JP2022114713A - Plasmid for expression of recombinant adeno-associated virus binding protein, and method for producing the protein by using the same - Google Patents

Abstract

To provide a plasmid that can efficiently produce adeno-associated virus binding protein by genetic transformation, and a method for efficiently producing the protein by using the same.SOLUTION: The present invention discloses a plasmid for expression of adeno-associated virus binding protein, wherein: as a polynucleotide encoding a signal peptide for secreting of the protein to the periplasm, used is a polynucleotide encoding MalE signal peptide, or a polynucleotide encoding an oligopeptide in which one or several residues are substituted, deleted or inserted in the MalE signal peptide.SELECTED DRAWING: Figure 2

Description

TECHNICAL FIELD The present invention relates to a recombinant adeno-associated virus-binding protein expression plasmid and a method for producing the protein using the same. In particular, the present invention relates to a plasmid capable of efficiently producing the protein by using a signal peptide that secretes the protein into the host periplasm, and a method for producing the protein using the plasmid.

It is known that after a protein is synthesized in a cell, one remains inside the cell as it is, and another is released outside the cell as a secretory protein. In addition, in recombinant protein production using E. coli or the like as a host, a method of secreting and expressing the protein in the periplasmic region between the intracellular membrane and the outer membrane is known (Patent Document 1). An oligopeptide called a signal peptide plays an important role in transporting a recombinant protein from the cell to the periplasmic region (Patent Document 1). The following three characteristics of the amino acid sequence that constitutes the signal peptide are mentioned (Patent Document 1).
(i) The N-terminal region contains at least one basic amino acid such as arginine or lysine, and the ionic bond between the positively charged side chain of the basic amino acid and the negatively charged surface of the cell membrane causes the signal peptide to bind to the cell membrane. move inside.
(ii) The central region contains many hydrophobic amino acids such as alanine, leucine, isoleucine and valine, and is involved in penetration into the cell membrane.
(iii) In the C-terminal region, a specific amino acid that is cleaved by signal peptidase after cell membrane penetration exists as a recognition site, and cleavage at the recognition site releases the mature protein to the periplasmic region and outside the cell. be done.

In order to secretly express a recombinant protein into the periplasm of the host, it is necessary to add a signal peptide to the N-terminal side of the protein to promote secretory expression into the periplasm. However, when the above method is used, there is a problem that the productivity as an active protein is low. There have also been reports of improved productivity by modification of the signal peptide (Patent Documents 1 to 4), but these were not sufficient for industrial production. In particular, no example has yet been known in which an adeno-associated virus-binding protein could be expressed in large amounts as an active recombinant protein.

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

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

In order to solve the above problems, the present inventors have made intensive studies and found that, by optimizing a signal peptide for secreting a protein into the host periplasm, an adeno-associated virus (AAV)-binding protein by genetic recombination technology The inventors have found that the production amount of can be improved, leading to the completion of the present invention.

That is, the present invention includes the following inventions:
(A) A plasmid for expressing the protein, comprising a polynucleotide encoding an AAV binding protein and a polynucleotide encoding a signal peptide for secreting the protein into the periplasm,
The above 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 are substituted, deleted or inserted.

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

(C) The transformant according to (B), wherein the host is E. coli.

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

The present invention will be described in detail below.

The present invention provides a polynucleotide that encodes a signal peptide for secreting the protein into the periplasm, contained in a plasmid that expresses an AAV-binding protein,
(I) a polynucleotide encoding a native MalE signal peptide (region from 1 to 30 of UniProt No. P0AEX9) consisting of the amino acid sequence set forth in SEQ ID NO: 1, or (II) the polynucleotide described in (I) above A polynucleotide encoding an oligopeptide in which one or several residues are substituted, deleted or inserted (hereinafter collectively referred to as "alteration") in the signal peptide,
is characterized by efficient production of AAV-binding proteins.
In (II) above, the number of amino acid residues to be modified (modification of one or several residues) is the objective of the present invention, the recombinant protein expressed in the host, efficiently translocating to the periplasmic region is not particularly limited, and one example is 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, one amino acid residue may be modified multiple times, two or more amino acid residues may be modified continuously, or a combination thereof may be used.

