JP7622451B2 - Plasmid for expressing recombinant adeno-associated virus-binding protein and method for producing said protein using the same - Google Patents

Description

The present invention relates to a plasmid for expressing a recombinant adeno-associated virus binding protein and a method for producing the protein using the same. In particular, the present invention relates to a plasmid that can efficiently produce the protein by using a signal peptide that causes the protein to be secreted into the host periplasm, and a method for producing the protein using the same.

It is known that after synthesis within a cell, some proteins remain within the cell and some are released outside the cell as secreted proteins. In addition, in recombinant protein production using Escherichia coli or the like as a host, a method is known in which the protein is secreted and expressed in the periplasmic region between the intracellular membrane and the outer membrane (Patent Document 1). When a recombinant protein is transported from within the cell to the periplasmic region, the function of an oligopeptide called a signal peptide is important (Patent Document 1). The amino acid sequence that constitutes a signal peptide has the following three characteristics (Patent Document 1).
(i) The N-terminal region contains at least one basic amino acid such as arginine or lysine, and the signal peptide is translocated into the cell membrane through ionic bonds between the positive charge of the side chain of the basic amino acid and the negative charge on the surface of the cell membrane.
(ii) The central region contains many hydrophobic amino acids such as alanine, leucine, isoleucine, and valine, and is involved in penetrating into the cell membrane.
(iii) In the C-terminal region, a specific amino acid exists as a recognition site that is cleaved by a signal peptidase after penetration into the cell membrane, and cleavage at this recognition site results in the release of the mature protein into the periplasmic space or outside the cell.

In order to secrete and express a recombinant protein in the periplasm of a host, the protein must be expressed in a state in which a signal peptide that promotes secretory expression into the periplasm has been added to the N-terminus of the protein. However, when using this method, there is a problem that the productivity of the protein as an active protein is low. There have also been reports of cases in which productivity has been improved by modifying the signal peptide (Patent Documents 1 to 4), but these have not been sufficient for industrial production. In particular, there are no known cases in which an adeno-associated virus binding protein has been mass-expressed as an active recombinant protein.

JP 2000-175686 A JP 2008-073044 A WO2008/032659 JP 2014-223064 A

The object of the present invention is to provide a plasmid capable of efficiently producing an adeno-associated virus binding protein using recombinant gene technology, and a method for efficiently producing said protein using the same.

As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that the production amount of adeno-associated virus (AAV) binding protein can be improved by optimizing the signal peptide for secreting the protein into the host periplasm using recombinant gene technology, which led to the completion of the present invention.

That is, the present invention includes the following inventions:
(A) a plasmid for expressing an AAV binding protein, the plasmid comprising a polynucleotide encoding the protein and a polynucleotide encoding a signal peptide for secreting the protein into the periplasm,
The above-mentioned plasmid, wherein the signal peptide is an oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or an oligopeptide in which one or several residues of the oligopeptide have been substituted, deleted or inserted.

(B) A transformant obtained by transforming a host with the plasmid described in (A).

(C) The transformant described in (B), wherein the host is Escherichia coli.

(D) A method for producing the protein, comprising the steps of: culturing the transformant described in (B) or (C) to express the AAV-binding protein from the transformant; and isolating the protein from the transformant.

The present invention is described in detail below.

The present invention relates to a polynucleotide encoding a signal peptide for secreting a protein into the periplasm, which is contained in a plasmid expressing an AAV binding protein, comprising:
(I) a polynucleotide encoding a native MalE signal peptide consisting of the amino acid sequence set forth in SEQ ID NO:1 (the region from the 1st to 30th residues of UniProt No. P0AEX9), or (II) a polynucleotide encoding an oligopeptide in which one or several residues of the signal peptide set forth in (I) have been substituted, deleted or inserted (hereinafter, these are also collectively referred to as "modified");
The present invention is characterized in that the AAV binding protein is efficiently produced by using the above.
In the above (II), the number of amino acid residues to be modified (modification of one or several residues) is not particularly limited as long as the recombinant protein expressed in the host can be efficiently transferred to the periplasmic region, which is the object of the present invention, and means, for example, any of 1 to 5 residues, 1 to 4 residues, 1 to 3 residues, 1 or 2 residues, and 1 residue. When modifying two or more residues, multiple modifications of one amino acid residue may be made, two or more amino acid residues may be modified consecutively, or a combination thereof may be used.

