the tag peptide is TSQNGQHPT-COOH (5) The tag peptide is GQHGT- COOH, tag peptide eluting peptide is GQHPT-COOH (6) tag peptide is GQHGT-COOH, tag peptide eluting peptide is TSQNGQHPT-COOH (7) tag peptide is GQHLT-COOH, Tag peptide elution peptide is GQHPT-COOH (8) Tag peptide is GQHLT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (9) Tag peptide is GQHFT-COOH, for tag peptide elution The peptide is GQHPT-COOH (10) The tag peptide is GQHFT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (11) The tag peptide is GQHMT-COOH, and the peptide for eluting tag peptide is GQHPT- COOH (12) Tag peptide is GQHMT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (13) Tag peptide is QHPT-COOH, Tag peptide elution peptide is GQHPT-COOH ( 14) The tag peptide is QHPT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (15) The tag peptide is HPT-COOH, and the peptide for eluting tag peptide is GQHPT-COOH (16) Is HPT-COOH and the peptide for eluting the tag peptide is TSQNGQHPT-COOH A method for producing a protein, comprising the following steps.
(1) A step of producing a protein in which the tag peptide according to any one of 1 to 4 above is fused or the tag peptide fusion protein according to the above 5 with a cell-free protein synthesis solution,
(2) contacting a cell-free protein synthesis solution containing the tag peptide fusion protein with an antibody or antibody fragment that recognizes DRD1, and
(3) Step of adding tag peptide eluent 13. The method for producing a protein according to item 12, wherein the protein is a membrane protein.
14 14. The method for producing a protein according to item 12 or 13, wherein the tag peptide eluent contains the tag peptide elution peptide according to any one of items 1 to 4.
15. 15. The method for producing a protein according to 14 above, wherein X is Pro, Val, Trp, Gly, Leu, Phe or Met.
16. 16. The method for producing a protein according to item 14 or 15, wherein the tag peptide elution peptide further has an amino acid sequence of Thr-Ser-Gln-Asn on the N-terminal side.
17. 17. The method for producing a protein according to any one of the above items 14 to 16, wherein the tag peptide elution peptide has a higher binding affinity for an antibody or antibody fragment that recognizes DRD1 than the tag peptide.
18. 18. The method for producing a protein according to any one of items 14 to 17, wherein a combination of the tag peptide and the peptide for eluting the tag peptide is as follows.
(1) Tag peptide is GQHVT-COOH, tag peptide elution peptide is GQHPT-COOH (2) Tag peptide is GQHVT-COOH, tag peptide elution peptide is TSQNGQHPT-COOH (3) Tag The peptide is GQHWT-COOH and the peptide for eluting the tag peptide is GQHPT-COOH (4) The tag peptide is GQHWT-COOH and the peptide for eluting the tag peptide is TSQNGQHPT-COOH (5) The tag peptide is GQHGT- COOH, tag peptide eluting peptide is GQHPT-COOH (6) tag peptide is GQHGT-COOH, tag peptide eluting peptide is TSQNGQHPT-COOH (7) tag peptide is GQHLT-COOH, Tag peptide elution peptide is GQHPT-COOH (8) Tag peptide is GQHLT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (9) Tag peptide is GQHFT-COOH, for tag peptide elution The peptide is GQHPT-COOH (10) The tag peptide is GQHFT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (11) The tag peptide is GQHMT-COOH, and the peptide for eluting tag peptide is GQHPT- COOH (12) Tag peptide is GQHMT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (13) Tag peptide is QHPT-COOH, Tag peptide elution peptide is GQHPT-COOH ( 14) The tag peptide is QHPT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (15) The tag peptide is HPT-COOH, and the peptide for eluting tag peptide is GQHPT-COOH (16) Is HPT-COOH and the peptide for peptide peptide elution is TSQNGQHPT-COOH

The present invention has at least one of the following effects. Moreover, in this invention, it is not limited to the following effect.
(1) In the protein purification method of the present invention, a DRD1-derived peptide (a peptide derived from an epitope of an antibody that recognizes DRD1) having low tissue intrinsicity is used, so the background is low (Example 1).
(2) The antibody recognizing DRD1 of the present invention does not show binding affinity to subtypes other than DRD1 (Example 2).
(3) The antibody that recognizes DRD1 of the present invention is a high-affinity antibody against DRD1 (Example 3).
(4) The tag peptide of the present invention is shorter than the known His tag (6 to 10 residues). Because it is as short as 5 residues, it can be easily inserted into the target protein by PCR. Moreover, since the tag size is small, it does not interfere with the structure of the fused protein (Example 6).
(5) In the protein purification method of the present invention, a protein fused with a tag peptide can be purified with high purity in one-step (Example 7).
(6) In the protein purification method of the present invention, the immobilized antibody can be washed under severe dissociation conditions and can be easily regenerated (Example 8).
(7) In the protein purification method of the present invention, the tag fusion protein can be purified with high purity (Example 10).
(8) In the protein purification method of the present invention, a fluorescent protein that maintains fluorescence activity can be purified with high purity (Example 11).
(9) The protein purification method of the present invention can purify an enzyme maintaining its enzyme activity with high purity (Example 12).
(10) The protein purification method of the present invention can purify a membrane protein in a state of maintaining the membrane protein structure with high purity (Example 13).
(11) The protein production method of the present invention can purify a membrane protein from a cell membrane with high purity (Example 14).

Results of expression pattern analysis of human DRD1 gene {Source: COXPRESdb (coxpresdb.jp/)}. Confirmation of specificity of Ra48 and Ra62 antibody subtypes. A: Confirmation of binding affinity between Ra48 antibody and C-terminal region of DRD1. B: Confirmation of affinity between Ra62 and D1CE sequences. C: Confirmation of affinity between Ra62 and CP5 sequences. The result of the binding detection of Ra48 antibody or Ra62 antibody and DRD1 swap mutant using AlphaScreen is shown. The result of the binding detection of Ra48 antibody or Ra62 antibody and biotinylated fusion protein using AlphaScreen is shown. Determination of the epitope sequence of Ra62 antibody. A: Epitope mapping using alanine-substituted mutant. B: Epitope mapping using deletion mutants. In A and B, the upper row shows the detection results with the Ra62 antibody. The lower row shows the detection result (positive control) with the anti-GEP antibody. “Venus” in the figure means Venus fluorescent protein, “Q (glutamine)”, “N (asparagine)”, “G (glycine)”, “H (histidine)”, “P (proline)”, “T (threonine)” and “A (alanine)” represent amino acid residues in the C-terminal region of DRD1 fused. C: Amino acid sequences of human DRD1, mouse DRD1, and rabbit DRD1. FIG. 6C shows the position of the D1CE sequence in the human, mouse and rabbit DRD1 amino acid sequences. Confirmation of specificity of Ra62 antibody. Western blotting using Ra62 antibody was performed on extracts of various cultured cells of HeLa, Hek293T, MCF7, Huh7, Jurkat, H1299 and U2OS. As a result, it was confirmed that noise was low and antibody specificity was high. A: Blueprint of a protein fused with the C-terminal region of DRD1 containing the D1CE sequence. B: FLAG-GST-DRD1 Among the C-terminal region fusion protein, the detection result by Ra62 antibody of a mutant in which the C-terminal 3 amino acid residues of the D1CE sequence are converted one by one. Among the D1CE sequences (GQHPT), for “H”, “P” or “T”, “R”, “K”, “G”, “V”, “L”, “F”, “M”, “ A construct converted to “I”, “W”, “D”, “E”, “S”, “N”, “Q”, “Y” or “C” was used. The upper row shows the detection results using Ra62 antibody, and the lower row shows the detection results using anti-FLAG antibody for confirming the amount of protein synthesis. C: Biacore analysis results of 16 kinds of amino acid substitution mutants of D1CE sequence and Ra62 antibody. An enlarged view of a part of the Biacore analysis result is shown on the right side of the figure. Conceptual diagram of CP5 tag cloning. The base sequence encoding the CP5 tag is shown on the left side of the figure. Based on this base sequence, primers were designed and cloned as shown on the right side of the figure. Purification of GST protein using CP5 tag. The CBB dyeing | staining image of each sample of a purification process is shown. Shiroyajiri indicates a band of a GST fusion protein having a CP5 tag. Purification of Venus protein using CP5 tag. The CBB dyeing | staining image of each sample of a purification process is shown. White arrowheads indicate the position of the Venus protein band to which the CP5 tag is added. Purification of DRD1 protein using CP5 tag. A: A CBB-stained image of each sample in the purification process is shown. Black arrowheads indicate the positions of the heavy and light chain bands of the Ra62 antibody contained in the DRD1 protein to which the CP5 tag is added and the resin fraction. Shiroyajiri indicates lipid. B: Absorbance (280 nm wavelength) of each fraction during the purification process. Purification of CLDN1 protein using CP5 tag. A: A CBB-stained image of each sample in the purification process is shown. White arrowheads indicate the positions of the dimer and monomer bands of the CLDN1 protein to which the CP5 tag was added. B: Absorbance (280 nm wavelength) of each fraction during the purification process. Purification of M2 protein using CP5 tag. A: CBB stained image of each sample in the purification process. White arrowhead indicates the position of the band of the M2 protein to which the CP5 tag is added.

(Terms in this specification)
The polynucleotide (DNA molecule) in this specification includes not only double-stranded DNA but also single-stranded DNA of sense strand and antisense strand constituting it, and is not limited to the length. .
Moreover, the polynucleotide (DNA molecule) in this specification does not ask | require the distinction of a functional region, and can contain at least one of an expression suppression area | region, a coding region, a leader sequence, an exon, and an intron. The polynucleotide includes RNA and DNA.
Polynucleotide variants (mutant DNA) include naturally occurring allelic variants, non-naturally occurring variants, variants with deletions, substitutions, additions and insertions. However, these variants shall encode a polypeptide having substantially the same function as that of the polypeptide encoded by the polynucleotide before mutation.

(Dopamine D1 receptor)
The dopamine D1 receptor (dopamine receptor D1, DRD1) has an open reading frame (ORF) of 1341 bases, and is a putative 50 kDa protein consisting of 446 amino acid residues.
DRD1 is one of dopamine receptors belonging to a family of membrane proteins called GPCR (G protein coupled receptor). By binding dopamine, which is a ligand molecule, the action of dopamine is transmitted into the cell.