The plasmid of the present invention is a polynucleotide (polynucleotide C) obtained by adding a polynucleotide (polynucleotide B) encoding the above-described signal peptide to the 5' end of a polynucleotide (polynucleotide A) encoding an AAV-binding protein. is obtained by inserting it into the 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 is inserted as a linker between the polynucleotide A and the polynucleotide B. You may The plasmid into which the polynucleotide C is inserted is not particularly limited as long as it can stably exist and replicate in the host to be transformed. , and the pBBR plasmid. The above-mentioned suitable position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, and the regions involved in transmissibility. Transformation of a host with the plasmid of the present invention can be carried out by a method commonly used by those skilled in the art.

The AAV-binding protein produced using the 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. Examples include laminin receptors such as integrins, anti-AAV antibodies and AAV receptors (AAVR). A preferred embodiment when the AAV-binding protein is AAVR includes a polypeptide shown in any of the following (i) to (iii);
(i) a polypeptide comprising at least amino acid residues from serine 312 to aspartic acid 500 in the amino acid sequence set forth in SEQ ID NO: 2;
(ii) containing at least amino acid residues from serine 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 2, provided that the amino acid residues from position 312 to 500 are further one or several A polypeptide having an amino acid sequence containing one or several amino acid residue substitutions, deletions, insertions, or additions at the position of and having AAV binding activity;
(iii) contains at least amino acid residues from serine 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 2, but is 70% or more homologous to the amino acid sequence from 312 to 500 and AAV-binding activity.

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

The polypeptide shown in any one of (i) to (iii) above may contain at least the regions corresponding to PKD1 and PKD2 of KIAA0319L described above, for example, other extracellular regions on the C-terminal side of PKD2 It may contain all or part of the region corresponding to the domain (domain 3 (PKD3), domain 4 (PKD4) and domain 5 (PKD5)), or MANSC (Motif At N terminus with Seven Cysteines) domain corresponding to the signal sequence and all or part of the cysteine-rich region, the transmembrane region on the N-terminal side and / or C-terminal side of the extracellular region and the intracellular region All or part may be included.

In (ii) above, "one or several" varies depending on the position of amino acid substitution in the AAVR conformation and the type of amino acid residue, but examples include 1 to 50, 1 to 30, 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, 1 means either