The plasmid of the present invention can be obtained by inserting a polynucleotide (polynucleotide C) in which a polynucleotide (polynucleotide B) encoding the above-mentioned signal peptide is added to the 5'-end of a polynucleotide (polynucleotide A) encoding an AAV binding protein, into an appropriate position of the plasmid. When the polynucleotide B is added to the polynucleotide A, an oligonucleotide encoding an arbitrary oligopeptide consisting of one to several amino acid residues may be inserted as a linker between the polynucleotide A and the polynucleotide B. The plasmid into which the polynucleotide C is inserted is not particularly limited as long as it is stable and can be replicated in the host to be transformed. When the host is Escherichia coli, examples of the plasmid include pET plasmid, pUC plasmid, pTrc plasmid, pCDF plasmid, and pBBR plasmid. The appropriate position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, and the region related to transmissibility. A host can be transformed using the plasmid of the present invention by a method commonly used by those skilled in the art.

The AAV-binding protein produced using a transformant obtained by transforming a host with the plasmid of the present invention (hereinafter simply referred to as the transformant of the present invention) is not particularly limited, and examples thereof include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVRs). When the AAV-binding protein is AAVR, preferred embodiments include polypeptides shown in any of (i) to (iii) below:
(i) a polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 2;
(ii) A polypeptide having an amino acid sequence including at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid sequence includes one or more substitutions, deletions, insertions, or additions of amino acid residues at one or more positions in the amino acid residues from the 312th to the 500th positions, and having AAV binding activity;
(iii) A polypeptide comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:2, and having 70% or more homology to the amino acid sequence from positions 312 to 500, and having AAV-binding activity.

The amino acid sequence described in SEQ ID NO:2 is the amino acid sequence of KIAA0319L (official database: UniProt No. Q8IZA0), which is one embodiment of AAVR, and the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence described in SEQ ID NO:2 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

The polypeptides shown in any of (i) to (iii) above may contain at least the regions corresponding to PKD1 and PKD2 of KIAA0319L described above. For example, they may contain all or part of the regions corresponding to other extracellular domains (domain 3 (PKD3), domain 4 (PKD4) and domain 5 (PKD5)) on the C-terminal side of PKD2, all or part of the regions corresponding to signal sequences such as the MANSC (Motif At N terminus with Seven Cysteines) domain on the N-terminal side of PKD1, or all or part of the cysteine-rich region, or all or part of the transmembrane region and intracellular region on the N-terminal and/or C-terminal side of the extracellular region.

In (ii) above, "one or several" may mean any of the following, although it may vary depending on the position of the amino acid substitution in the three-dimensional structure of the AAVR and the type of amino acid residue: 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