Polypeptide or polynucleotide homology can be analyzed by measurement using the FASTA program {Clustal, V., Methods Mol. Biol., 25, 307-318 (1994)}.
As the most preferable and simple method of homology analysis, the following known sequence database can be used.
・ DNA Database of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/)
・ Genebank (http://www.ncbi.nlm.nih.gov/web/Genebank/Index.htlm)
・ The European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL) (http://www.ebi.ac.uk/ebidocs/embl db.html)
Many search algorithms are available for homology analysis. One example is a program called a BLAST program.

(Antibodies of the present invention)
The antibodies of the present invention are intended for the two antibodies (Ra48 antibody and Ra62 antibody) obtained in the following Examples. The Ra48 antibody of the present invention has a molecular weight of about 150,000 daltons and is IgG. The Ra62 antibody of the present invention has a molecular weight of about 150,000 daltons and is IgG.

(Ra48 antibody of the present invention)
An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a heavy chain variable region sequence in an antigen recognition site.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 3 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 3 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 (5) the amino acid sequence set forth in SEQ ID NO: 1 (6) A deletion or insertion of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1. A polypeptide represented by an amino acid sequence inserted, substituted or added (7) a polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence of SEQ ID NO: 1

An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a heavy chain sequence at the antigen recognition site.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 4 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 4 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 4 (5) the amino acid sequence set forth in SEQ ID NO: 2 (6) In the amino acid sequence of SEQ ID NO: 2, one or more amino acids are deleted or inserted A polypeptide represented by an amino acid sequence inserted, substituted or added (7) a polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence of SEQ ID NO: 2

An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a light chain variable region sequence in an antigen recognition site.
(1) A polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 7 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 7 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 7 (4) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 7 (5) The amino acid sequence set forth in SEQ ID NO: 5 (6) In the amino acid sequence of SEQ ID NO: 5, one or more amino acids are deleted or inserted A polypeptide represented by the amino acid sequence inserted, substituted or added (7) a polypeptide having a homology of 95% or more with the polypeptide represented by the amino acid sequence of SEQ ID NO: 5

An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a light chain sequence at the antigen recognition site.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 8 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 8 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 (4) Polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 (5) Amino acid sequence set forth in SEQ ID NO: 6 (6) A deletion or insertion of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 6 A polypeptide represented by an amino acid sequence inserted, substituted or added (7) a polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence of SEQ ID NO: 6

A polypeptide selected from the following (1) to (14) is used as a heavy chain variable region sequence or heavy chain sequence, and a polypeptide selected from the following (15) to (28) is used as a light chain variable region sequence or light chain. An antibody or antibody fragment that recognizes DRD1, which is contained in the antigen recognition site as a chain sequence.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 3 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 3 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 3 (5) the amino acid sequence set forth in SEQ ID NO: 1 (6) A deletion or insertion of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1. A polypeptide represented by the amino acid sequence inserted, substituted or added (7) A polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence described in SEQ ID NO: 1 (8) A polypeptide encoded by the polynucleotide represented by the nucleotide sequence described above (9) represented by a nucleotide sequence in which one or more bases are deleted, inserted, substituted or added in the nucleotide sequence described in SEQ ID NO: 4 Polypeptide encoded by polynucleotide (10) Polypeptide encoded by polynucleotide represented by nucleotide sequence having 95% or more homology with the polynucleotide represented by SEQ ID NO: 4 (11) Sequence A polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide represented by the nucleotide sequence of No. 4 is (12) Polypeptide represented by the amino acid sequence described in SEQ ID NO: 2 (13) Amino acid in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence described in SEQ ID NO: 2 Polypeptide represented by sequence (14) Polypeptide having 95% or more homology with polypeptide represented by amino acid sequence described in SEQ ID NO: 2 (15) Represented by base sequence described in SEQ ID NO: 7 Polypeptide encoded by polynucleotide (16) Polypeptide encoded by a polynucleotide represented by a nucleotide sequence in which one or more bases are deleted, inserted, substituted or added in the nucleotide sequence of SEQ ID NO: 7 17) A polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 7 Polypeptide encoded by Otide (18) Polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide represented by the base sequence described in SEQ ID NO: 7 (19) described in SEQ ID NO: 5 (20) A polypeptide represented by an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence of SEQ ID NO: 5. (21) Sequence A polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence of No. 5 (22) The polypeptide encoded by the polynucleotide represented by the base sequence of SEQ ID No. 8 (23) Sequence A base in which one or more bases are deleted, inserted, substituted or added in the base sequence of No. 8. Polypeptide encoded by polynucleotide represented by sequence (24) Polypeptide encoded by polynucleotide represented by base sequence having 95% or more homology with polynucleotide represented by base sequence described in SEQ ID NO: 8 Peptide (25) Polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with the complementary strand of the polynucleotide represented by the base sequence set forth in SEQ ID NO: 8 (26) Expressed by the amino acid sequence set forth in SEQ ID NO: 6 (27) A polypeptide represented by an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence described in SEQ ID NO: 6 (28) described in SEQ ID NO: 6 A polypeptide having a homology of 95% or more with a polypeptide represented by an amino acid sequence

(Ra62 antibody of the present invention)
An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a heavy chain variable region sequence in an antigen recognition site.
(1) A polypeptide encoded by a polynucleotide represented by the base sequence described in SEQ ID NO: 11 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 11 (3) A polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 11 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 11 (5) the amino acid sequence set forth in SEQ ID NO: 9 (6) In the amino acid sequence set forth in SEQ ID NO: 9, one or more amino acids are Polypeptide represented by amino acid sequence deleted, inserted, substituted or added (7) Polypeptide having homology of 95% or more with polypeptide represented by amino acid sequence of SEQ ID NO: 9

An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a heavy chain sequence at the antigen recognition site.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 12 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 12 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 12 (4) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 12 (5) The amino acid sequence set forth in SEQ ID NO: 10 (6) one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 10 Polypeptide represented by amino acid sequence in which acid is deleted, inserted, substituted or added (7) Polypeptide having 95% or more homology with polypeptide represented by amino acid sequence of SEQ ID NO: 10

An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a light chain variable region sequence in an antigen recognition site.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 15 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 15 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 15 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 15 (5) the amino acid sequence set forth in SEQ ID NO: 13 (6) one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 13 Polypeptide represented by amino acid sequence in which acid is deleted, inserted, substituted or added (7) Polypeptide having 95% or more homology with polypeptide represented by amino acid sequence of SEQ ID NO: 13

An antibody or antibody fragment that recognizes DRD1, comprising a polypeptide selected from the following group as a light chain sequence at the antigen recognition site.
(1) Polypeptide encoded by the polynucleotide represented by the base sequence described in SEQ ID NO: 16 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 16 (3) a polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 16 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 16 (5) the amino acid sequence set forth in SEQ ID NO: 14 (6) one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 14 Polypeptide represented by amino acid sequence in which acid is deleted, inserted, substituted or added (7) Polypeptide having 95% or more homology with polypeptide represented by amino acid sequence of SEQ ID NO: 14

A polypeptide selected from the following (1) to (14) is used as a heavy chain variable region sequence or heavy chain sequence, and a polypeptide selected from the following (15) to (28) is used as a light chain variable region sequence or light chain. An antibody or antibody fragment that recognizes DRD1, which is contained in the antigen recognition site as a chain sequence.
(1) A polypeptide encoded by a polynucleotide represented by the base sequence described in SEQ ID NO: 11 (2) One or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID NO: 11 (3) A polynucleotide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 11 (4) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 11 (5) the amino acid sequence set forth in SEQ ID NO: 9 (6) In the amino acid sequence set forth in SEQ ID NO: 9, one or more amino acids are Polypeptide represented by amino acid sequence deleted, inserted, substituted or added (7) Polypeptide having 95% or more homology with the polypeptide represented by amino acid sequence of SEQ ID NO: 9 (8) Sequence A polypeptide encoded by the polynucleotide represented by the base sequence described in No. 12. (9) A base sequence in which one or more bases are deleted, inserted, substituted or added in the base sequence described in SEQ ID No. 12. (10) a polypeptide encoded by a polynucleotide represented by a nucleotide sequence having a homology of 95% or more with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 12 11) A polynucleotide that hybridizes under stringent conditions with a complementary strand of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 12 Polypeptide encoded by leotide (12) Polypeptide represented by the amino acid sequence shown in SEQ ID NO: 10 (13) One or more amino acids in the amino acid sequence shown in SEQ ID NO: 10 are deleted, inserted, substituted or added (14) A polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence described in SEQ ID NO: 10 (15) The base sequence described in SEQ ID NO: 15 Polypeptide Encoded by Polynucleotide Represented (16) Encoded by a polynucleotide represented by a base sequence in which one or more bases are deleted, inserted, substituted or added in the base sequence set forth in SEQ ID NO: 15 Polypeptide (17) Has 95% or more homology with the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 15 Polypeptide encoded by polynucleotide represented by base sequence (18) Polypeptide encoded by polynucleotide hybridizing under stringent conditions with complementary strand of polynucleotide represented by base sequence described in SEQ ID NO: 15 ( 19) a polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 13 (20) represented by an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 13 (21) A polypeptide having 95% or more homology with the polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 13 (22) The polynucleotide represented by the base sequence set forth in SEQ ID NO: 16 is encoded (23) one or more polypeptides in the nucleotide sequence set forth in SEQ ID NO: 16 Polypeptide encoded by a polynucleotide represented by a base sequence in which a base is deleted, inserted, substituted or added (24) 95% or more homology with the polynucleotide represented by the base sequence described in SEQ ID NO: 16 A polypeptide encoded by a polynucleotide represented by the nucleotide sequence having the polypeptide (25) a polypeptide encoded by a polynucleotide that hybridizes with a complementary strand of the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 16 under stringent conditions (26) a polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 14 (27) represented by an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 14 (28) A polypeptide represented by the amino acid sequence of SEQ ID NO: 14 and 95% Polypeptides having homology of above

  The present Ra48 antibody comprises a humanized antibody or humanized antibody fragment, a single chain antibody or a single chain antibody fragment, or a Fab antibody or Fab antibody fragment. Furthermore, the antibody can be made into a humanized antibody by a method known per se.