As an example of the above (ii), at least the amino acid residues from the 312nd serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 2, provided that the amino acid residues from the 312nd to the 500th , Gly390Ser (this notation indicates that glycine at position 390 in SEQ ID NO: 2 is replaced with serine, the same applies below), Ile319Phe, Leu321Pro, Pro322Leu, Asn324Asp, Asn324Ser, Gln327Arg, Asn329Asp, Asn329Tyr, Ala330Cysr331Gly, Ｖａｌ３３２Ｇｌｎ、Ｌｅｕ３３３Ｖａｌ、Ｌｅｕ３３３Ｐｒｏ、Ｇｌｎ３３４Ａｒｇ、Ｐｒｏ３３７Ｌｅｕ、Ｌｙｓ３３８Ａｒｇ、Ｌｙｓ３３８Ｇｌｕ、Ｇｌｕ３４０Ｇｌｙ、Ｔｈｒ３４１Ａｌａ、Ｔｙｒ３４２Ｃｙｓ、Ｔｙｒ３４２Ｈｉｓ、Ｔｙｒ３４２Ａｓｎ、Ｔｈｒ３４３Ｍｅｔ、Ｔｙｒ３４４Ｈｉｓ、Ａｓｐ３４５Ａｓｎ、Ｔｒｐ３４６Ｌｅｕ、Ｔｒｐ３４６Ａｒｇ、Ｇｌｎ３４７Ｌｅｕ、Ｇｌｎ３４７Ｐｒｏ、Ｌｅｕ３４８Ｐｒｏ、Ｉｌｅ３４９Ｔｈｒ、Ｔｈｒ３５０Ｍｅｔ、Ｈｉｓ３５１Ｌｅｕ、Ｐｒｏ３５２Ｌｅｕ、Ａｒｇ３５３Ｃｙｓ、 Ａｓｐ３５４Ｇｌｙ、Ｔｙｒ３５５Ｈｉｓ、Ｔｙｒ３５５Ａｓｎ、Ｔｙｒ３５５Ｃｙｓ、Ｓｅｒ３５６Ｃｙｓ、Ｇｌｙ３５７Ｃｙｓ、Ｈｉｓ３６３Ａｒｇ、Ｈｉｓ３６３Ｌｅｕ、Ｓｅｒ３６４Ｐｒｏ、Ｇｌｎ３６５Ａｒｇ、Ｉｌｅ３６６Ｔｈｒ、Ｌｅｕ３６７Ｐｒｏ、Ｌｙｓ３６８Ａｒｇ、Ｌｅｕ３６９Ｇｌｎ、Ｌｅｕ３６９Ｐｒｏ、Ｓｅｒ３７０Ａｌａ、Ｌｙｓ３７１Ｇｌｕ、Ｔｈｒ３７３Ａｌａ、Ｌｅｕ３７６Ｐｒｏ、Ｔｙｒ３７７Ｃｙｓ、Ｐｈｅ３７９Ｓｅｒ、Ｖａｌ３８１Ａｌａ、Ｇｌｕ３８４Ｇｌｙ、Ａｓｎ３８７Ｓｅｒ、Ｈｉｓ３８９Ｇｌｎ、 Ｈｉｓ３８９Ｌｅｕ、Ｈｉｓ３８９Ａｒｇ、Ｇｌｕ３９１Ｌｙｓ、Ｔｙｒ３９３Ｃｙｓ、Ｖａｌ３９６Ａｌａ、Ｌｙｓ３９９Ｇｌｕ、Ｌｙｓ３９９Ａｒｇ、Ａｒｇ４０６Ｓｅｒ、Ｖａｌ４１２Ａｌａ、Ｇｌｎ４１５Ｌｅｕ、Ｐｈｅ４１６Ｓｅｒ、Ｇｌｎ４４１Ｌｅｕ、Ｔｙｒ４４２Ｐｈｅ、Ｌｙｓ４４８Ａｒｇ、Ｇｌｕ４５３Ｇｌｙ、Ｌｙｓ４５５Ａｒｇ、Ｇｌｕ４５８Ｇｌｙ、Ａｌａ４６１Ｓｅｒ、Ｓｅｒ４７６Ａｒｇ、Ｌｅｕ４７７Ｐｒｏ、Ａｓｎ４８７Ａｓｐ、Ａｓｎ４９２Ａｓｐ、Ｉｌｅ３１９Ａｓｎ、Ｉｌｅ３１９Ｓｅｒ、 Ｔｈｒ３２０Ｉｌｅ、Ｌｙｓ３２３Ｇｌｕ、Ｌｅｕ３２８Ｇｌｎ、Ｌｅｕ３２８Ｐｒｏ、Ｔｙｒ３３１Ｈｉｓ、Ｖａｌ３３２Ａｌａ、Ｖａｌ３３２Ｇｌｕ、Ｐｒｏ３３６Ｇｌｎ、Ｐｒｏ３３７Ｇｌｎ、Ｌｙｓ３３８Ａｓｎ、Ｔｙｒ３４２Ａｒｇ、Ｔｈｒ３５０Ｓｅｒ、Ｉｌｅ３６６Ｐｈｅ、Ｖａｌ３８３Ａｌａ、Ｇｌｎ３８６Ａｒｇ、Ｈｉｓ３８９Ａｓｐ、Ｇｌｙ３９２Ｃｙｓ、Ｖａｌ３９４Ａｌａ、Ｉｌｅ４０９Ｖａｌ、Ｓｅｒ４７６Ｇｌｙ、Ｔｈｒ４９０Ｓｅｒ、Ｓｅｒ３１２Ｐｒｏ、Ａｌａ３１３Ｓｅｒ、Ｖａｌ３１７Ａｓｐ、Ｇｌｎ３１８Ｐｒｏ、 Ｔｈｒ３２０Ａｌａ、Ｌｙｓ３２３Ａｒｇ、Ｖａｌ３２６Ａｌａ、Ｖａｌ３２６Ｇｌｕ、Ｇｌｎ３２７Ｈｉｓ、Ｇｌｎ３２７Ｌｅｕ、Ａｓｎ３２９Ｈｉｓ、Ａｓｎ３２９Ｉｌｅ、Ｖａｌ３３２Ａｌａ、Ｇｌｕ３３５Ｇｌｙ、Ｇｌｕ３３５Ｖａｌ、Ｇｌｕ３４０Ｖａｌ、Ｔｈｒ３４１Ｐｒｏ、Ｔｈｒ３４３Ｓｅｒ、Ｔｙｒ３４４Ｐｈｅ、Ｔｒｐ３４６Ｃｙｓ、Ｍｅｔ３５９Ｌｅｕ、Ｇｌｕ３６０Ｌｙｓ、Ｇｌｕ３６０Ｖａｌ、Ｇｌｙ３６１Ｃｙｓ、Ｌｙｓ３６２Ｇｌｕ、Ｌｙｓ３６２Ａｓｎ、Ｌｙｓ３６２Ｇｌｙ、Ｓｅｒ３６４Ｌｅｕ、Ｌｙｓ３７１Ａｓｎ、 Ｌｙｓ３７１Ａｓｐ、Ｌｅｕ３７２Ｐｒｏ、Ｌｅｕ３７２Ｇｌｎ、Ｐｒｏ３７４Ｌｅｕ、Ｇｌｕ３７８Ｇｌｙ、Ｇｌｕ３７８Ｖａｌ、Ｐｈｅ３７９Ｔｙｒ、Ｐｈｅ３７９Ｃｙｓ、Ｌｙｓ３８０Ｇｌｕ、Ｖａｌ３８１Ａｓｐ、Ｉｌｅ３８２Ｖａｌ、Ｇｌｕ３８４Ｖａｌ、Ｖａｌ３９４Ａｓｐ、Ｖａｌ３９４Ｉｌｅ、Ａｓｎ３９５Ｓｅｒ、Ｔｈｒ３９７Ｓｅｒ、Ｇｌｕ４０１Ｖａｌ、Ａｒｇ４０３Ｈｉｓ、Ａｒｇ４０６Ｈｉｓ、Ｇｌｎ４１５Ａｒｇ、Ｔｈｒ４２６Ａｌａ、Ｇｌｎ４３２Ｌｅｕ、Ｇｌｎ４３２Ａｒｇ、Ｇｌｎ４４１Ａｒｇ、Ｈｉｓ４４３Ｌｅｕ、 Examples thereof include polypeptides in which at least one of Lys448Glu, Ile456Val, Asp483Asn, Ser488Leu, Val499Glu and Val499Ile has been substituted with an amino acid. Among them, Gly390Ser is an amino acid substitution with particularly improved stability to heat and acid. Therefore, at least the amino acid residues from the 312nd serine to the 500th aspartic acid in the amino acid sequence set forth in SEQ ID NO: 2, provided that the 312nd to the 500th amino acid residues are substituted with Gly390Ser At least the resulting polypeptide is a preferred embodiment of (ii) above.