As an example of (ii) above, there is provided a method for producing a peptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence set forth in SEQ ID NO: 2, wherein the amino acid residues from the 312th to the 500th amino acid residues are selected from the group consisting of Gly390Ser (this notation indicates that the 390th glycine in SEQ ID NO: 2 is replaced with serine, the same applies below), Ile319Phe, Leu321Pro, Pro322Leu, Asn324Asp, Asn324Ser, Gln327Arg, Asn329Asp, A sn329Tyr, Ala330Gly, Tyr331Cys, Val332Gln, Leu333Val, Leu333Pro, Gln334Arg, Pro337Leu, Lys338Arg, Lys338Glu, Glu340Gly, Thr341 Ala, Tyr342Cys, Tyr342His, Tyr342Asn, Thr343Met, Tyr344His, Asp3 45Asn, Trp346Leu, Trp346Arg, Gln347Leu, Gln347Pro, Leu348Pro, Il e349Thr, Thr350Met, His351Leu, Pro352Leu, Arg353Cys, Asp354Gly, Tyr355His, Tyr355Asn, Tyr355Cys, Ser356Cys, Gly357Cys, His363A rg, His363Leu, Ser364Pro, Gln365Arg, Ile366Thr, Leu367Pro, Lys36 8Arg, Leu369Gln, Leu369Pro, Ser370Ala, Lys371Glu, Thr373Ala, Leu 376Pro, Tyr377Cys, Phe379Ser, Val381Ala, Glu384Gly, Asn387Ser, His389Gln, His389Leu, His389Arg, Glu391Lys, Tyr393Cys, Val396Al a, Lys399Glu, Lys399Arg, Arg406Ser, Val412Ala, Gln415Leu, Phe416 Ser, Gln441Leu, Tyr442Phe, Lys448Arg, Glu453Gly, Lys455Arg, Glu4 58Gly, Ala461Ser, Ser476Arg, Leu477Pro, Asn487Asp, Asn492Asp, Ile319Asn, Ile319Ser, Thr320Ile, Lys323Glu, Leu328Gln, Leu328Pro , Tyr331His, Val332Ala, Val332Glu, Pro336Gln, Pro337Gln, Lys338A sn, Tyr342Arg, Thr350Ser, Ile366Phe, Val383Ala, Gln386Arg, His38 9Asp, Gly392Cys, Val394Ala, Ile409Val, Ser476Gly, Thr490Ser, Ser312Pro, Ala313Ser, Val317Asp, Gln318Pro, Thr320Ala, Lys323Arg, Val326Ala, Val326Glu, Gln327His, Gln327Leu, Asn329His, Asn329Il e, Val332Ala, Glu335Gly, Glu335Val, Glu340Val, Thr341Pro, Thr343 Ser, Tyr344Phe, Trp346Cys, Met359Leu, Glu360Lys, Glu360Val, Gly361Cys, Lys362Glu, Lys362Asn, Lys362Gly, Ser364Leu, Lys371Asn, L ys371Asp, Leu372Pro, Leu372Gln, Pro374Leu, Glu378Gly, Glu378Val , Phe379Tyr, Phe379Cys, Lys380Glu, Val381Asp, Ile382Val, Glu384V Examples of such polypeptides include polypeptides having at least one amino acid substitution among al, Val394Asp, Val394Ile, Asn395Ser, Thr397Ser, Glu401Val, Arg403His, Arg406His, Gln415Arg, Thr426Ala, Gln432Leu, Gln432Arg, Gln441Arg, His443Leu, Lys448Glu, Ile456Val, Asp483Asn, Ser488Leu, Val499Glu, and Val499Ile. Among these, Gly390Ser is an amino acid substitution that is particularly improved in stability against heat and acid. Therefore, a polypeptide that contains at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO:2, with at least the amino acid substitution Gly390Ser occurring in the amino acid residues at positions 312 to 500, is a preferred embodiment of (ii) above.

The "substitution of one or several amino acid residues" in (ii) above may include, in addition to the amino acid substitution at the specific position described above, conservative substitutions in which substitution occurs between amino acids with similar physical and/or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of a protein between those with and without substitution. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). In addition, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (ii) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAVR or differences in species.

The homology of the amino acid sequence in (iii) above may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homology of amino acid sequence" means homology with respect to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31 (3), Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, 31 (3), Yodosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

Examples of hosts used in producing the transformant of the present invention include animal cells such as COS (CV-1 Origin, SV40) cells and CHO (Chinese Hamster Ovary) cells, bacteria such as bacteria of the genus Bacillus (including bacteria of the genus Bacillus in the broad sense, such as bacteria of the genus Brevibacillus and Paenibacillus) and Escherichia coli, yeasts such as those of the genus Saccharomyces, Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus oryzae, but it is preferable to use Escherichia coli as a host because it is easy to handle.