In the present invention, “a plurality of amino acids are deleted, inserted, substituted or added” means that 2 to 20 amino acids are deleted, inserted, substituted or added. The plurality of amino acids is preferably 2 or more and 10 or less, more preferably 2 or more and 7 or less, and even more preferably 2 or more and 5 or less. Further, this modified amino acid sequence has a homology with the amino acid sequence described in SEQ ID NO: of, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more, and even more preferably about 98%. % Or better.
Means for introducing mutations such as deletion, substitution, addition or insertion are known per se, for example using Ulmer's technology (KM Ulmer, "Science" 1983, Vol. 219, pp. 666-671) it can. From the viewpoint of not changing the basic properties (physical properties, functions, immunological activity, etc.) of the polypeptide containing the modified amino acid in the introduction of such mutations, for example, homologous amino acids (polar amino acids, nonpolar amino acids) Mutual substitution between amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned. Furthermore, these usable polypeptides can be altered to the extent that they do not undergo significant changes in function, such as modification of the constituent amino group or carboxyl group, for example, by amidation.

In the present invention, “a plurality of bases are deleted, inserted, substituted or added” means that 2 to 20 bases have been deleted, inserted, substituted or added. The plurality of bases is preferably 2 or more and 20 or less, more preferably 2 or more and 10 or less, and even more preferably 2 or more and 5 or less. Further, this modified base sequence has a homology with the base sequence described in SEQ ID NO: of, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more, and still more preferably about 98. % Or better.
Here, the “stringent conditions” are conditions in which hybridization is performed at 50 ° C. in 2 × SSC containing 0.1% SDS, and is not detached even by washing at 60 ° C. in 1 × SSC containing 0.1% SDS. Can be mentioned.

{Immunogen (antigen) used for preparation of Ra48 antibody and Ra62 antibody of the present invention}
As an immunogen for producing the Ra48 antibody and Ra62 antibody of the present invention, DRD1 (or DRD1 fragment) proteoliposome containing a peptide containing all or part of the amino acid sequence of DRD1 can be used.
Preferably, DRD1 is synthesized by a wheat germ cell-free protein synthesis system known per se, in particular, a synthesis system capable of synthesizing a membrane protein as a proteoliposome (reference: ProteoLiposome ExpressionKit (manufacturing and sales: Cell Free Science Co., Ltd.)).
The production of proteoliposomes for immune antigens can be carried out by genetic engineering techniques, chemical synthesis, cell-free protein synthesis, and the like. Artificial lipid vesicles (liposomes) contained in proteoliposomes are described in Nozawa et al. 2011 (BMC Biotechnol. 2011 Apr 11; 11: 35. Doi: 10.1186 / 1472-6750-11-35.) And Takeda et al. 2015 ( ScientificReports 2015: http://www.nature.com/articles/srep11333). After the proteoliposome is produced, it can be further purified and used.
In addition, purification and / or separation of proteoliposomes can be performed by various separation operation methods utilizing their physical properties, chemical properties, and the like. Examples of the separation operation method include known methods such as ammonium sulfate precipitation, ultrafiltration, gel chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, and dialysis. These methods can be used alone or in appropriate combination.

(Immune method)
A purified proteoliposome as an immunogen described above is dissolved or suspended in an appropriate buffer such as a phosphate buffer (PBS) as an antigen solution. In addition, when the antigenicity of the proteoliposome alone is low, it can be used after being crosslinked with an appropriate carrier protein such as albumin or keyhole limpet hemocyanin (KLH).
Examples of animals (immunized animals) immunized with the antigen include mice, rats, hamsters, horses, goats, rabbits, and the like. Rabbits are preferred.

  In order to enhance the responsiveness of the immunized animal to the antigen, the antigen solution can be mixed with an adjuvant and administered. The adjuvants that can be used here are Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), and Ribi (MPL + TDM). Examples include pertussis vaccine (Boredetella pertussis vaccine), muramyl dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof, but combinations using FCA for the first immunization and FIA or Ribi adjuvant for the additional immunization are particularly preferable. .

  The immunization method can appropriately change the injection site, schedule, etc. depending on the type of antigen used and the presence or absence of adjuvant mixing. For example, when a rabbit is used as the immunized animal, Freund's complete adjuvant and antigen ( HEL or KLH conjugated phosphorylated DRD1 proteoliposomes) is made and injected into the rabbit intraperitoneally, subcutaneously, intramuscularly or (tail) intravenously. A Freund's incomplete adjuvant and antigen cocktail are made 2, 4, and 6 weeks after the first immunization, and injected similarly subcutaneously. Rabbits are sacrificed one week after the final immunization.

(Acquisition of specific antibody-secreting cells by ISAAC method)
In the method for obtaining an antibody of the present invention, the ISAAC method is preferably used. The ISAAC technology can easily produce a rabbit monoclonal antibody that has been difficult to produce in the past (see Patent Document 1).
Specifically, IgG ⁺ cells obtained from rabbits immunized with DRD1 proteoliposomes were arrayed on a chip coated with anti-rabbit IgG antibody, and DRD1-specific antibody-secreting cells using biotinylated DRD1 proteoliposome and Cy3 conjugated streptavidin Is detected.

(Preparation of monoclonal antibodies using antibody cDNA-introduced CHO cells)
Single rabbit lymphocytes are recovered from the chip where DRD1-specific antibody-secreting cells are detected. Collect single cells that generated immunospots of DRD1-specific IgG in the rabbit-ISAAC system and separate individual cells to amplify immunoglobulin heavy (H) and light (L) variable region cDNAs Transfer into a microtube.
The variable regions of antibody heavy chain (H chain) and light chain (L chain) variable regions are amplified from a single rabbit lymphocyte in a microtube by a single cell 5′-RACE method. The obtained antibody heavy chain and light chain cDNAs are incorporated into H chain and L chain expression vectors, respectively, and introduced into CHO cells (cells derived from Chinese hamster ovary tissue) to produce antibody proteins.

(Other monoclonal antibody production methods)
In addition to the above, monoclonal antibodies (hereinafter may be abbreviated as “mAb”) can be obtained by a method known per se, for example, Kohler G, Milstein C. (1975) Continuous cultures of fused cells secretingantibody of Predefined specificity. Nature 256, 495-497.).
For example, a tissue (for example, spleen or lymph node) containing antibody-producing cells is collected from an immunized animal, and a hybridoma is prepared by fusing the antibody-producing cells with known tumor cells (for example, bone tumor cells). Subsequently, after the hybridoma is cloned, the hybridoma producing the desired antibody is selected, and the antibody is recovered from the culture medium of this hybridoma.
As myeloma cells, cells derived from mice, rats, humans, etc. are used. Examples are myeloma cells. Some myeloma cells produce an immunoglobulin light chain, and if this is used as a fusion target, the immunoglobulin heavy chain and light chain produced by the antibody-producing cell may be bound randomly. In particular, it is preferable to use myeloma cells that do not produce an immunoglobulin light chain, such as P3X63-Ag8 · 653 and SP2 / o-Ag14.
The antibody-producing cells and the myeloma cells are preferably derived from the same species, particularly the same strain. The myeloma cells may be stored according to a method known per se. For example, those subcultured in a general medium supplemented with horse, rabbit or fetal calf serum are stored by freezing. For cell fusion, cells in the logarithmic growth phase are preferably used.

(Production of hybridoma)
Examples of methods for producing hybridomas by fusing antibody-producing cells and myeloma cells include methods using polyethylene glycol (PEG), methods using Sendai virus, and methods using an electrofusion device. For example, in the case of the PEG method, splenocytes and myeloma cells are placed in an appropriate medium or buffer containing about 30 to 60% PEG (average molecular weight 1,000 to 6,000), 1 to 10: 1, preferably 5 to 10: 1. And the reaction may be carried out for about 30 seconds to 3 minutes under conditions of a temperature of about 25 to 37 ° C. and a pH of 6 to 8. After completion of the reaction, the cells are washed, removed from the PEG solution, resuspended in the medium, seeded in a microtiter plate, and the culture is continued.
Cells after the fusion operation are cultured in a selective medium to select hybridomas. The selection medium is a medium in which the parent cell line can be killed and only fused cells can grow, and hypoxanthine-aminopterin-thymidine (HAT) medium is usually used. Selection of hybridomas is usually carried out by exchanging a part of the medium, preferably about half of the medium, with the selective medium 1 to 7 days after the fusion operation, and further culturing while repeating the same medium exchange every 2 or 3 days. The well where the hybridoma colony is growing is confirmed by microscopic observation.

In order to know whether the growing hybridoma is producing the desired antibody, the culture supernatant is collected and an antibody titer assay may be performed by a method known per se.
Further, single clones are separated by a limiting dilution method, a soft agar method, or a method using a fluorescence excitation cell sorter.

  The immunoglobulin subclass of the antibody produced by the hybridoma is obtained by culturing the hybridoma under general conditions and analyzing the antibody secreted in the culture supernatant using a commercially available antibody class / subclass determination kit or the like. I can know.

(Method for obtaining mAb from hybridoma)
The method for obtaining mAb from the hybridoma can be appropriately selected depending on the required amount and the properties of the hybridoma. For example, a method of obtaining from mouse ascites transplanted with the hybridoma, a method of obtaining from the culture supernatant by cell culture, and the like are exemplified. A hybridoma capable of growing in the mouse abdominal cavity can obtain a high concentration mAb of several mg / mL from ascites. For hybridomas that cannot grow in vivo, mAbs are obtained from the culture supernatant of the cell culture.
Obtaining mAbs by cell culture has the advantage that the amount of mAb produced is lower than in vivo, but there is little contamination with immunoglobulins and other contaminants contained in the mouse abdominal cavity, and purification is easy.
When mAb is obtained from the abdominal cavity of a mouse transplanted with a hybridoma, for example, the intraperitoneal cavity of a BALB / c mouse previously administered with an immunosuppressive substance such as pristane (2, 6, 10, 14-tetramethylpentadecane) is used. Hepaticoma (about 10 ⁶ or more) is transplanted, and ascites collected after about 1 to 3 weeks is collected. In the case of heterologous hybridomas (for example, mice and rats), it is preferable to use nude mice or radiation-treated mice.
When obtaining the mAb from the cell culture supernatant, for example, in addition to the stationary culture method used for cell maintenance, the hybridoma is cultured using a culture method such as a high-density culture method or a spinner flask culture method, and the mAb is obtained. A culture supernatant containing is obtained.
Purification of mAb from ascites or culture supernatant can be performed by a method known per se. For example, the conventionally known fractionation method by salting out using ammonium sulfate or sodium sulfate, polyethylene glycol fractionation method, ethanol fractionation method, DEAE ion exchange chromatography method, gel filtration method, etc. are applied as the purification method of immunoglobulin. This is easily achieved.
Furthermore, when the mAb is mouse IgG, it can be easily purified by affinity chromatography using a protein A binding carrier or an anti-mouse immunoglobulin binding carrier.

