Method for producing recombinant adeno-associated virus-binding proteins
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- TOSOH CORP
- Filing Date
- 2025-01-17
- Publication Date
- 2026-07-30
Smart Images
Figure 2026123662000001_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to a method for producing an adeno-associated virus (AAV)-binding protein using genetically engineered Escherichia coli.
Background Art
[0002] Adeno-associated virus (AAV) is a non-enveloped virus classified in the family Parvoviridae, genus Dependovirus. The AAV outer shell particles are composed of three types of proteins (VP1, VP2, and VP3), and approximately 60 protein molecules are mixed and assembled at a ratio of approximately VP1:VP2:VP3 = 1:1:10, forming an icosahedral shape with a diameter of 20 nm to 30 nm.
[0003] AAV in nature lacks the ability to grow independently, and replication depends on helper viruses such as adenovirus and herpesvirus. When the helper virus is present, the AAV genome is replicated in the host cell, complete AAV particles containing the AAV genome are formed, and AAV particles are released from the host cell. On the other hand, when the helper virus is absent, the AAV genome remains in an episomal state or is integrated into the host chromosome (latent state).
[0004] AAV can infect cells of a wide range of species including humans, can also infect non-dividing cells that have completed differentiation such as blood cells, muscle, and nerve cells, has no pathogenicity to humans and thus has a low risk of side effects, and the virus particles are physically and chemically stable. Therefore, its utility value as a vector for gene transfer for the treatment of congenital genetic diseases has attracted attention.
[0005] GPR108 (G protein-coupled receptor 108) has been reported to be important for AAV infection (Non-Patent Literature 1). GPR108 is thought to be localized in the Golgi apparatus and is believed to play a role in one of the pathways necessary for various AAVs that have entered the cell to translocate into the nucleus and express their genes.
[0006] The production of recombinant AAV vectors (hereinafter also simply referred to as "AAV vectors") is typically carried out by introducing nucleic acids encoding elements essential for AAV particle formation into cells to create cells capable of producing AAV (hereinafter also referred to as AAV-producing cells), and then culturing these cells to express the elements essential for AAV particle formation. The manufactured AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector preparations.
[0007] The recovered and purified AAV vectors are evaluated for their performance using various analytical methods. For example, monodispersity in solution can be analyzed using size exclusion chromatography or dynamic light scattering; AAV vector concentration using quantitative PCR or ELISA; the full ratio of the AAV vector (the ratio of the vector containing the gene) using anion exchange chromatography or analytical ultracentrifugation; and the infectivity of the AAV vector using cell infection assays. By analyzing these methods, the quality of the manufactured AAV vectors can be evaluated from various perspectives.
[0008] Furthermore, the performance of AAV vectors can also be evaluated by affinity chromatography using an AAV adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier. Specifically, the infectivity of a manufactured AAV vector can be analyzed using an AAV adsorbent containing an insoluble carrier and a polypeptide containing extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0) immobilized on the carrier (Patent Document 1).
[0009] When using AAV-binding proteins for the purposes described above, it is necessary to mass-produce the proteins industrially. A suitable method for mass production includes culturing genetically modified E. coli containing a polynucleotide encoding the protein and expressing the protein, and recovering the protein expressed in the expression step. [Prior art documents] [Patent Documents]
[0010] [Patent Document 1] WO2023 / 140197 issue [Non-patent literature]
[0011] [Non-Patent Document 1] Amanda M. Dudek et. al., Mol. Ther, Vol.28, No.2, 367-381, 2020 [Overview of the project] [Problems that the invention aims to solve]
[0012] When heterologous proteins are overexpressed in E. coli, inclusion bodies can form, leading to aggregation and insolubility. In such cases, it is necessary to refold the insoluble protein to restore its higher-order structure.
[0013] The object of this disclosure is to provide a method for efficiently producing an adeno-associated virus (AAV)-binding protein that has been properly refolded by solubilizing the adeno-associated virus (AAV)-binding protein expressed in genetically modified Escherichia coli with a denaturing agent, and then refolding the solubilized AAV-binding protein. [Means for solving the problem]
[0014] To solve the above problems, the inventors diligently investigated the conditions for refolding and found conditions that enable the efficient production of adeno-associated virus (AAV)-binding proteins that have undergone appropriate refolding.