To recover the expressed protein from the culture medium of the transformant of the present invention, a suitable method may be selected depending on the form of expression. For example, when the expressed protein is expressed in the periplasm of the host, the culture medium is centrifuged, the host obtained is suspended in an appropriate buffer, and the cells are disrupted (physical disruption, chemical disruption, etc.), and then the disruption residue is removed by centrifugation to obtain a cell-free extract containing the expressed protein. When the expressed protein leaks from the periplasm of the host into the culture supernatant, the culture medium is centrifuged to recover the expressed protein from the culture supernatant obtained. When disrupting host cells with a drug, for example, 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1 mM EDTA, 6 mM MgSO ₄ , 250 U / L Benzonase (trade name), 0.3 g / L Lysozyme, 0.4% (w / v) Triton X-100 (trade name), 0.5% (w / v) CTAB (hexadecyltrimethylammonium bromide) and 50 mM CaCl ₂ (JP Patent Publication 2013-252099 A) or BugBuster Protein extraction kit (manufactured by Takara Bio Inc.) or other commercially available extraction reagents may be used for disruption.

The recovered protein-containing solution can be purified and isolated by cation exchange chromatography, hydrophobic chromatography, etc. Carriers for cation exchange chromatography have cation exchange groups such as carboxymethyl groups and sulfopropyl groups bound to them, and examples of such carriers include TOYOPEARL CM-650 and TOYOPEARL SP-650 (both manufactured by Tosoh Corporation) and CM Sepharose FastFlow (manufactured by Cytiva). Carriers for hydrophobic chromatography have hydrophobic functional groups such as ether groups, phenyl groups, and butyl groups bound to them, and examples of such carriers include TOYOPEARL Ether-650, TOYOPEARL Phenyl-650, and TOYOPEARL Butyl-650 (both manufactured by Tosoh Corporation). When purifying using various chromatography carriers, the amount of carrier determined based on the amount of application solution introduced and the protein adsorption performance of the carrier can be packed into an appropriate open column, etc.

In addition, the chromatography carrier is equilibrated with an appropriate buffer (Tris/Tris hydrochloride buffer, glycine/sodium hydroxide buffer, phosphate buffer, etc.) before the application solution is introduced. The protein-containing solution recovered by the above-mentioned method is introduced into the equilibrated chromatography carrier to adsorb the protein onto the carrier, which is then washed with the same buffer used for equilibration. The adsorbed protein is then eluted using an elution buffer to obtain a solution containing the purified protein.

The present invention uses a polynucleotide encoding a MalE signal peptide or a polynucleotide encoding an oligopeptide in which one or several residues of the MalE signal peptide have been substituted, deleted or inserted as a polynucleotide contained in a plasmid expressing an adeno-associated virus binding protein and which encodes a signal peptide for secreting the protein into the periplasm. This allows the amount of protein produced to be significantly increased compared to when a PelB signal peptide is used, and therefore the efficiency of producing an industrially useful recombinant protein can be significantly improved.

A diagram showing the structure of an adeno-associated virus (AAV) binding protein consisting of the amino acid sequence set forth in SEQ ID NO:16. FIG. 1 is a diagram comparing the productivity of AAV binding protein when a PelB signal peptide was added to the N-terminus of the AAV binding protein (Comparative Example 1) and when a MalE signal peptide was added to the N-terminus of the AAV binding protein (Example 4).

The present invention will be described in detail below using examples and comparative examples, but the present invention is not limited to these examples.

Example 1 Preparation of Expression Vector An expression vector pTrcpel was prepared by inserting a polynucleotide encoding the native PelB signal peptide (amino acid sequence: MKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 3) into plasmid pTrc99a (manufactured by Cytiva) using the method described below.