(Peptide of the present invention)
The peptides of the present invention are intended for both tag peptides and / or peptides for eluting tag peptides.
The tag peptide of the present invention is a peptide (preferably a peptide sequence introduced at the C-terminus) fused to an expressed (target / target) protein. More specifically, the tag peptide has an action of binding to an antibody or antibody fragment that recognizes DRD1.
The peptide for eluting tag peptides of the present invention has the effect of releasing the tag peptide from the antibody or antibody fragment that recognizes DRD1.

The tag peptide and / or peptide for eluting tag peptide of the present invention is represented by the amino acid sequence represented by the following formula (I), the amino acid sequence represented by Gln-His-Pro-Thr, or the His-Pro-Thr. Contains or consists of an amino acid sequence.
(I) Gly-Gln-His-X-Thr (wherein X represents a hydrophobic amino acid)
In the formula, X represents a hydrophobic amino acid, preferably Pro, Val, Trp, Gly, Leu, Phe or Met, more preferably Pro or Val.
In addition, 5 residues (GQHPT-COOH) containing the C-terminal carboxyl group of DRD1 were named as DRD1 C-Terminus Epitope (D1CE) sequences. In addition, a mutant sequence (GQHVT-COOH) in which proline at the second residue from the C-terminus of the D1CE sequence was replaced with valine was named C-terminus Peptide 5 (CP5) sequence.
In addition, in the peptide for eluting tag peptides, about 1 to 10 arbitrary amino acid sequences (eg, Thr-Ser-Gln-Asn) may be added to the N-terminal side for solubility and / or charge adjustment. .

The combination of the tag peptide and tag peptide eluting peptide of the present invention is preferably such that the tag peptide eluting peptide has a higher binding affinity for an antibody or antibody fragment that recognizes DRD1 than the tag peptide, There is no particular limitation, and the following can be exemplified (left: tag peptide, right: peptide for eluting tag peptide).
(1) GQHVT-COOH and GQHPT-COOH
(2) GQHVT-COOH and TSQNGQHPT-COOH
(3) GQHWT-COOH and GQHPT-COOH
(4) GQHWT-COOH and TSQNGQHPT-COOH
(5) GQHGT-COOH and GQHPT-COOH
(6) GQHGT-COOH and TSQNGQHPT-COOH
(7) GQHLT-COOH and GQHPT-COOH
(8) GQHLT-COOH and TSQNGQHPT-COOH
(9) GQHFT-COOH and GQHPT-COOH
(10) GQHFT-COOH and TSQNGQHPT-COOH
(11) GQHMT-COOH and GQHPT-COOH
(12) GQHMT-COOH and TSQNGQHPT-COOH
(13) QHPT-COOH and GQHPT-COOH
(14) QHPT-COOH and TSQNGQHPT-COOH
(15) HPT-COOH and GQHPT-COOH
(16) HPT-COOH and TSQNGQHPT-COOH
In the present invention, “binding affinity for an antibody or antibody fragment recognizing DRD1” can be represented by a dissociation constant (Kd) measured and calculated by a method known per se. For example, a peptide containing a D1CE sequence has a higher binding affinity for an antibody or antibody fragment that recognizes DRD1 than a peptide containing a CP5 sequence. Therefore, the combination of the tag peptide and the peptide for eluting the tag peptide is preferably a tag peptide containing a CP5 sequence and a peptide containing a D1CE sequence as a peptide for eluting a tag peptide.

(Tag peptide fusion protein)
The tag peptide of the present invention can be combined with an expressed (target / target) protein to obtain a fusion protein by a method known per se. The tag peptide can be bound to either the C-terminus or the N-terminus of the expressed protein, but is preferably the C-terminus.
In addition, the tag peptide fusion protein can be purified in one-step with high purity by using a peptide for eluting the tag peptide and an antibody or antibody fragment that recognizes DRD1.
Furthermore, tag peptide fusion proteins (especially tag peptide fusion membrane proteins) include wheat germ cell-free protein synthesis systems known per se (especially synthesis systems that can synthesize membrane proteins as proteoliposomes), peptide for peptide peptide elution and DRD1. If the antibody or antibody fragment to be recognized is used, a tag peptide fusion protein (particularly a tag peptide fusion membrane protein) can be produced and purified in one-step.

The tag peptide fusion protein of the present invention can be produced by a known method. For example, a tag peptide may be added to the expressed protein by chemical bonding or physical adsorption.
In addition, by adding a DNA encoding a tag peptide fusion protein in which a DNA encoding a tag peptide is linked to the 3 ′ end or 5 ′ end of the DNA encoding the expressed protein to a known protein expression / synthesis system, Peptide fusion proteins can be produced. A spacer peptide known per se may be contained between the tag peptide and the expressed protein. The spacer peptide is not particularly limited as long as it does not affect the binding with an antibody or antibody fragment that recognizes DRD1, and may be a spacer known per se (eg, a repeating sequence of glycine), but a peptide sequence having a protease cleavage sequence, etc. But it ’s okay.
More specifically, DNA encoding the tag peptide fusion protein is inserted into a known expression vector as necessary. Then, an expression vector carrying DNA encoding the tag peptide fusion protein is introduced into a known protein synthesis system (cell-free / cell synthesis system). In the cell synthesis system, although not particularly limited, microorganisms such as Escherichia coli and yeast, and animal cells such as insect cells can be used.
It is also possible to use a cell-free protein synthesis system (a wheat or E. coli-derived synthesis system), in particular, a wheat germ cell-free protein synthesis system (see: wheat cell-free protein synthesis kit, production and sales: Cell Free Science Co., Ltd.). preferable.

(An antibody or antibody fragment that recognizes DRD1 for purification of tag peptide fusion protein)
The antibody or antibody fragment that recognizes DRD1 for purification of the tag peptide fusion protein of the present invention is not particularly limited as long as it can bind to the tag peptide of the tag peptide fusion protein, but examples include the Ra48 antibody and Ra62 antibody described above. Particularly preferred is Ra62 antibody.

(Method for purifying the protein of the present invention)
The protein purification method of the present invention includes at least the following steps.
(1) Contacting a solution containing a tag peptide fusion protein with an antibody or antibody fragment that recognizes DRD1 (2) Adding a tag peptide eluent

In the above step (1), the solution containing the tag peptide fusion protein is not particularly limited, but in consideration of purifying the protein, it means a protein synthesis solution or the like that synthesizes the protein. By the contacting step, the tag peptide fusion protein and the antibody or antibody fragment recognizing DRD1 are bound.
In addition, the antibody or antibody fragment recognizing DRD1 may be immobilized on a carrier or the like, or may be attached and immobilized on beads or the like.
The carrier for immobilizing the antibody is not particularly limited, and a known carrier can be used. For example, Sepharose, Affigel, etc. can be illustrated. As a preferred example, the Ra62 antibody of the present invention can be immobilized on NHS-Sepharose by amine coupling to produce an affinity resin.
In addition, the antibody or antibody fragment binding bead (antibody binding bead) that recognizes DRD1 is prepared by immobilizing or attaching the antibody or antibody fragment recognizing DRD1 of the present invention to a known bead (eg, magnetic bead) or the like. can do.

Examples of the protein purification method of the present invention include a column method in which the above-described immobilized antibody is packed in a column and a batch method in which a solution containing antibody-bound beads and a tag peptide fusion protein is mixed.
In the column method, an immobilized antibody is packed in a column, a solution containing the tag peptide fusion protein is allowed to flow through the column, and the tag peptide fusion protein is bound to the antibody or antibody fragment.
In the batch method, beads bound with an antibody or antibody fragment are added to a solution containing the tag peptide fusion protein, gently mixed to bind the tag peptide fusion protein to the antibody or antibody fragment, and the beads are recovered. To do.

In the above-mentioned step (2), in the column method, the tag peptide eluent is passed through the column to release the tag peptide fusion protein from the antibody or antibody fragment. Thereby, the tag peptide fusion protein is contained in the eluent of the tag peptide and further recovered from the column.
In the batch method, the tag peptide eluate is added to the solution containing the collected beads to release the tag peptide fusion protein from the antibody or antibody fragment. Thereby, the tag peptide fusion protein is contained in the eluate of the tag peptide.

The eluent of the tag peptide of the present invention is not particularly limited as long as the tag peptide fusion protein can be released from the antibody or antibody fragment. For example, a solution containing the peptide for eluting the tag peptide of the present invention, a hydrophilic organic solvent, Examples thereof include sodium thiocyanate.
When a solution containing the peptide for eluting tag peptides of the present invention is used as an eluent for tag peptides, it is preferable that about 150 μM of the peptide for eluting tag peptides is contained in water, buffer solution or the like.

(Method for producing the protein of the present invention)
The method for producing a protein of the present invention includes at least the following steps.
(1) Step of producing a tag peptide fusion protein with a cell-free protein synthesis solution (2) Step of contacting a cell-free protein synthesis solution containing the tag peptide fusion protein with an antibody or antibody fragment that recognizes DRD1 (3) Tag peptide In the protein production method of the present invention, it has been confirmed that the membrane protein can be directly produced and purified from the cell membrane in one-step from the results of the following examples. Therefore, the protein production method of the present invention is particularly suitable for a membrane protein production method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

<Expression pattern analysis of DRD1>
The expression pattern of human DRD1 gene in human tissues was analyzed as follows.
DRD1 gene microarray data were obtained from BioGPS (http://biogps.org/). GeneAtlas U133A was used for the data set, 214652_at was used for the probe set, and GCRMA was used for data standardization. From the microarray results, the mRNA expression level of DRD1 is low in any tissue, and as a result, when a tag system is constructed using the antibody (Ra62 antibody) prepared in the following Example, it reacts with endogenous DRD1. It was confirmed that the fear was low (background was low) (FIG. 1).