In addition, the "replacement of one or several amino acid residues" in (ii) above includes substitutions between amino acids with similar physical and/or chemical properties in addition to amino acid substitutions at specific positions described above. Conservative substitutions may occur. Conservative substitutions are generally known to those skilled in the art to maintain protein function between those with and without substitutions. 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 includes naturally occurring mutations ( mutants or variants) are also included.

The homology of the amino acid sequences in (iii) above may be 70% or more, and may be higher (eg, 80% or more, 85% or more, 90% or more, or 95% or more). As used herein, "homology" may mean similarity or identity, and may mean identity in particular. "Amino acid sequence homology" means homology to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same kind 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 and the ratio of amino acid residues with similar side chain properties in those amino acid sequences (Jikken Igaku, 31 (3 ), Youdosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

Examples of the host used for producing the transformant of the present invention include animal cells such as COS (CV-1 Origin, SV40) cells and CHO (Chinese Hamster Ovary) cells, Bacillus (Brevibacillus and Paenibacillus) cells. Bacillus in a broad sense such as genus bacteria), bacteria typified by Escherichia coli, yeast typified by the genus Saccharomyces, genus Pichia, and Schizosaccharomyces, and filamentous fungi typified by Aspergillus oryzae. Escherichia coli, which is easy to handle, is preferably used as a host.