[0015] In other words, the disclosure encompasses the following [1] to [3] aspects: [1] A step of culturing genetically modified Escherichia coli containing a polynucleotide encoding an adeno-associated virus (AAV) binding protein and expressing the AAV binding protein, A step of solubilizing the AAV-binding protein with a denaturing agent, and a step of refolding the solubilized AAV-binding protein, A method for producing AAV-binding proteins, The aforementioned refolding process, The manufacturing method, comprising the step of gradually reducing the concentration of the denaturant contained in the solution of the solubilized AAV-binding protein by performing multiple dialysis operations. [2] The method for producing the AAV-binding protein according to [1], wherein the AAV-binding protein is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the 33rd to 262nd amino acid residues, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, provided that it has 70% or more identity with the amino acid sequence consisting of the 33rd to the 262nd amino acid residues, and has AAV binding activity. [3] The denaturant used in the solubilization step is a buffer solution containing 5 mol / L or more of guanidine salt, The refolding step is a step (first dialysis step) of dialyzing the solution of the solubilized AAV-binding protein with a buffer solution containing 0.5 mol / L or more and 2.0 mol / L or less of guanidine salt, and a step of dialyzing the solution of the AAV-binding protein after the first dialysis step with a buffer solution not containing guanidine salt, the production method according to [1] or [2].
Advantages of the Invention
[0016] According to the present disclosure, an adeno-associated virus (AAV)-binding protein in which refolding has been appropriately performed can be efficiently produced.
[0017] Also, according to one aspect of the present disclosure, the ratio of the AAV-binding protein present as a monomer after refolding can be improved, and in other words, the monodispersity of the AAV-binding protein after refolding can be improved. Therefore, the present disclosure can contribute to the efficient production of the AAV-binding protein.
Brief Description of the Drawings
[0018] [Figure 1] It is a diagram for confirming the purity of the adeno-associated virus (AAV)-binding protein GPR wild purified in Example 1 by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). [Figure 2] It is a diagram (chromatogram) showing the results of SEC analysis of refolded GPR wild. (a) shows the results of SEC analysis of GPR wild refolded by the method of Example 2 (stepwise dialysis method), and (b) shows the results of SEC analysis of GPR wild refolded by the method of Comparative Example 1 (dilution method).
Embodiments for Carrying Out the Invention
[0019] The details of this disclosure are described below.
[0020] The AAV-binding protein to be manufactured in this disclosure is not particularly limited as long as it is a polypeptide capable of binding to AAV, and examples include the AAV receptor (AAVR), GPR108 (G protein-coupled receptor 108), anti-AAV antibodies, and laminin receptors such as integrins. The AAV-binding protein may be a native type, a mutant (also called a variant), or the full-length or partial amino acid sequence of a native AAV-binding protein or a variant of an AAV-binding protein. Examples of native AAV-binding proteins include polypeptides consisting of the sequence UniProt No. Q9NPR9 and polypeptides consisting of the sequence UniProt No. Q8IZA0. A variant of an AAV-binding protein may mean a protein containing an amino acid sequence in which at least one or more amino acid residues are substituted, deleted, inserted, or added at least one or more positions in the amino acid sequence of a native AAV-binding protein.
[0021] Examples of AAV-binding proteins include polypeptides containing at least the N-terminal extramembrane region of GPR108, and polypeptides containing at least the extracellular domains 1 and 2 of KIAA0319L. In particular, the manufacturing method disclosed herein may be used to produce polypeptides containing at least the N-terminal extramembrane region of GPR108.
[0022] Specific examples of polypeptides containing at least the N-terminal extramembrane region of GPR108 include polypeptides selected from any of the following (i) to (iii). (i) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the 33rd to 262nd amino acid residues, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, provided that it has 70% or more identity with the amino acid sequence consisting of the 33rd to the 262nd amino acid residues, and has AAV binding activity.
[0023] In (ii) above, "one or several" means one of the following, although it varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue. Specifically, it means, for example, one to 20, one to 15, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, one or two, or one.