<Preparation of antibodies that recognize DRD1>
DRD1 proteoliposomes were synthesized by a large-scale synthesis method of GPCR antigen using a wheat cell-free protein synthesis system. Details are as follows.
It is known that membrane proteins such as GPCRs can be stably synthesized as proteoliposomes by adding artificial lipid vesicles to a wheat cell-free protein synthesis system and performing a translation reaction (Nozawa et al. 2011 , Takeda et al. Scientific Reports 2015).
In this example, DRD1 proteoliposomes are produced using a dialysis layer method (see Takeda et al. Scientific Reports 2015) in which the substrate solution and reaction solution are layered in a dialysis cup and the dialysis cup is then immersed in the substrate solution. did.
Next, using DRD1 proteoliposome as an antigen, an anti-DRD1 antibody was prepared using the ISAAC method (Reference: Non-Patent Document 1) (Reference: Takeda et al. Scientific Reports 2015). More specifically, cell-free synthesized proteoliposomes containing 1 mg of DRD1 were mixed with Freund's adjuvant to immunize rabbits. After 5 immunizations, lymphocyte cells were obtained from rabbit blood, spleen and bone marrow tissue. Isolation of specific antibody-secreting cells by ISAAC was performed by partially modifying the method described in Patent Document 1.
Specifically, lymphocyte cells were seeded on a microwell chip on which an anti-rabbit antibody was immobilized, and allowed to stand for several hours to capture antibodies secreted from the cells. Thereafter, the cells were treated with cell-free synthesized biotinylated DRD1 proteoliposomes and fluorescently labeled streptavidin, respectively. Anti-DRD1 antibody-secreting cells were identified under fluorescence microscope observation, and the cells were collected with a microcapillary. The heavy chain and light chain antibody genes were isolated from the collected cells by RT-PCR, and recombined into cultured cell expression vectors. Antibody expression was performed transiently using Expi293F cells, and the obtained antibody was purified using Protein G Sepharose. Finally, a specific antibody having two unique sequences was obtained.
In this example, the obtained anti-DRD1 monoclonal antibodies Ra62 antibody and Ra48 antibody were used in the following examples.
Furthermore, the binding affinity between Ra48 antibody and Ra62 antibody was analyzed by AlphaScreen using biotinylated proteoliposome expressing human DRD1 homolog. Neither antibody showed binding affinity to subtypes other than human DRD1 and mouse Drd1 (FIG. 2).
In addition, the heavy chain sequence, heavy chain variable region sequence, light chain sequence and light chain variable region sequence of the following two antibodies were determined.

(Ra48 antibody heavy chain sequence, heavy chain variable region sequence, light chain sequence and light chain variable region sequence)
Ra48 antibody heavy chain amino acid sequence: SEQ ID NO: 2
Ra48 antibody heavy chain base sequence: SEQ ID NO: 4
Ra48 antibody heavy chain variable region amino acid sequence: SEQ ID NO: 1
Ra48 antibody heavy chain variable region nucleotide sequence: SEQ ID NO: 3
Ra48 antibody light chain amino acid sequence: SEQ ID NO: 6
Ra48 antibody light chain base sequence: SEQ ID NO: 8
Ra48 antibody light chain variable region amino acid sequence: SEQ ID NO: 5
Ra48 antibody light chain variable region nucleotide sequence: SEQ ID NO: 7

(Ra62 antibody heavy chain sequence, heavy chain variable region sequence, light chain sequence and light chain variable region sequence)
Ra62 heavy chain amino acid sequence of antibody: SEQ ID NO: 10
Ra62 antibody heavy chain base sequence: SEQ ID NO: 12
Ra62 antibody heavy chain variable region amino acid sequence: SEQ ID NO: 9
Ra62 antibody heavy chain variable region nucleotide sequence: SEQ ID NO: 11
Ra62 antibody light chain amino acid sequence: SEQ ID NO: 14
Ra62 antibody light chain base sequence: SEQ ID NO: 16
Ra62 antibody light chain variable region amino acid sequence: SEQ ID NO: 13
Ra62 antibody light chain variable region nucleotide sequence: SEQ ID NO: 15

<Binding affinity of antibody>
-Confirmation of binding affinity between Ra48 antibody and C-terminal region of DRD1
The confirmation of the binding affinity between the Ra48 antibody and the C-terminal region of DRD1 was analyzed by Biacore. The C-terminal region of FLAG-GST-DRD1 was synthesized using a wheat cell-free system as a fusion protein and purified using glutathione sepharose. The concentration of the purified FLAG-GST-DRD1 C-terminal region fusion protein was determined by the molar extinction coefficient at 280 nm determined from the amino acid sequence. The following experiment was performed using BiacoreX100. Protein G was immobilized on the sensor chip CM5 using amine coupling. Approximately 100 RU of Ra48 antibody was captured, and then various concentrations of FLAG-GST-DRD1 C-terminal region fusion protein were run. Result of obtaining kinetic parameters by curve fitting, K _D value of a binding affinity between the C-terminal region of Ra48 antibody ^{DRD1 = 4.90 × 10 -9 M (} K a = 3.18 × 10 4 1 / Ms, K d = 1.56 × 10 ^-4 1 / s) (FIG. 3A).
○ Confirmation of affinity between Ra62 and D1CE sequences
The affinity between Ra62 and D1CE sequences was analyzed by Biacore. The FLAG-GST-D1CE fusion protein was synthesized using a wheat cell-free system and purified using glutathione sepharose. The concentration of the purified FLAG-GST-D1CE fusion protein was determined by the molar extinction coefficient. Using a sensor chip CM5 immobilized with Protein G, about 200 RU of Ra62 antibody was captured, and then various concentrations of FLAG-GST-D1CE fusion protein were run. As a result of obtaining the speed parameter by curve fitting, the binding affinity between Ra62 antibody and FLAG-GST-D1CE was calculated as follows: K _D value = 1.00 × 10 ^-8 M (K _a = 3.65 × 10 ⁴ 1 / Ms, K _d = 3.65 × 10 ^-4 1 / s) (FIG. 3B).
-Confirmation of affinity between Ra62 and CP5 sequences
The affinity between Ra62 and CP5 sequences was analyzed by Biacore. The FLAG-GST-CP5 fusion protein was synthesized using a wheat cell-free system and purified using glutathione sepharose. The concentration of the purified FLAG-GST-CP5 fusion protein was determined by the molar extinction coefficient. Using a sensor chip CM5 immobilized with Protein G, about 200 RU of Ra62 antibody was captured, and then various concentrations of FLAG-GST-CP5 fusion protein were run. Result of obtaining kinetic parameters by curve fitting, K _D value of a binding affinity between Ra62 antibody and FLAG-GST-CP5 = 1.46 × 10 -7 M (K a = 1.28 × 10 4 1 / Ms, K d = 1.87 × 10 ^-3 1 / s) (FIG. 3C).
Compared to the binding with FLAG-GST-D1CE, the binding rate of FLAG-GST-CP5 and Ra62 antibody did not change greatly, but the dissociation rate increased.

<Ra48 antibody and Ra62 antibody binding site determination using swap mutant and BiLIA method>
In order to determine the binding site of Ra48 antibody and Ra62 antibody, and to construct a tag system using Ra48 antibody and Ra62 antibody, the epitope region of the antibody was determined using a swap mutant. Specifically, a cell-free expression plasmid of a mutant (swap mutant) in which the loop region or terminal region of DRD1 is replaced with a homologous region of ADRB2 is prepared, and further, DRD1 Swap mutants were synthesized as biotinylated proteoliposomes.
Next, DRD1 swap mutant was reacted with Ra48 antibody or Ra62 antibody, and the interaction was detected by BiLIA method (see Takeda et al. Scientific Reports 2015). Antibody binding was not observed only in the mutant in which the C-terminal region of DRD1 was swapped (FIG. 4).
From this result, it was confirmed that Ra48 antibody and Ra62 antibody bind to the C-terminal (337-447) region of DRD1.

<Determination of Ra48 antibody and Ra62 antibody binding site in C-terminal region>
DRD1 has a relatively long C-terminal intracellular region. Of the C-terminal region (337-447) of DRD1, in order to determine where the Ra48 antibody and Ra62 antibody were bound, the region was divided and binding to the antibody was confirmed by AlphaScreen. Specifically, the biotinylated fusion protein is cell-free protein by fusing the full-length or partial sequence of DRD1 C-terminal region (337-447) to the N-terminus of SrtA protein and the biotin ligase recognition site bls. It was synthesized in a synthetic system (reference: Sawasaki et al. 2007: FEBS Lett. 2008 Jan 23; 582 (2): 221-8.). Next, binding between the biotinylated fusion protein and Ra62 antibody was detected using AlphaScreen (see Takeda et al. Scientific Reports 2015).
The Ra62 antibody was confirmed to bind to the C-terminal 1/3 region (411-447) of the region. When the sequence of this region of human DRD1 was compared with the homologous region of mouse or rabbit, the Ra-terminal antibody was predicted to recognize the C-terminus of DRD1 because the C-terminal region was not particularly conserved. In fact, the Ra62 antibody bound to a fusion protein fused with 7 C-terminal residues (441-447) (FIG. 5).
The Ra48 antibody was confirmed to bind to the C-terminal region (374-413) (FIG. 5).