In order to recover the expressed protein from the culture medium of the transformant of the present invention, it may be appropriately selected according to the mode of expression. For example, when the expressed protein is expressed in the periplasm of the host, the host obtained by centrifuging the culture medium is suspended in an appropriate buffer and the cells are disrupted (physical disruption, disruption with a drug, etc.), followed by centrifugation. A cell-free extract containing the expressed protein can be obtained by removing the crushed residue. If the expressed protein leaks from the periplasm of the host into the culture supernatant, the culture obtained by centrifuging the culture solution can be obtained. The expressed protein may be recovered from the supernatant. When disrupting host cells with a drug, for example, 150 mM NaCl, 1 mM EDTA, ₆ mM MgSO4, 250 U/L Benzonase (trade name), 0.3 g/L Lysozyme, and 0.4% (w/v) Triton X -100 (trade name), 50 mM Tris-HCl (pH 8.0) containing 0.5% (w/v) CTAB (hexadecyltrimethylammonium bromide) and 50 mM CaCl ₂ (JP 2013-252099 A) Alternatively, it may be crushed using a commercially available extraction reagent such as BugBuster Protein extraction kit (manufactured by Takara Bio Inc.).

A solution containing the recovered protein can be purified and isolated by cation exchange chromatography, hydrophobic chromatography, or the like. The carrier for cation exchange chromatography is obtained by binding a cation exchange group such as a carboxymethyl group or a sulfopropyl group to the carrier. CM Sepharose FastFlow (manufactured by Cytiva) is exemplified. The carrier for hydrophobic chromatography is obtained by binding a hydrophobic functional group such as an ether group, a phenyl group, or a butyl group to the carrier. manufactured by Tosoh Corporation). When purification is carried out using various chromatographic carriers, an appropriate open column or the like may be packed with an amount of the carrier determined according to the amount of the applied liquid to be introduced and the protein adsorption performance of the carrier.

In addition, the chromatography carrier is pre-equilibrated with an appropriate buffer (Tris/Tris hydrochloride buffer, glycine/sodium hydroxide buffer, phosphate buffer, etc.) before introducing the application liquid. . The protein-containing solution recovered by the above-described method is introduced into the equilibrated chromatography carrier so that the protein is adsorbed on the carrier, and the carrier is washed with the same buffer as that used for equilibration. Thereafter, the adsorbed protein is eluted using an elution buffer to obtain a solution containing the purified protein.

The present invention uses a MalE signal peptide-encoding polynucleotide or a MalE signal peptide as a polynucleotide encoding a signal peptide for secreting the adeno-associated virus-binding protein into the periplasm, contained in a plasmid that expresses the adeno-associated virus-binding protein. By using a polynucleotide encoding an oligopeptide in which one or several residues are substituted, deleted or inserted, the production amount of the protein can be greatly improved compared to the use of the PelB signal peptide. It is possible to greatly improve the production efficiency of recombinant proteins useful in the field.

Fig. 2 shows the structure of the adeno-associated virus (AAV)-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 16; Comparison of productivity of AAV-binding protein between addition of PelB signal peptide (Comparative Example 1) and addition of MalE signal peptide (Example 4) to the N-terminal side of AAV-binding protein figure.

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 By inserting a polynucleotide encoding a native PelB signal peptide (amino acid sequence: MKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 3) into a plasmid pTrc99a (manufactured by Cytiva) by the method shown below, the expression vector pTrcpel was made.

(1) SEQ ID NO: 4 (5'-CATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGC-3') and SEQ ID NO: 5 (5'-CATGGCCATCGCCGGCTGGGCAGCGAGGAGCAGCAGACCAGCAGCAGCGGTCGGCAGCAGGTATTT-3') after heating for 5 minutes with an equal amount of mixed oligonucleotides consisting of the sequences set forth in The double-stranded oligonucleotide PelBp1 was prepared by lowering the temperature in 1°C increments for 1 minute until 15°C. PelBp1 was kept at 15° C. until use.

