Method for producing adeno-associated virus binding protein

Abstract

To provide a method for efficiently producing adeno-associated virus (AAV)-binding protein obtained by a genetic engineering procedure using a coliform bacillus capable of expressing the protein.SOLUTION: A method for producing AAV-binding protein includes: a process of culturing a gene recombinant coliform bacillus including an expression vector into which an inductive promoter and polynucleotide encoding AAV-binding protein are inserted; a process of adding isopropyl-β-thiogalactopyronoside so as to become 0.04 mM or more and 4 mM or less as the final concentration at the point where the bacterial concentration of the coliform bacillus reaches 70 or more and 120 or less at the absorbance 600nm, and expressing AAV-binding protein from the coliform bacillus; and a process of collecting the expressed AAV-binding protein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently producing an adeno-associated virus (AAV) binding protein using Escherichia coli obtained by genetic engineering techniques and capable of expressing the protein, and in particular to a method for efficiently producing the protein by expressing the AAV binding protein from the Escherichia coli cultured at high density. [Background technology]

[0002] Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus of the Dependovirus genus in the Parvoviridae family. Wild-type AAV is an icosahedral particle with a diameter of approximately 20 to 30 nm, composed of approximately 60 molecules of three capsid proteins (VP1, VP2, and VP3) mixed together in a VP1:VP2:VP3 ratio of 1:1:10. It is not capable of autonomous replication and requires a helper virus such as adenovirus or herpesvirus for replication. Approximately 5 kb of single-stranded genomic DNA is stored within the virus particle. The genomic DNA is composed of T-shaped hairpin structures called ITRs (inverted terminal repeats) at both ends, which are involved in replication and packaging of genomic DNA into viral particles. Sandwiched between the two ITRs are polynucleotides encoding Rep proteins (Rep78, Rep68, Rep52, Rep40) that regulate replication and transcription, capsid proteins (VP1, VP2, VP3) that form viral particles, and AAP (Assembly Activating Protein), which promotes viral particle formation.

[0003] In recent years, AAVs have been rapidly developed as vectors for gene therapy. Examples include Glybera (manufactured by uniQure), the first gene therapy drug approved by the European Medicines Agency (EMD) in 2012 to treat lipoprotein lipase deficiency, and Luxturna (manufactured by Spark Therapeutics), the first gene therapy drug approved by the Food and Drug Administration (FDA) in 2017 to treat the rare disease hereditary retinal dystrophy, both of which are attracting attention as new treatments.

[0004] The advantages of AAV as a gene therapy vector include the ability to efficiently transfer genes into non-dividing cells (neuronal cells, muscle cells, liver cells, etc.), long-lasting gene expression in target cells, and the fact that AAV is a non-pathogenic virus, which means it is expected to be safer than other viral vectors. However, its drawback is that it requires a huge amount of vector to achieve therapeutic effects due to its low gene expression efficiency.

[0005] Recombinant AAV vectors (hereafter simply referred to as AAV vectors) are typically produced by introducing a polynucleotide encoding elements essential for AAV particle formation into cells to generate cells capable of producing AAV (hereafter also referred to as AAV-producing cells), and then culturing the cells to express the elements essential for AAV particle formation. The produced AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector formulations.

[0006] A known method for purifying AAV vectors involves affinity chromatography using an AAV adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier (Patent Document 1). However, when the AAV-binding protein used in the adsorbent is produced using Escherichia coli capable of expressing the protein, which has been obtained by genetic engineering techniques, there is a problem in that the expression level of the protein per culture medium is low. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2021 / 106882 issue Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for efficiently producing an adeno-associated virus binding protein using Escherichia coli capable of expressing said protein, which has been obtained by genetic engineering techniques. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors have conducted extensive research into the culture conditions for Escherichia coli capable of expressing an adeno-associated virus (AAV) binding protein, as well as the expression conditions for said protein, and as a result have completed the present invention.