<Epitope sequencing of Ra62 antibody>
In order to determine the epitope sequence of Ra62 antibody, epitope mapping using an alanine mutant was performed. In order to determine the detailed epitope sequence, the C-terminal region of 7 residues of DRD1 was fused with Venus fluorescent protein (modified GFP protein) to produce a mutant in which a residue was substituted with alanine. Mutants were synthesized by a cell-free protein synthesis system, and binding of Ra62 antibody was confirmed by Western blotting (FIG. 6A). In the protein fused to the C-terminus of Venus, Ra62 antibody did not bind to the mutant in which the terminal 3 residues (HPT) were substituted with alanine, and these residues were found to be important for antibody recognition. In addition, Ra62 antibody does not bind to mutants (Venus-QNGQHP) from which the C-terminal threonine residue has been deleted or proteins in which 7 residues of the C-terminal region of DRD1 are fused to the N-terminus of Venus (QNGQHPT-Venus). It was. Therefore, it was confirmed that the terminal 3 amino acids including the C-terminal carboxyl group are important for antibody recognition.
In addition, the minimal epitope sequence was determined. More specifically, for Venus-QNGQHPT, a deletion mutant was prepared in which the C-terminal sequence of DRD1 (QNGQHPT) was deleted one by one from the N-terminal side. The fusion protein was synthesized using a wheat cell-free protein synthesis system, and binding to the Ra62 antibody was confirmed by Western blotting (FIG. 6B). The antibody bound only by the fusion of the terminal 3 residues (HPT-COOH) or 4 residues (QHPT-COOH). Furthermore, when the terminal 5 residues (GQHPT-COOH) were fused (Venus-GQHPT), the antibody binding ability was high, and no signal decrease was observed.
From these results, the minimum epitope of Ra62 antibody was determined to be 3 residues including the C-terminal carboxyl group of DRD1. Furthermore, 5 residues containing the C-terminal carboxyl group of DRD1 were named as D1CE (DRD1 C-Terminus Epitope) sequence.
Based on these results, the tag of the present invention is shorter than the known His tag (6-10 residues). Since it is as short as 3-5 residues, it can be easily inserted into the target protein by PCR or the like. Further, since the tag of the present invention has a small tag size, it does not interfere with the structure of the fused protein.

<Specificity of Ra62 antibody>
Whether or not the endogenous protein reacts with the Ra62 antibody was confirmed using general cultured cells. Specifically, various cultured cells and SDS-PAGE sample buffer were mixed, and the obtained cell extract was subjected to SDS-PAGE and Western blotting was performed. Ra62 antibody was used as the primary antibody, and anti-rabbit IgG-HRP antibody was used as the secondary antibody. As a result, a band corresponding to DRD1 expected to be 40 to 50 kDa was not detected, and non-specific binding to the endogenous protein was negligible (FIG. 7). The noise level was clearly lower than when the same sample was detected using the FLAG M2 antibody.
From these results, using the Ra62 antibody of the present invention and a tag, the protein fused with the tag peptide can be purified in one-step with high purity. Furthermore, using the Ra62 antibody of the present invention and a tag, a protein fused with a tag peptide can be detected specifically and with high sensitivity.

<Analysis of binding affinity between modified D1CE and Ra62 antibody>
13 types of constructs in which the C-terminal 110 residues of DRD1, including the D1CE sequence, are fused to the C-terminal of the FLAG-GST protein (Fig. 8A), and the C-terminal 3 residues of the D1CE sequence are converted one by one. And then synthesized using a wheat cell-free protein synthesis system. As a result of SDS-PAGE and CBB staining, it was confirmed that the affinity was lowered by amino acid substitution at the C-terminal part of the D1CE sequence (FIG. 8B).
Next, the binding affinity with the Ra62 antibody was analyzed by D1CE sequence amino acid substitution. Since the Ra62 antibody binds strongly to the D1CE sequence, elution by competition with the peptide is difficult as it is with the FLAG antibody as it is. Therefore, amino acid substitutions were introduced into the D1CE sequence in order to moderately reduce the binding affinity between the antibody and the D1CE sequence. That is, the terminal 3 residues of the D1CE sequence, which is important for antibody binding, were converted to amino acids having a relatively close structure.
Specifically, 16 types of constructs in which the C-terminal 3 residues of the D1CE sequence of the Venus-D1CE protein were converted one by one were prepared and synthesized using a wheat cell-free system. About each synthesized amino acid substitution product, the binding affinity with the antibody was analyzed by Biacore. Biacore analysis was performed using Biacore X100. Protein G was immobilized on 6,000 RU on Biacore sensor chip CM5 using amine coupling. 1,500 RU of Ra62 antibody was captured on a protein G-immobilized chip. Thereafter, either Venus-D1CE having a wild type or amino acid-substituted D1CE sequence was added for 120 seconds. After observing the dissociation from the antibody for 300 seconds, the sensor chip was regenerated by treating with a regeneration solution containing 10 mM NaOH and 500 mM sodium thiocyanate for 30 seconds. As a result of the Biacore assay, many of the D1CE sequences with amino acid substitution did not bind to the Ra62 antibody. In addition, except for one mutation that did not lose its binding ability, the dissociation rate did not change. Only a slight increase in dissociation rate was observed in the mutant (GQHVT) in which the second proline residue from the end was substituted with valine ("P445V" in Fig. 8C).

From these results, it was confirmed that the sequence in which P of D1CE (GQHPT-COOH) was substituted with Val, Trp, Gly, Leu, Phe, or Met also had the ability to bind to the Ra antibody. That is, GQHGT-COOH, GQHVT-COOH, GQHLT-COOH, GQHFT-COOH, GQHWT-COOH and GQHMT-COOH can be used as a tag peptide or a peptide for eluting tag peptides.
Furthermore, the following protein purification method was established.
1) Fusing a CP5 tag with a relatively fast dissociation rate to the C-terminus of the target protein 2) Binding the CP5 tag fusion protein to the Ra62 antibody and washing with a buffer 3) More binding affinity for the Ra62 antibody Add a peptide containing a high D1CE sequence, and 4) release and recover the CP5 tag fusion protein by exchange reaction between the CP5 tag and the D1CE sequence peptide.
In addition, in the protein purification method described above, the immobilized antibody can be washed under severe dissociation conditions and can be regenerated more easily.

<Cloning of CP5 tag>
Since the CP5 tag is as short as 5 residues, it can be easily inserted into a target protein incorporated in any expression vector using inverse PCR and ligation in E. coli. The CP5 tag was cloned using a known kit {PrimeSTAR Max DNA Polymerase (Takara Bio): http://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100005117}.
Primers were designed as shown in FIG. 9 in order to insert a 15-base sequence encoding CP5 into the C-terminus of the target ORF. Primers in the forward and reverse directions were designed immediately before the stop codon of the target protein, and a sequence encoding CP5 was added to the terminal position. Inverse PCR was performed using a highly accurate Pfu-based PCR enzyme such as PrimeStar Max to produce linear DNA with CP5 sequences at both ends. The template plasmid DNA was digested with DpnI or the like, purified after column purification, and transformed into E. coli. In E. coli, linear DNA was circularized as shown in FIG. After transformation, the plasmid was cloned according to a conventional method, and the insertion of the CP5 sequence was confirmed by sequencing.

<Purification of tag fusion protein>
Among the fusion proteins synthesized in Example 8 above, in which the C-terminal 110 residue of DRD1 is fused to the C-terminus of FLAG-GST protein, a mutant in which the second proline residue from the C-terminus is substituted with valine ( FLAG-GST-DRD1_Cterminal mutant) was purified. That is, the mutant has GQHVT-COOH (CP5 tag) at the C-terminus. For affinity purification, Ra62 antibody was immobilized on NHS-Sepharose by amine coupling {anti-Ra62 monoclonal antibody (mAb) Sepharose}. Purification was performed by a batch method using a spin column. 4 mL of FLAG-GST-CP5 synthesis solution was added to 100 μL of Ra62 antibody covalently-bonded sepharose and mixed by inversion at 4 ° C. for 1 hour using a rotator. The slurry was centrifuged at 2000 × g for 2 minutes to precipitate the resin, leaving a small amount of liquid and removing the unbound fraction. The resin was resuspended with the remaining liquid, transferred to an empty MicroSpin column (GE healthcare), and the flow-through fraction was collected by centrifugation for 10 seconds using a tabletop centrifuge. The operation of adding 600 μL of PBS buffer and collecting the washed fraction by centrifugation for 10 seconds using a tabletop centrifuge was repeated 5 times. Thereafter, 200 μL of elution buffer {150 μM D1CE peptide (TSQNGQHPT) -containing PBS buffer} was added, and the mixture was mixed by inverting at room temperature for 2 hours using a rotator. Elution fraction 1 was recovered by centrifugation at 15,000 rpm for 1 minute. Next, 200 μL of elution buffer {150 μM D1CE peptide (TSQNGQHPT) and Mg 2 ⁺ -containing PBS buffer} was added, and the mixture was mixed by inverting at room temperature for 2 hours using a rotator, and the elution fraction 2 was collected by centrifugation. Furthermore, 200 μL of elution buffer {150 μM D1CE peptide (TSQNGQHPT), Mg 2 ⁺ -containing 0.1 M acetate buffer pH 4.0} was added, shaken at room temperature for 5 minutes, and elution fraction 3 was collected by centrifugation. Finally, 400 μL of SDS-PAGE sample buffer was added, and the protein remaining on the column was eluted (resin fraction). Finally, purity was confirmed by SDS-PAGE and CBB staining (FIG. 10). Most of the FLAG-GST-DRD1_Cterminal mutant (Shirayajiri in FIG. 10) was eluted in elution fraction 1. Therefore, it was confirmed that the FLAG-GST-DRD1_Cterminal mutant was dissociated from the antibody and eluted by the addition of only 150 μM D1CE peptide.
As described above, the protein purification method of the present invention can purify tag fusion proteins (particularly antibodies).