(2) The PelBp1 prepared in (1) was ligated to the plasmid pTrc99a previously digested with the restriction enzyme NcoI using a DNA Ligation kit (manufactured by Takara Bio Inc.). company) was transformed.

(3) After culturing the obtained transformant in LB (Luria-Bertani) medium containing 100 μg/mL carbenicillin sodium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), QIAprep Spin Miniprep kit (manufactured by Qiagen) was used. was used to extract the expression vector pTrcpel.

(4) Of the expression vector pTrcpel prepared in (3), the nucleotide sequence of the polynucleotide encoding the PelB signal peptide and its surrounding region was analyzed with a fully automatic DNA sequencer Genetic Analyzer 3500 (manufactured by Thermo Fisher Scientific). , was confirmed to be the desired sequence (that is, the sequence of the polynucleotide encoding the oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO:3). In the analysis, either the oligonucleotide shown 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) is added to the C-terminal side of the adeno-associated virus (AAV) binding protein AVR8g consisting of the amino acid sequence set forth in SEQ ID NO: 8, It was inserted into the expression vector pTrcpel prepared in Example 1. AVR8g corresponds to the extracellular domain 1 (PKD1) and domain 2 (PKD2) regions of KIAA0319L (UniProt No. Q8IZA0), amino acid residues 312 to 500 in the amino acid sequence set forth in SEQ ID NO: 2. However, at the 312nd to 500th amino acid residues, Val317Asp (this notation represents that the 317th valine is substituted with aspartic acid, the same applies hereinafter), Tyr342Cys, Lys362Glu, Lys371Asn , Gly390Ser, Lys399Glu, Ser476Arg and Asn487Asp amino acid substitutions.

(1) Plasmid pET-AVR8g (SEQ ID NO: 9) containing a polynucleotide encoding AAV-binding protein AVR8g consisting of the amino acid sequence set forth in SEQ ID NO: 8 was used as a template DNA, and SEQ ID NO: 10 (5'-TAATACGACTCACTATAGGG-3' ) and SEQ ID NO: 11 (5'-TTTT [AAGCTT] CATCCGCAGGTATCGTTGCGGCAGTCTACCGCTTTGTTGACGGTAAG-3') oligonucleotides (the square brackets in SEQ ID NO: 11 indicate the restriction enzyme HindIII recognition sequence) as PCR primers, PCR was performed. For PCR, after preparing a reaction solution having the composition shown in Table 1, the reaction solution was subjected to the first step at 98 ° C. for 10 seconds, the second step at 50 ° C. for 5 seconds, and the third step at 72 ° C. for 1 minute. It implemented by repeating 30 cycles of reaction made into 1 cycle. A polynucleotide encoding AVR8g-CRNDTCG was obtained by purifying the resulting PCR product.

(2) The obtained polynucleotide encoding AVR8g-CRNDTCG was digested with restriction enzymes NcoI and HindIII, and then ligated to the expression vector pTrcpel prepared in Example 1, which had been previously digested with restriction enzymes NcoI and HindIII. E. coli strain W3110 was transformed using the

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

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

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

Example 3 Construction of AAV binding protein expression vector (Part 2)
Design a polypeptide (SEQ ID NO: 16) in which the signal peptide of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 12 is replaced with the MalE signal peptide (amino acid sequence: MKIKTGARILALSALTTMMFSASALAKIEE, SEQ ID NO: 1) from PelB (SEQ ID NO: 3) Then, a vector capable of expressing the polypeptide was constructed.