[0010] That is, the first aspect of the present invention is Culturing a genetically modified E. coli containing an expression vector into which an inducible promoter and a polynucleotide encoding an AAV binding protein have been inserted; adding an inducer when the E. coli cell concentration reaches a certain concentration, thereby expressing the AAV binding protein from the E. coli; and recovering the expressed AAV binding protein. 1. A method for producing an AAV binding protein, comprising: In the production method, the constant concentration is 70 to 120 in terms of absorbance at 600 nm, and the inducer is isopropyl-β-thiogalactopyranoside (IPTG) at a final concentration of 0.04 mM to 4 mM.

[0011] A second aspect of the present invention is The production method according to the first aspect, wherein the step of culturing the recombinant E. coli is carried out by fed-batch culture in which a feed solution containing a carbon source and a nitrogen source is added during the culture, and the medium at the start of the culture and the carbon source and the nitrogen source contained in the feed solution are each of the following aspects: [Culture medium at the start of cultivation] Carbon source: glucose 20g / L or less, nitrogen source: yeast extract 80g / L or less [Fed-batch liquid] Carbon source: 300 g / L or more and 900 g / L or less of glucose, nitrogen source: 100 g / L or more and 500 g / L or less of yeast extract.

[0012] A third aspect of the present invention is The method according to the first or second aspect, wherein the AAV binding protein is a polypeptide selected from any one of the following (i) to (iii): (i) a polypeptide containing 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: 1; (ii) A polypeptide having an amino acid sequence containing 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: 1, provided that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, 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: 1, wherein the polypeptide has 70% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0013] The present invention will be described in detail below.

[0014] In the present invention, the Escherichia coli strain is not particularly limited, and examples thereof include the E. coli JM109 strain, the E. coli W3110 strain, and the E. coli BL21(DE3) strain.

[0015] In the present invention, the expression vector used for genetic recombination of E. coli and into which an inducible promoter and a polynucleotide encoding an AAV-binding protein are inserted may be any vector that is capable of expressing a heterologous protein in E. coli, and examples include pUC plasmid vectors, pCDF plasmid vectors, pTrc plasmid vectors, and pET plasmid vectors.

[0016] The AAV-binding protein produced by the production method of the present invention is not particularly limited as long as it is a polypeptide that can bind to AAV, and examples include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVR).

[0017] When the AAV binding protein is AAVR, preferred embodiments include polypeptides shown in any of (i) to (iii) below. (i) a polypeptide containing 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: 1; (ii) A polypeptide having an amino acid sequence containing 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: 1, provided that the amino acid sequence contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, 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: 1, wherein the polypeptide has 70% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0018] The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence of KIAA0319L (official database: UniProt, accession number: 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 set forth in SEQ ID NO: 1 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

[0019] The polypeptides shown in any one of (i) to (iii) above may contain at least the regions corresponding to PKD1 and PKD2 of the above-mentioned KIAA0319L, and may be, for example, It may include all or part of the region corresponding to other extracellular domains (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)) located on the C-terminal side of PKD2, or all or part of the region corresponding to a signal sequence such as the MANSC (Motif At N terminus with Seven Cysteines) domain located on the N-terminal side of PKD1, or the cysteine-rich region, or it may include all or part of the transmembrane region and intracellular region located on the N-terminal and / or C-terminal side of the extracellular region.

[0020] Examples of (ii) include a polypeptide comprising at least the amino acid sequence set forth in SEQ ID NO: 2 and the AAV-binding protein disclosed in WO 2021 / 106882. Examples of the substitution, deletion, insertion, or addition described in (ii) include the substitution of amino acid residues disclosed in WO 2021 / 106882.

[0021] In (ii), "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of AAVR and the type of amino acid residue, but for example, it means 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. The substitution of "one or several" amino acid residues may occur at positions other than those of the amino acid residues disclosed in WO2021 / 106882, for example, as long as it has AAV-binding activity.

[0022] The "substitution of one or several amino acid residues" in (ii) above may include not only the amino acid substitution at the specific position described above, but also conservative substitutions in which amino acids with similar physical and / or chemical properties are substituted. Those skilled in the art know that conservative substitutions generally maintain protein function between substituted and unsubstituted amino acids. 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). Furthermore, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (ii) above also includes naturally occurring mutations (mutants or variants) due to differences in the origin of AAVR or differences in species.