<Purification of fluorescent protein>
In this example, a GP5 fusion protein in which a CP5 tag was fused to a Venus fluorescent protein, which is a fluorescent protein, was purified. Specifically, for affinity purification, Ra62 antibody was immobilized on NHS-Sepharose by amine coupling (anti-Ra62 mAb Sepharose). An expression plasmid for cell-free protein synthesis in which a CP5 tag was fused to the C-terminus of Venus was prepared and synthesized using a wheat cell-free protein synthesis system (Venus-CP5 protein). Purification was performed by an open column and a column method by natural drop. To 200 μL of anti-Ra62 mAb Sepharose, 600 μL of Venus-CP5 was added and mixed by inverting at 4 ° C. for 1 hour using a rotator. The slurry was added to the open column, and the flow-through fraction was collected by natural fall. Washing buffer (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl) was added 5 times by 500 μL to wash the column. The washing solution was collected for each fraction and used as a washing fraction. Thereafter, elution buffer {50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, D1CE peptide (TSQNGQHPT)} was added 15 times by 100 μL, and elution was performed (elution fraction). Each collected fraction was measured for absorbance at 280 nm and observed for fluorescence under blue light. Finally, the purity was confirmed by SDS-PAGE and CBB staining (FIG. 11). Venus-CP5 is hardly dissociated by washing with a washing buffer containing 750 mM NaCl, indicating that the CP5 tag and Ra62 antibody are tightly bound. On the other hand, Venus-CP5 was dissociated from the antibody and eluted by the addition of 150 μM D1CE peptide. The completion of elution of Venus-CP5 in only a few fractions is not only that the affinity of the D1CE sequence for the Ra62 antibody is higher than that of the CP5 tag, but the affinity of the D1CE sequence and the Ra62 antibody is 6.91 × 10 ⁻¹⁰ M and very strong have contributed. That is, since the D1CE peptide replaced with the CP5 tag does not dissociate and binds irreversibly to the Ra62 antibody, the released Venus-CP5 was eluted again without being captured by the Ra62 antibody. The eluted Venus-CP5 was observed as almost a single band, the endogenous protein of the wheat cell-free protein synthesis system and the Ra62 antibody did not cross-react, and ion-binding by a washing buffer containing a high concentration of salt, etc. This shows that contaminating proteins that bind nonspecifically were effectively removed. Further, since the eluted Venus-CP5 emitted strong fluorescence, it was confirmed that the Venus protein was purified while maintaining the activity.
As described above, the method for purifying a protein of the present invention can purify a fluorescent protein maintaining its fluorescent activity.

<Purification of enzyme>
Affinity purification using a CP5 tag elutes under physiological conditions using a low concentration of peptide, so it is considered that protein purification is less likely to occur during the purification process. Thus, it is considered suitable for purification of functional proteins such as enzymes and receptors. As an example of an enzyme that is a functional protein, a CP5 tag was added to human CYLD, which is a deubiquitinase, and purification was attempted.
Purification was performed by a batch method using a spin column. To 200 μL of anti-Ra62 mAb sepharose, 3 mL of wheat-free cell-synthesized CYLD-CP5 was added and mixed by inversion at 4 ° C. for 1 hour using a rotator. The slurry was transferred to a spin column, and the flow-through fraction was collected by centrifugation at 2000 rpm for 30 seconds. Wash the resin by adding 500 μL of washing buffer (50 mM Hepes-NaOH, pH 7.5, 500 mM NaCl, 1 mM DTT) to the resin and collecting the washing fraction by centrifugation at 2000 rpm for 30 seconds 4 times. did. Then add 100 μL of eluate (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1 mM DTT, 150 μM D1CE peptide), let stand for 5 minutes, and collect the eluted fraction by centrifugation at 15,000 rpm for 1 minute. did. The elution operation was repeated 4 times. As a result of SDS-PAGE and CBB staining, the CYLD protein was a relatively large enzyme of 130 kDa, but was well purified by CP5 purification. Since the batch method was used, it was confirmed that most CYLD-CP5 was eluted in the first elution fraction.
Next, a deubiquitination assay was performed using the eluted purified CYLD-CP5. 100 nM CYLD-CP5 purified with CP5 was mixed with 1 μM of a tetrameric linear ubiquitin chain and allowed to stand at 30 ° C. for 3 hours. As a negative control, D1CE peptide or CP5 purified Venus-CP5 was used and mixed with the ubiquitin chain and reacted. The reaction solution was subjected to SDS-PAGE, and the degradation of the ubiquitin chain was confirmed by SYPRO Ruby staining. Only when reacted with purified CYLD-CP5, monomer, dimer and trimer ubiquitin, which are degradation products of ubiquitin chains, were observed. From these results, it was confirmed that CYLD-CP5 maintained its activity even after CP5 purification, and that CP5 purification was suitable for enzyme purification. The wheat germ extract used in the wheat cell-free protein synthesis system contains a large amount of deubiquitin enzyme, but CP5-purified Venus-CP5 did not show deubiquitin activity. This indicates that CP5 purification completely removed the deubiquitinase present in the wheat germ extract.
As described above, the protein purification method of the present invention can purify an enzyme that maintains the enzyme activity.

<Purification of membrane protein>
Using the CP5 tag, DRD1 protein synthesized in a cell-free protein synthesis system was purified. Ra62 antibody is a rabbit high affinity antibody. Rabbit antibodies generally have a strong structure, so that the binding ability can be maintained even under severe conditions such as the presence of a surfactant. This suggested that CP5 purification is suitable for the purification of membrane proteins solubilized with surfactants. Therefore, a DRD1 construct (DRD1-CP5) having a CP5 sequence in which one amino acid substitution was introduced into the D1CE sequence, which is the terminal sequence of DRD1, was prepared. DRD1-CP5 was synthesized as a proteoliposome using a wheat cell-free protein synthesis system (Takeda et al. Scientific Reports 2015). After purification of proteoliposomes by centrifugation and buffer washing, a solubilized solution containing DDM as a surfactant (150 mM carbonate buffer, 750 mM NaCl, 4% (w / v) DDM, 10% glycerol, 1 mM DTT) DRD1-CP5 was solubilized with (Solubilized DRD1). The CP5 purification operation was performed by a batch method using a spin column. Dilute 500 μL of solubilized DRD1-CP5 4 times with washing solution (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 2% Glycerol, 0.1% DDM, 0.02% CHS) (DDM concentration 1%), 200 μL Was added to the anti-Ra62 mAb Sepharose and mixed by inversion at 4 ° C. for 1 hour using a rotator. The slurry was transferred to a spin column, and the flow-through fraction was collected by centrifugation at 2000 rpm for 30 seconds. The operation of adding 500 μL of the washing solution and collecting the washing fraction by centrifugation at 2000 rpm for 30 seconds was repeated 6 times. Then add 100 μL eluate (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 2% Glycerol, 0.1% DDM, 0.02% CHS, 150 μM D1CE peptide), let stand for 5 minutes, 15,000 rpm The eluted fraction was collected by centrifugation for 1 minute. The elution operation was repeated 5 times. Finally, 100 μL of SDS-PAGE sample buffer was added to elute the remaining protein on the column (resin fraction). As a result of SDS-PAGE and CBB staining, solubilized DRD1-CP5 bound well to anti-Ra62 mAb sepharose, and no DRD1 band was confirmed in the flow-through fraction (FIG. 13A). FIG. 13B shows the absorbance (280 nm wavelength) of each fraction. Competitive elution with the D1CE peptide was successful, and almost all DRD1-CP5 was eluted in 100 μL × 3 fractions. The bands other than the target protein were reduced by purification. Moreover, distortion of the electrophoretic image (white arrowhead in FIG. 13A) due to the liposome-derived lipid seen in the solubilized fraction was not observed after purification, and it was confirmed that the lipid was removed by CP5 purification. In addition, no residual DRD1-CP5 was observed in the purified Sepharose (resin fraction).

<Purification of CLDN1 protein>
As another example of purification of membrane protein, we attempted to purify CLDN1, which is a constituent protein of tight junction with 4 times transmembrane protein. A CP5 tag was inserted into the C-terminus of CLDN1 (CLDN1-CP5) and synthesized as proteoliposomes in a wheat cell-free system (unpurified fraction). Proteoliposomes are purified by centrifugation and buffer washing (centrifugation fraction), and CLDN1 is obtained using a lysate (150 mM carbonate buffer, 750 mM NaCl, 4% (w / v) DDM, 10% glycerol, 1 mM DTT). -CP5 was solubilized (solubilized fraction). The CP5 purification operation was performed by an open column and a column method by natural drop. 200 μL of anti-Ra62 mAb Sepharose equilibrated with washing solution (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 2% Glycerol, 0.1% DDM, 0.02% CHS) is packed in an open column and 500 μL of solubilized CLDN1 -CP5 was added. After collecting the flow-through fraction by natural fall, 500 μL of the washing solution was added four times each and collected as a washing fraction. Then, the eluate (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 2% Glycerol, 0.1% DDM, 0.02% CHS, 150 μM D1CE peptide) was added 12 times by 100 μL, and the eluted fraction was collected. . Finally, 100 μL of 10 mM NaOH was added 6 times to elute the protein remaining on the column (NaOH fraction). As a result of SDS-PAGE and CBB staining, good purification of CLDN1-CP5 was confirmed (FIG. 14A). From the absorbance at 280 nm shown in FIG. 14B, the eluted target protein is almost concentrated in the three fractions of fractions 3 to 5 of the eluted fraction. This indicates that peptide elution was performed efficiently. After elution of the peptide, washing with 10 mM NaOH slightly eluted the remaining CLDN1-CP5, but it was confirmed that most of the CLDN1-CP5 was eluted with a very small amount of peptide-eluted protein. did.