(1) Equal amounts of oligonucleotides consisting of the sequences described in SEQ ID NO: 4 (5'-CATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGC-3') and SEQ ID NO: 5 (5'-CATGGCCATCGCCGGCTGGGCAGCGAGGAGCAGCAGACCAGCAGCAGCAGCGGTCGGCAGCAGGTATTT-3') were mixed and heated at 95°C for 5 minutes, and the temperature was then lowered in 1°C increments over a 1 minute period until the temperature reached 15°C, thereby preparing the double-stranded oligonucleotide PelBp1. PelBp1 was kept at 15°C until use.

(2) The PelBp1 prepared in (1) was ligated to the plasmid pTrc99a, which had been digested with the restriction enzyme NcoI, using a DNA Ligation kit (Takara Bio Inc.), and the ligation product was used to transform E. coli JM109 strain (Takara Bio Inc.).

(3) The obtained transformant was cultured in LB (Luria-Bertani) medium containing 100 μg/mL carbenicillin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.), and the expression vector pTrcpel was extracted using a QIAprep Spin Miniprep kit (Qiagen).

(4) The nucleotide sequence of the polynucleotide encoding the PelB signal peptide and its surrounding region of the expression vector pTrcpel prepared in (3) was analyzed using a fully automated DNA sequencer, Genetic Analyzer 3500 (Thermo Fisher Scientific), and it was confirmed to be the desired sequence (i.e., the sequence of the polynucleotide encoding the oligopeptide consisting of the amino acid sequence described in SEQ ID NO: 3). In this analysis, either the oligonucleotide described in SEQ ID NO: 6 (5'-TGTGGTATGGCTGTGCAGG-3') or SEQ ID NO: 7 (5'-TCGGCATGGGGTCAGGTG-3') was used as a sequencing primer.

Example 2: Construction of AAV binding protein expression vector (part 1)
A polynucleotide encoding a polypeptide (AVR8g-CRNDTCG) in which a cysteine tag (amino acid sequence: CRNDTCG) has been added to the C-terminus of the adeno-associated virus (AAV) binding protein AVR8g consisting of the amino acid sequence set forth in SEQ ID NO:8 was inserted into the expression vector pTrcpel prepared in Example 1. AVR8g is a polypeptide consisting of amino acid residues 312 to 500 of the amino acid sequence set forth in SEQ ID NO:2, which corresponds to the regions of extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0), with the following amino acid substitutions occurring in the 312 to 500 amino acid residues: Val317Asp (this notation indicates that the 317th valine has been substituted with aspartic acid, the same applies below), Tyr342Cys, Lys362Glu, Lys371Asn, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp.

(1) PCR was performed using plasmid pET-AVR8g (SEQ ID NO: 9) containing a polynucleotide encoding the AAV binding protein AVR8g consisting of the amino acid sequence set forth in SEQ ID NO: 8 as template DNA, and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 10 (5'-TAATACGACTCACTATAGGG-3') and SEQ ID NO: 11 (5'-TTTT[AAGCTT]CATCCGCAGGTATCGTTGCGGCAGTCTACCGCTTTGTTGACGGTAAG-3') (brackets in SEQ ID NO: 11 indicate the restriction enzyme HindIII recognition sequence) as PCR primers. After preparing a reaction solution with the composition shown in Table 1, PCR was performed by repeating 30 cycles of reaction with the reaction solution, in which one cycle is a first step at 98°C for 10 seconds, a second step at 50°C for 5 seconds, and a third step at 72°C for 1 minute. The resulting PCR product was purified to obtain a polynucleotide encoding AVR8g-CRNDTCG.

(2) The obtained polynucleotide encoding AVR8g-CRNDTCG was digested with restriction enzymes NcoI and HindIII and then ligated into the expression vector pTrcpel prepared in Example 1 that had been previously digested with the restriction enzymes NcoI and HindIII, and the ligation product was used to transform Escherichia coli W3110 strain.

(3) The obtained transformant was cultured in LB medium containing 100 μg/mL carbenicillin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.), and the expression vector pTrcAVR8g-CRNDTCG containing the polynucleotide encoding AVR8g-CRNDTCG was extracted using a QIAprep Spin Miniprep kit (Qiagen).