[0023] The homology of the amino acid sequences in (iii) above may be 70% or more, but may be even higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). As used herein, "homology" may refer to similarity or identity, and may particularly refer to identity. "Amino acid sequence homology" refers to homology across the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences refers to the sum of the proportion of amino acid residues of the same type and the proportion of amino acid residues with similar side chain properties in those amino acid sequences (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.

[0024] The AAV-binding protein produced by the production method of the present invention may further have an oligopeptide attached to its N-terminus or C-terminus, which is useful for accelerating analysis and purification from solutions containing contaminants, stabilizing the protein, etc. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and C-myc tag.

[0025] Furthermore, a signal peptide may be added to the N-terminus of the AAV-binding protein produced by the production method of the present invention to promote efficient expression in a host. Examples of signal peptides that direct protein secretion into the periplasm include PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Application Laid-Open No. 2011-097898).

[0026] In the present invention, the polynucleotide encoding the AAV binding protein to be inserted into the expression vector is, for example, (I) A method of converting the amino acid sequence of an AAV binding protein into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence; (II) A method in which polynucleotides containing the entire or partial sequence of an AAV binding protein are prepared artificially directly or from cDNA of the AAV binding protein using a DNA amplification method such as PCR, and the prepared polynucleotides are then ligated by an appropriate method; It can be made with.

[0027] In the method (I), when converting an amino acid sequence to a nucleotide sequence, it is preferable to consider the frequency of codon usage in the E. coli to be transformed. Specifically, the following codons are used infrequently (so-called rare codons): AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro). Therefore, conversion should be performed to avoid these codons.

[0028] In the present invention, any medium can be used to culture recombinant E. coli as long as it allows the E. coli to grow and the AAV binding protein to be expressed. Examples of carbon sources include glucose, fructose, maltose, sucrose, raw sugar, and molasses, with glucose being preferred. Yeast extract is preferred as a nitrogen source, but polypeptone, casein and its metabolites, corn steep liquor, soy protein, meat extract, and fish extract may also be used. Examples of inorganic salts include phosphates such as sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, sodium chloride, magnesium chloride, magnesium sulfate, iron(II) sulfate, iron(III) sulfate, iron(II) chloride, iron(III) chloride, iron citrate, ammonium iron sulfate, calcium chloride, calcium sulfate, zinc sulfate, zinc chloride, copper(II) sulfate, copper(II) chloride, manganese(II) sulfate, and manganese(II) chloride. Examples of vitamins include biotin, nicotinic acid, thiamine, riboflavin, inositol, and pyridoxine.

[0029] In the present invention, the method for culturing recombinant E. coli is not particularly limited, and the culture may be performed by any of batch culture, semi-batch culture (also known as fed-batch culture), and perfusion culture, or a combination of these. However, adding nutrient sources such as a carbon source and a nitrogen source to the medium all at once at the start of culture may inhibit the growth of E. coli and the expression of the AAV binding protein by the E. coli, and may also produce by-products such as organic acids, which may adversely affect the expression efficiency of the protein and the quality of the obtained protein. Therefore, it is preferable to culture recombinant E. coli by fed-batch culture, in which the amount of nutrient sources added at the start of culture is minimized and nutrient sources are appropriately supplied (fed-batch) during culture.

[0030] In the present invention, when culturing recombinant Escherichia coli by fed-batch culture, the concentrations of the carbon source and nitrogen source added at the start of culture are preferably 20 g / L or less when the carbon source is glucose, and 80 g / L or less when the nitrogen source is yeast extract. It is preferable to use high-concentration solutions of the carbon source and nitrogen source to be fed, as this can prevent an increase in the volume of the culture medium. Specifically, When the carbon source is glucose, the concentration is preferably 300 g / L or more and 900 g / L or less, and when the nitrogen source is yeast extract, the concentration is preferably 100 g / L or more and 500 g / L or less. The aforementioned inorganic salts may also be added.