<Purification of MARCH3 protein>
A CP5 tag was inserted at the C-terminus of MARCH3 (MARCH3-CP5) and synthesized as a proteoliposome (membrane protein) in a wheat cell-free protein synthesis system (unpurified). Proteoliposomes were purified by centrifugation and buffer washing, and MARCH3-CP5 was solubilized with a solubilizing solution (150 mM carbonate buffer, 750 mM NaCl, 4% (w / v) DDM, 10% glycerol, 1 mM DTT) . The CP5 purification operation was performed by an open column and a column method by natural drop. Add 100 μL of anti-Ra62 mAb Sepharose equilibrated with washing solution (50 mM Hepes-NaOH, pH 7.4, 750 mM NaCl, 2% Glycerol, 0.1% DDM, 1 mM DTT) to 500 μL of solubilized MARCH3-CP5, Rotated for 1 hour. The mixture was poured into an open column, and the flow-through fraction was collected by natural fall. The washing solution was added 4 times by 500 μL and collected as a washing fraction. Thereafter, an eluate (50 mM Hepes-NaOH, pH 7.4, 750 mM NaCl, 2% Glycerol, 0.1% DDM, 1 mM DTT, 150 μM D1CE peptide) was added 6 times by 100 μL, and the eluted fraction was collected. SDS-PAGE and CBB staining were performed together with the control BSA to confirm the elution purification of MARCH3. The elution fraction with the highest MARCH3 concentration was used to evaluate the E3 ubiquitin ligase activity of MARCH3.
The E3 ubiquitin ligase activity evaluation procedure is as follows. 5 μL of CP5 purified MARCH3-CP5 or Venus-CP5 was added to 4 μM recombinant HA-ubiquitin (Boston Biochem), 3 mM ATP, 40 nM recombinant E1 (Boston Biochem), and 300 nM recombinant E2 (UbcH6, Enzo Life Sciences). After reacting at 30 ° C. for 3 hours, the reaction solution was subjected to SDS-PAGE and Western blotting to confirm self-ubiquitination of MARCH3-CP5. Western blotting was detected by chemiluminescence using an anti-HA antibody conjugated with HRP. As a result, only when all of HA-ubiquitin, E1, E2, and MARCH3-CP5 were mixed, a ubiquitinated MARCH3-CP5 ladder-like band was detected in the range of 40 kDa to 300 kDa. However, no ladder band was observed when any of the above proteins was missing.
From the above, it was confirmed that the purified MARCH3-CP5 has E3 ubiquitin ligase activity.

  As described above, the protein purification method of the present invention can purify the membrane protein while maintaining the membrane protein activity.

<Production of membrane protein from cell membrane>
It was confirmed that the membrane protein expressed in large quantities on the cell membrane of cultured insect cells could be purified with the CP5 tag as follows. CP5 was fused to the C-terminal of muscarinic acetylcholine receptor M2 which is GPCR (M2-CP5) and introduced into insect cultured cell Sf9 using baculovirus, and M2-CP5 was overexpressed on the cell membrane. Cells were collected, disrupted, washed with buffer, and the cell membrane fraction was purified. Suspend the membrane fraction in a solubilization buffer containing DDM (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 4% Glycerol, 0.1 μM atropine, 1% DDM, 0.2% CHS) and sonicate for 5 minutes Thereafter, the supernatant containing solubilized M2-CP5 was collected by centrifugation at 15000 rpm for 10 minutes at 4 ° C. (solubilized cell membrane fraction). The CP5 purification operation was performed by a batch method. To 200 μL of anti-Ra62 mAb Sepharose, 15 mL of solubilized M2-CP5 was added and mixed by inverting at 4 ° C. for 1 hour using a rotator. The supernatant and the resin were separated by centrifugation at 5000 rpm for 5 minutes, and the supernatant was collected as a flow-through fraction. Next, mix the resin with 10 mL wash buffer (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 2% Glycerol, 0.1 μM atropine, 0.1% DDM, 0.02% CHS), and centrifuge at 5000 rpm for 5 minutes. The operation of recovering the washing fraction was repeated 9 times to wash the resin. The resin is then transferred to a spin column and 100 μL of elution buffer (50 mM Hepes-NaOH, pH 7.5, 750 mM NaCl, 2% Glycerol, 0.1 μM atropine, 0.1% DDM, 0.02% CHS, 150 μM D1CE peptide) The mixture was allowed to stand for 5 minutes, and the eluted fraction was collected by centrifugation at 15,000 rpm for 1 minute. The elution operation was repeated 5 times. Finally, 300 μL of SDS-PAGE sample buffer was added to elute the remaining protein on the column (resin fraction). From the results of SDS-PAGE and CBB staining, most contaminating proteins and lipids contained in the solubilized cell membrane fraction were removed by CP5 purification, and M2-CP5 was successfully purified (FIG. 16). In the resin fraction, no M2-CP5 band was observed, and it was confirmed that M2-CP5 was eluted very efficiently (white arrowhead in FIG. 16). The protein production method of the present invention was confirmed to be effective for the production of membrane proteins used for crystallization and biochemical analysis because the target membrane protein with high purity was obtained by one-step purification. .
As described above, the protein production method of the present invention can produce a membrane protein from a cell membrane.

  The present invention can provide a protein purification method using an antibody that recognizes DRD1 and a peptide derived from an epitope of the antibody.

Claims (18)

The following formula (I);
Gly-Gln-His-X-Thr (I)
A tag peptide comprising an amino acid sequence represented by (wherein X represents a hydrophobic amino acid), an amino acid sequence represented by Gln-His-Pro-Thr, or an amino acid sequence represented by His-Pro-Thr; Peptide for tag peptide elution.
The tag peptide or tag peptide elution peptide according to claim 1, wherein X is Pro, Val, Trp, Gly, Leu, Phe or Met.
The tag peptide or tag peptide elution peptide according to claim 1 or 2, wherein X is Pro or Val.
Furthermore, the tag peptide of Claim 1 or the peptide for tag peptide elution which has an amino acid sequence of Thr-Ser-Gln-Asn in the N terminal side of the peptide for tag peptide elution.
A tag peptide fusion protein in which the tag peptide according to claim 1 is fused.
A protein purification method comprising the following steps.
(1) A step of contacting a protein containing the tag peptide according to any one of claims 1 to 4 or a solution containing the tag peptide fusion protein according to claim 5 with an antibody or antibody fragment that recognizes DRD1 ( 2) A step of adding an eluent of tag peptide
The method for purifying a protein according to claim 6, wherein the elution solution of the tag peptide contains the peptide for eluting the tag peptide according to any one of claims 1 to 4.
The method for purifying a protein according to claim 7, wherein X is Pro, Val, Trp, Gly, Leu, Phe or Met.
The method for purifying a protein according to claim 7 or 8, wherein the peptide for eluting the tag peptide further has an amino acid sequence of Thr-Ser-Gln-Asn on the N-terminal side.
The method for purifying a protein according to any one of claims 7 to 9, wherein the tag peptide elution peptide has a higher binding affinity for an antibody or antibody fragment recognizing DRD1 than the tag peptide.
The method for purifying a protein according to any one of claims 7 to 10, wherein the combination of the tag peptide and the peptide for eluting the tag peptide is as follows.
(1) Tag peptide is GQHVT-COOH, tag peptide elution peptide is GQHPT-COOH (2) Tag peptide is GQHVT-COOH, tag peptide elution peptide is TSQNGQHPT-COOH (3) Tag The peptide is GQHWT-COOH and the peptide for eluting the tag peptide is GQHPT-COOH (4) The tag peptide is GQHWT-COOH and the peptide for eluting the tag peptide is TSQNGQHPT-COOH (5) The tag peptide is GQHGT- COOH, tag peptide eluting peptide is GQHPT-COOH (6) tag peptide is GQHGT-COOH, tag peptide eluting peptide is TSQNGQHPT-COOH (7) tag peptide is GQHLT-COOH, Tag peptide elution peptide is GQHPT-COOH (8) Tag peptide is GQHLT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (9) Tag peptide is GQHFT-COOH, for tag peptide elution The peptide is GQHPT-COOH (10) The tag peptide is GQHFT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (11) The tag peptide is GQHMT-COOH, and the peptide for eluting tag peptide is GQHPT- COOH (12) Tag peptide is GQHMT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (13) Tag peptide is QHPT-COOH, Tag peptide elution peptide is GQHPT-COOH ( 14) The tag peptide is QHPT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (15) The tag peptide is HPT-COOH, and the peptide for eluting tag peptide is GQHPT-COOH (16) Is HPT-COOH and the peptide for peptide peptide elution is TSQNGQHPT-COOH
A method for producing a protein, comprising the following steps.
(1) A step of producing a protein in which the tag peptide according to any one of claims 1 to 4 is fused or the tag peptide fusion protein according to claim 5 in a cell-free protein synthesis solution,
(2) contacting a cell-free protein synthesis solution containing the tag peptide fusion protein with an antibody or antibody fragment that recognizes DRD1, and
(3) Step of adding tag peptide eluent
The method for producing a protein according to claim 12, wherein the protein is a membrane protein.
The method for producing a protein according to claim 12 or 13, wherein the tag peptide eluent comprises the peptide for peptide tag elution according to any one of claims 1 to 4.
The method for producing a protein according to claim 14, wherein X is Pro, Val, Trp, Gly, Leu, Phe or Met.
The method for producing a protein according to claim 14 or 15, wherein the peptide for eluting the tag peptide further has an amino acid sequence of Thr-Ser-Gln-Asn on the N-terminal side.
The method for producing a protein according to any one of claims 14 to 16, wherein the tag peptide elution peptide has a higher binding affinity for an antibody or antibody fragment that recognizes DRD1 than the tag peptide.
The method for producing a protein according to any one of claims 14 to 17, wherein the combination of the tag peptide and the peptide for eluting the tag peptide is as follows.
(1) Tag peptide is GQHVT-COOH, tag peptide elution peptide is GQHPT-COOH (2) Tag peptide is GQHVT-COOH, tag peptide elution peptide is TSQNGQHPT-COOH (3) Tag The peptide is GQHWT-COOH and the peptide for eluting the tag peptide is GQHPT-COOH (4) The tag peptide is GQHWT-COOH and the peptide for eluting the tag peptide is TSQNGQHPT-COOH (5) The tag peptide is GQHGT- COOH, tag peptide eluting peptide is GQHPT-COOH (6) tag peptide is GQHGT-COOH, tag peptide eluting peptide is TSQNGQHPT-COOH (7) tag peptide is GQHLT-COOH, Tag peptide elution peptide is GQHPT-COOH (8) Tag peptide is GQHLT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (9) Tag peptide is GQHFT-COOH, for tag peptide elution The peptide is GQHPT-COOH (10) The tag peptide is GQHFT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (11) The tag peptide is GQHMT-COOH, and the peptide for eluting tag peptide is GQHPT- COOH (12) Tag peptide is GQHMT-COOH, Tag peptide elution peptide is TSQNGQHPT-COOH (13) Tag peptide is QHPT-COOH, Tag peptide elution peptide is GQHPT-COOH ( 14) The tag peptide is QHPT-COOH, the peptide for eluting tag peptide is TSQNGQHPT-COOH (15) The tag peptide is HPT-COOH, and the peptide for eluting tag peptide is GQHPT-COOH (16) Is HPT-COOH and the peptide for peptide peptide elution is TSQNGQHPT-COOH