(4) The nucleotide sequence of the polynucleotide encoding AVR8g-CRNDTCG and its surrounding region in the expression vector pTrcAVR8g-CRNDTCG was analyzed in the same manner as in Example 1(4) and confirmed to be the desired sequence.

The amino acid sequence of the polypeptide expressed by the expression vector pTrcAVR8g-CRNDTCG is shown in SEQ ID NO: 12, and the sequence of the polynucleotide encoding said polypeptide is shown in SEQ ID NO: 13. In SEQ ID NO: 12, the amino acid sequence from the 1st methionine to the 22nd alanine is the PelB signal peptide (SEQ ID NO: 3), the sequence from the 25th serine to the 213th aspartic acid is the AAV binding protein AVR8g (SEQ ID NO: 8, corresponding to the region from the 312th to the 500th amino acid of the amino acid sequence described in SEQ ID NO: 2 (PKD1 and PKD2 of KIAA0319L)), and the sequence from the 214th cysteine to the 220th glycine is the cysteine tag. In addition, in SEQ ID NO:12, the aspartic acid of Val317Asp is at the 30th position, the cysteine of Tyr342Cys is at the 55th position, the glutamic acid of Lys362Glu is at the 75th position, the asparagine of Lys371Asn is at the 84th position, the serine of Gly390Ser is at the 103rd position, the glutamic acid of Lys399Glu is at the 112th position, the arginine of Ser476Arg is at the 189th position, and the aspartic acid of Asn487Asp is at the 200th position.

Example 3: Construction of AAV binding protein expression vector (part 2)
A polypeptide (SEQ ID NO: 16) was designed in which the signal peptide of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 12 was replaced from PelB (SEQ ID NO: 3) to MalE signal peptide (amino acid sequence: MKIKTGARILALSALTTMFSASALAKIEE, SEQ ID NO: 1), and a vector capable of expressing said polypeptide was prepared.

(1) Using the expression vector pTrcAVR8g-CRNDTCG prepared in Example 2 as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NO: 14 (5'-TTTT[GGTCTC]ACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAAAATCGAAGAAGCCATGGGCTCTGCAGGCG-3') and SEQ ID NO: 15 (5'-TTTT[GGTCTC]AGTCGTTAATGCGGATAATGCGAGGATGCGTGCCACCTGTTTTTATTTTCATGGTCTGTTTCCTGTG-3') as PCR primers (the brackets in SEQ ID NOs: 14 and 15 indicate the restriction enzyme BsaI recognition sequences), a reaction solution having the composition shown in Table 1 was prepared. The reaction solution was heat-treated at 98°C for 5 minutes, and 30 cycles of reaction were carried out, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 6 minutes, and finally a heat treatment at 72°C for 5 minutes to perform PCR.

(2) The amplified PCR product was subjected to agarose gel electrophoresis and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was named V2p.

(3) The obtained V2p was treated with the restriction enzyme DpnI (NEB) at 37°C for 1.5 hours, and then at 80°C for 20 minutes. The obtained polynucleotide was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).

(4) A reaction solution with the composition shown in Table 2 was prepared, and the DpnI-treated PCR product purified in (3) was digested with the restriction enzyme BsaI (NEB) while a ligation reaction was carried out at 25°C for 1.0 hour using T4 DNA ligase (NEB).

(5) The ligation product was used to transform E. coli W3110 strain. The obtained transformant was cultured in LB medium supplemented with 50 μg/mL kanamycin. A plasmid was extracted from the recovered cells (transformant) to obtain plasmid AVR8g-CRNDTCG-V2 containing a polynucleotide encoding a polypeptide in which the signal peptide in the AAV binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 12 was replaced with the MalE signal peptide (SEQ ID NO: 1).

(6) The nucleotide sequence of pTrcAVR8g-CRNDTCG-V2 was analyzed in the same manner as in Example 1(4) and confirmed to be the desired sequence.