[0031] When culturing recombinant E. coli in fed-batch culture, the carbon and nitrogen sources must be fed while maintaining the concentrations of the carbon and nitrogen sources in the medium at a predetermined low level. As used herein, "a predetermined low concentration" refers to a concentration at which the carbon source is not depleted and by-products such as organic acids are not produced. For example, when glucose is used as a carbon source, culturing at a carbon source concentration above 5 g / L may inhibit E. coli growth and AAV binding protein expression due to the accumulation of by-product organic acids. Therefore, the carbon source concentration should be at least 5 g / L or less, preferably 1 g / L or less, more preferably 0.5 g / L or less, and most preferably 0.1 g / L or less. Carbon source depletion can be monitored by any method, including, for example, a decrease in respiratory activity. A decrease in respiratory activity can manifest itself, for example, as an increase in dissolved oxygen concentration (DO) in the culture medium, an increase in oxygen concentration in the exhaust gas, a decrease in carbon dioxide concentration, or an increase in pH. In particular, DO is a preferred indicator for monitoring carbon source depletion because it responds quickly, as microbial respiratory activity decreases and then rises sharply when the carbon source is depleted.

[0032] In the present invention, the culture conditions for the recombinant E. coli are not particularly limited as long as they allow the E. coli to grow and express the AAV binding protein, but the culture temperature is preferably 15°C or higher and 50°C or lower, and particularly preferably 20°C or higher and 33°C or lower. The pH is preferably 6 or higher and 8 or lower. The culture time can be set as desired, but is usually set to several hours or higher and 100 hours or lower.

[0033] The production method of the present invention is characterized in that, when an AAV binding protein is produced by culturing recombinant E. coli containing an expression vector into which an inducible promoter and a polynucleotide encoding an AAV binding protein have been inserted, the AAV binding protein is expressed by adding the inducer IPTG to a final concentration of 0.04 mM to 4 mM when the concentration of the E. coli contained in the culture medium reaches a certain high concentration (high density). As used herein, "certain high concentration (high density)" refers to the concentration of bacterial cells reaching an absorbance at 600 nm (OD 600) refers to 70 to 120, and preferably OD 600 The final concentration of IPTG added is preferably 0.08 mM or more and 0.4 mM or less, since this particularly improves the expression level of the AAV binding protein.

[0034] To recover an AAV-binding protein expressed by the above-described method, the protein can be recovered by isolating and purifying it from the culture using a method appropriate for the expression form of the protein in the recombinant E. coli. For example, if the protein is expressed in the culture supernatant, the cells can be separated by centrifugation, and the AAV-binding protein can be purified from the resulting culture supernatant. Alternatively, if the protein is expressed intracellularly (including in the periplasm), the cells can be collected by centrifugation and then disrupted by adding an enzyme treatment agent, surfactant, or the like, and the AAV-binding protein can be extracted and purified.

[0035] The recovered AAV-binding protein can be purified by methods known in the art, such as separation / purification using liquid chromatography, which includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography. High-purity proteins can be prepared by combining these chromatographic methods for purification.

[0036] Methods for measuring the binding activity of the resulting AAV-binding protein to AAV include, for example, separating the AAV-binding protein using standard SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), staining it with dyes or immunological methods, and then colorimetrically quantifying the activity. Alternatively, AAV-binding activity may be measured using enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) or surface plasmon resonance. While activity quantification using ELISA may also be performed, the latter method is preferred for its simplicity. The combination of antibodies reacted with the AAV-binding protein in ELISA is not particularly limited, as long as the method can quantify the protein. The detection method for ELISA is also not particularly limited; for example, detection may be performed using commercially available colorimetric, fluorescent, or chemiluminescent reagents specific to the enzyme used for labeling. Specifically, when horseradish peroxidase (HRP) is used as the labeling enzyme, a colorimetric assay can be performed after oxidation of a chromogenic substrate such as TMB (3,3',5,5'-tetramethylbenzidine) with HRP and hydrogen peroxide. The AAV used to measure binding activity can be either an AAV vector or a VLP (virus-like particle). Furthermore, any serotype of AAV vector or VLP can be used as long as it exhibits binding activity to the AAV-binding protein. [Effects of the Invention]