（１）実施例２で作製した発現ベクターｐＴｒｃＡＶＲ８ｇ－ＣＲＮＤＴＣＧを鋳型とし、配列番号１４（５'－ＴＴＴＴ［ＧＧＴＣＴＣ］ＡＣＧＡＣＧＡＴＧＡＴＧＴＴＴＴＣＣＧＣＣＴＣＧＧＣＴＣＴＣＧＣＣＡＡＡＡＴＣＧＡＡＧＡＡＧＣＣＡＴＧＧＧＣＴＣＴＧＣＡＧＧＣＧ－３'）および配列番号１５（５'－ＴＴＴＴ［ＧＧＴＣＴＣ］ＡＧＴＣＧＴＴＡＡＴＧＣＧＧＡＴＡＡＴＧＣＧＡＧＧＡＴＧＣＧＴＧＣＡＣＣＴＧＴＴＴＴＴＡＴＴＴＴＣＡＴＧＧＴＣＴＧＴＴＴＣＣＴＧＴＧ－３' ) (square brackets in SEQ ID NOS: 14 and 15 indicate the restriction enzyme BsaI recognition sequence) as PCR primers, reaction solutions having the compositions shown in Table 1 were prepared. The reaction solution is heat-treated at 98 ° C. for 5 minutes, and the reaction is performed for 30 cycles, with the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 6 minutes. Finally, PCR was performed by heat treatment at 72° C. for 5 minutes.

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

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

(4) A reaction solution having 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 using T4 DNA ligase (NEB). Then, the ligation reaction was carried out at 25°C for 1.0 hour.

(5) Escherichia coli strain W3110 was transformed with the ligation product. The resulting transformants were cultured in LB medium supplemented with 50 μg/mL of kanamycin. A plasmid is extracted from the collected cells (transformants) to encode a polypeptide in which the signal peptide in the AAV-binding protein consisting of the amino acid sequence of SEQ ID NO: 12 is replaced with the MalE signal peptide (SEQ ID NO: 1). A plasmid AVR8g-CRNDTCG-V2 containing a polynucleotide was obtained.

(6) The nucleotide sequence of pTrcAVR8g-CRNDTCG-V2 was analyzed in the same manner as in Example 1(4) to confirm that it had 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 the polypeptide is shown in SEQ ID NO: 17, respectively.
In SEQ ID NO: 16, from the 1st methionine to the 30th glutamic acid is the MalE signal peptide (SEQ ID NO: 1), and from the 34th serine to the 222nd aspartic acid is the AAV binding protein AVR8g (SEQ ID NO: 8, It is a region from 312nd to 500th amino acid sequence of SEQ ID NO: 2 (corresponding to PKD1 and PKD2 of KIAA0319L), and 223rd cysteine to 229th glycine is a cysteine tag. In SEQ ID NO: 16, Val317Asp aspartic acid is 39th, Tyr342Cys cysteine is 64th, Lys362Glu glutamic acid is 84th, Lys371Asn asparagine is 93rd, Gly390Ser serine is 112th, Lys399Glu glutamic acid is 121st, Arginine of Ser476Arg is present at position 198, and aspartic acid of Asn487Asp is present at position 209, respectively.

Example 4 Expression of AAV binding protein (Part 1)
(1) AAV-binding protein obtained by transforming E. coli strain W3110 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 was inoculated into 3 mL of 2YT liquid medium containing 50 μg/mL of kanamycin, and precultured by aerobically shaking culture at 37° C. overnight.

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

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

(4) After completion of the culture, the culture solution was centrifuged at 8000 rpm at 4° C. for 20 minutes to collect the cells, and a protein extract was prepared using BugBuster Protein extraction kit (manufactured by Takara Bio Inc.).

(5) The resulting 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 protein (part 2)
As a transformant, 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 in the same manner as in Example 4. AAV binding proteins were expressed by the method and the resulting protein extracts were subjected to SDS-PAGE.

The results of Example 4 and Comparative Example 1 are shown together in FIG. By using the MalE signal peptide (SEQ ID NO: 1) (Example 4), the band corresponding to the AAV-binding protein (approximately 24 kDa) is darker, indicating that the expression level is significantly improved.

Claims (4)

A plasmid for expressing the protein, comprising a polynucleotide encoding an adeno-associated virus-binding protein and a polynucleotide encoding a signal peptide for secreting the protein into the periplasm,
The above 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 are substituted, deleted or inserted. A transformant obtained by transforming a host with the plasmid according to claim 1. 3. The transformant according to claim 2, wherein the host is E. coli. 4. The method comprising the steps of culturing the transformant according to claim 2 or 3 to express an adeno-associated virus-binding protein from the transformant, and isolating the protein from the transformant. A method for producing proteins.

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