The amino acid sequence of the polypeptide expressed by the expression vector pTrcAVR8g-CRNDTCG-V2 is shown in SEQ ID NO:16, and the sequence of the polynucleotide encoding said polypeptide is shown in SEQ ID NO:17.
In SEQ ID NO:16, the portion from the 1st methionine to the 30th glutamic acid is the MalE signal peptide (SEQ ID NO:1), the portion from the 34th serine to the 222nd aspartic acid is the AAV binding protein AVR8g (SEQ ID NO:8, corresponding to the region from the 312th to the 500th amino acid of the amino acid sequence described in SEQ ID NO:2 (PKD1 and PKD2 of KIAA0319L)), and the portion from the 223rd cysteine to the 229th glycine is a cysteine tag. In addition, in SEQ ID NO:16, the aspartic acid of Val317Asp is at the 39th position, the cysteine of Tyr342Cys is at the 64th position, the glutamic acid of Lys362Glu is at the 84th position, the asparagine of Lys371Asn is at the 93rd position, the serine of Gly390Ser is at the 112th position, the glutamic acid of Lys399Glu is at the 121st position, the arginine of Ser476Arg is at the 198th position, and the aspartic acid of Asn487Asp is at the 209th position.

Example 4 Expression of AAV binding proteins (part 1)
(1) Escherichia coli W3110 strain was transformed with plasmid pTrcAVR8g-CRNDTCG-V2 containing a polynucleotide (SEQ ID NO:17) encoding an AAV-binding protein consisting of the amino acid sequence set forth in SEQ ID NO:16, and the resulting transformant capable of expressing the AAV-binding protein was inoculated into 3 mL of 2YT liquid medium containing 50 μg/mL kanamycin and pre-cultured overnight at 37° C. under aerobic shaking.

(2) 0.2 mL of the preculture solution from (1) was inoculated into 20 mL of 2YT liquid medium containing 50 μg/mL kanamycin in a 200 mL baffled flask, and cultured aerobically at 37°C with shaking.

(3) 2.0 hours after the start of culture, the medium was cooled on ice, IPTG (IsoPropyl β-D-1-ThioGalactopyranoside) was added to a final concentration of 0.1 mM, and the medium was then cultured aerobically with shaking at 25°C overnight.

(4) After the cultivation was completed, the culture medium was centrifuged at 8,000 rpm at 4°C for 20 minutes to recover the bacterial cells, and a protein extract was prepared using a BugBuster Protein extraction kit (manufactured by Takara Bio Inc.).

(5) The obtained protein extract was subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and stained with CBB (Coomassie Brilliant Blue) staining solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

Comparative Example 1 Expression of AAV binding proteins (part 2)
The AAV-binding protein was expressed in the same manner as in Example 4, except that a transformant capable of expressing the AAV-binding protein obtained by transforming the E. coli W3110 strain with the expression vector pTrcAVR8g-CRNDTCG prepared in Example 2 was used as the transformant, and the obtained protein extract was subjected to SDS-PAGE.

The results of Example 4 and Comparative Example 1 are shown in Figure 2. By using the MalE signal peptide (SEQ ID NO: 1) (Example 4), the band corresponding to the AAV binding protein (approximately 24 kDa) is stronger than when PelB (SEQ ID NO: 3) is used as the signal peptide (Comparative Example 1), indicating a significant improvement in the expression level.

Claims (4)

A plasmid for expressing an adeno-associated virus (AAV) binding protein, comprising a polynucleotide encoding said protein and a polynucleotide encoding a signal peptide for secreting said protein into the periplasm,
The signal peptide is an oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO:1,
A plasmid, wherein the AAV binding protein is a polypeptide comprising at least the amino acid sequence set forth in SEQ ID NO:8 . A transformant obtained by transforming a host with the plasmid according to claim 1. The transformant according to claim 2, wherein the host is Escherichia coli. A method for producing the protein, comprising the steps of: culturing the transformant according to claim 2 or 3 to express the adeno-associated virus binding protein from the transformant; and isolating the protein from the transformant.