[0037] The present invention provides a method for producing an AAV binding protein by culturing recombinant Escherichia coli containing an expression vector incorporating an inducible promoter and a polynucleotide encoding an adeno-associated virus (AAV) binding protein until the cell density reaches a certain level, adding an inducer to express the AAV binding protein, and then recovering the expressed protein. The method is characterized in that the certain cell density is a high density, with an absorbance at 600 nm of 70 to 120, and the inducer added is isopropyl-β-thiogalactopyranoside at a final concentration of 0.04 mM to 4 mM. The present invention enables efficient mass production of AAV binding proteins. The AAV binding proteins obtained by the production method of the present invention can be used as pharmaceuticals, clinical diagnostic agents, biosensors, and ligands for AAV separation agents. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 shows a summary of the relationship between the amount of IPTG added during induction and the amount of adeno-associated virus binding protein produced per culture medium. [Example]

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0040] Example 1: Inducer concentration study (1) Escherichia coli W3110 was transformed with an expression vector containing a polynucleotide (SEQ ID NO: 3) encoding the adeno-associated virus (AAV) binding protein AVR10s (sequence number 2) and an inducible promoter. The resulting recombinant E. coli capable of expressing the AAV binding protein was inoculated into 2xYT medium (tryptone: 16 g / L, yeast extract: 10 g / L, NaCl: 5 g / L, kanamycin sulfate: 50 mg / L) and precultured at 30°C for 16 hours. AVR10s (SEQ ID NO: 2) is a polypeptide containing the amino acid substitutions (i) to (x) shown below in the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500, which correspond to the extracellular domains 1 and 2 (PKD1 and PKD2) of KIAA0319L (UniProt accession number: Q8IZA0, SEQ ID NO: 1). (i) Valine at position 317 of SEQ ID NO: 1 (position 6 of SEQ ID NO: 2) is substituted with aspartic acid (ii) tyrosine at position 342 of SEQ ID NO: 1 (position 31 of SEQ ID NO: 2) is substituted with serine (iii) Lysine at position 362 of SEQ ID NO: 1 (position 51 of SEQ ID NO: 2) is substituted with glutamic acid (iv) Lysine at position 371 of SEQ ID NO: 1 (position 60 of SEQ ID NO: 2) is substituted with asparagine (v) valine at position 381 of SEQ ID NO: 1 (position 70 of SEQ ID NO: 2) is substituted with alanine (vi) Isoleucine at position 382 of SEQ ID NO: 1 (position 71 of SEQ ID NO: 2) is substituted with valine (vii) Glycine at position 390 of SEQ ID NO: 1 (position 79 of SEQ ID NO: 2) is substituted with serine (viii) Lysine at position 399 of SEQ ID NO: 1 (position 88 of SEQ ID NO: 2) is substituted with glutamic acid (ix) Serine at position 476 of SEQ ID NO: 1 (position 165 of SEQ ID NO: 2) is substituted with arginine (x) Asparagine at position 487 of SEQ ID NO: 1 (position 176 of SEQ ID NO: 2) is substituted with aspartic acid

[0041] (2) 36 mL of the preculture solution (1) was added to 1.2 L of the medium having the composition shown in Table 1, and main culture was performed. The culture apparatus used was a BMS-03PI manufactured by Able Co., Ltd., with an agitation speed of 400 to 700 rpm, an air flow rate of 1.5 L / min, a culture temperature of 30°C, and a pH of 6.8 to 7.2. Fluctuations in pH during culture were controlled within the above range by adding 14% (w / v) aqueous ammonia or 50% (w / v) phosphoric acid.

[0042] [Table 1]

[0043] (3) When the DO (dissolved oxygen concentration) measured using the DO electrode attached to the BMS-03PI exceeded 40% saturation, the feed pump was started, and the operation of supplying a feed medium having the composition shown in Table 2 was continued until the DO again fell below 40% saturation, until the end of the culture.

[0044] [Table 2]

[0045] (4) 19 to 21 hours after the start of culture, the culture temperature was changed to 25°C, the stirring speed to 600 rpm, and IPTG was added to induce the expression of the AAV binding protein. At 19 to 21 hours after the start of culture, the absorbance at 600 nm (OD 600 ) was in the range of 70 to 120, and the final concentrations of IPTG added were 0.0044 mM, 0.023 mM, 0.051 mM, 0.11 mM, 0.25 mM, 0.55 mM, or 2.7 mM.

[0046] (5) After 48 hours from the start of the culture, the culture was terminated and the cultured cells were collected by centrifugation of the culture medium.

[0047] (6) The obtained bacterial cells were extracted using a commercially available extraction reagent (BugBuster, manufactured by Merck) according to the standard protocol attached to the reagent, and the supernatant (cell-free extract) was obtained by centrifugation.

[0048] (7) The obtained cell-free extract was subjected to SDS-PAGE alongside an AAV-binding protein standard of known concentration.

[0049] (8) The density of the band corresponding to the AAV-binding protein was quantified using image analysis software (ImageQuant TL 10.0, Cytiva). The AAV-binding protein contained in the cell-free extract was quantified by comparison with the density of the band in the standard sample, and the production amount per culture medium was calculated.

[0050] Comparative Example 1: Culture without the addition of an inducer The same procedure as in Example 1 was carried out, except that IPTG was not added in Example 1(4), and the culture was completed 43 hours after the start of culture in Example 1(5).

[0051] The culture conditions and results of Example 1 and Comparative Example 1 are shown in Table 3. The production amounts per culture medium of the AAV binding proteins produced in the cultures of Example 1 and Comparative Example 1 are summarized in Figure 1. OD 600 When expressing an AAV binding protein from recombinant E. coli containing an expression vector carrying an inducible promoter and a polynucleotide encoding the AAV binding protein, cultured at a high density of 70 to 120 kJ / ml, the addition of IPTG as an inducer at a final concentration of 0.055 mM or higher improved the amount of AAV binding protein produced per culture medium compared to when IPTG was added at a final concentration of 0.023 mM or less or when no IPTG was added. As shown in Figure 1, the amount of AAV binding protein produced per culture medium was significantly improved when IPTG was added at final concentrations of 0.11 mM and 0.25 mM.

[0052] [Table 3]

Claims

1. Culturing a genetically modified Escherichia coli strain containing an expression vector into which an inducible promoter and a polynucleotide encoding an adeno-associated virus binding protein have been inserted; adding an inducer when the concentration of the E. coli cells reaches a certain concentration, thereby expressing the adeno-associated virus binding protein from the E. coli; and recovering the expressed adeno-associated virus binding protein. A method for producing an adeno-associated virus binding protein, comprising: The step of culturing the recombinant E. coli is carried out by fed-batch culture in which a feed solution containing a carbon source and a nitrogen source is fed during the culture; The adeno-associated virus binding protein is a polypeptide selected from any one of the following (i) to (iii): (i) a polypeptide containing 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: 1; (ii) A polypeptide having an amino acid sequence containing 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: 1, with the proviso that the amino acid sequence contains a substitution, deletion, insertion, or addition of 1 to 10 amino acid residues in the amino acid residues at positions 312 to 500, and having adeno-associated virus-binding activity; (iii) a polypeptide comprising at least the amino acid residues from the serine at position 312 to the aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, and having 90% or more identity to the amino acid sequence consisting of the amino acid residues at positions 312 to 500, and having adeno-associated virus-binding activity; The above-mentioned production method, wherein the constant concentration is 90 or more and 120 or less in terms of absorbance at 600 nm, and the inducer is isopropyl-β-thiogalactopyranoside at a final concentration of 0.08 mM or more and 0.4 mM or less.

2. The method according to claim 1, wherein in the step of culturing the recombinant Escherichia coli, the carbon source and nitrogen source contained in the medium at the start of the culture and in the feed solution are respectively in the following forms: [Culture medium at the start of culture] Carbon source: 20 g / L or less of glucose, Nitrogen source: 80 g / L or less of yeast extract [Fed-batch liquid] Carbon source: 300 g / L or more and 900 g / L or less of glucose, nitrogen source: 100 g / L or more and 500 g / L or less of yeast extract.

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