[0024] Furthermore, the "substitution of one or more amino acid residues" in (ii) above may include not only the amino acid substitutions at specific positions mentioned above, but also conservative substitutions that occur between amino acids with similar physical and / or chemical properties. In the case of conservative substitutions, it is generally known to those skilled in the art that the function of the protein is maintained between the substituted and unsubstituted parts. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005).
[0025] Furthermore, the "substitution, deletion, insertion, or addition of one or more amino acid residues" in (ii) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin of AAV-binding proteins or differences in species.
[0026] The "identity" of amino acid sequences in (iii) above refers to the percentage obtained by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When aligning the sequences as necessary, gaps are inserted into one or both of the two sequences to be compared as appropriate. The method of aligning sequences is not particularly limited, but can be done using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, or CLUSTALW. When gaps are inserted, the total number of amino acid residues is the number of residues counted with each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences to be compared, the identity is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues of the longer sequence.
[0027] Furthermore, the AAV-binding protein may also include a portion of the sequence located at its N-terminal and / or C-terminal end.
[0028] Furthermore, the AAV-binding protein may have an oligopeptide added to its N-terminus or C-terminus that is useful for separating it from a solution in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Additionally, an oligopeptide containing cysteine, which is useful for immobilizing the AAV-binding protein on a solid phase such as a support for chromatography, may be added to the N-terminus or C-terminus of the AAV-binding protein.
[0029] The length of the oligopeptide to be attached to the N-terminus or C-terminus of the AAV-binding protein is not particularly limited, as long as it does not impair the AAV-binding ability or stability of the AAV-binding protein. When attaching the oligopeptide to the AAV-binding protein, the polynucleotide encoding the oligopeptide may be prepared and then genetically engineered to attach to the N-terminus or C-terminus of the AAV-binding protein using a method well known to those skilled in the art, or the oligopeptide may be chemically synthesized and chemically bonded to the N-terminus or C-terminus of the AAV-binding protein.
[0030] Furthermore, a signal peptide may be added to the N-terminus of the AAV-binding protein to promote efficient expression in the host. Examples of such signal peptides include signal peptides that induce protein secretion into the periplasm, such as PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Publication No. 2011-097898), and the translation amplification sequence (TEE) at the N-terminus of the CspA protein.
[0031] In this specification, polynucleotides encoding AAV-binding proteins are, for example, (I) A method for converting the amino acid sequence of an AAV-binding protein into a nucleotide sequence and artificially synthesizing a polynucleotide containing the said nucleotide sequence, (II) A method of directly and artificially preparing polynucleotides containing the whole or partial sequence of an AAV-binding protein, or preparing them from the cDNA of an AAV-binding protein using a DNA amplification method such as PCR, and then ligating the prepared polynucleotides by an appropriate method. It can be made with [this method].
[0032] In the method described in (I) above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to consider the frequency of codon use in the host E. coli being transformed. Specifically, for arginine (R), AGA / AGG / CGG / CGA, for isoleucine (I), ATA, for leucine (L), CTA, for glycine (G), GGA, and for proline (P), CCC are used infrequently (they are so-called rare codons), so the conversion should avoid these codons. Codon usage frequency analysis can also be performed using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website).
[0033] Genetically modified Escherichia coli containing a polynucleotide encoding an AAV-binding protein can be produced by transforming the host E. coli using the polynucleotide encoding the AAV-binding protein. When transforming the host E. coli using the polynucleotide encoding the AAV-binding protein, the polynucleotide itself may be used, but it is more preferable to use an expression vector (for example, a bacteriophage, cosmid, or plasmid commonly used for the transformation of prokaryotic or eukaryotic cells) into which the polynucleotide has been inserted at an appropriate position. The expression vector is not particularly limited as long as it can stably exist and replicate within the host (E. coli) to be transformed, and examples include pET plasmid vectors, pUC plasmid vectors, pTrc plasmid vectors, pCDF plasmid vectors, and pCold vectors.
[0034] Furthermore, the aforementioned appropriate position means a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region involved in transduction. When inserting a polynucleotide encoding an AAV-binding protein into the expression vector, it is preferable to insert it in a state where it is linked to a functional polynucleotide such as a promoter necessary for expression. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and cspA promoter.
[0035] Transforming the host E. coli using an expression vector containing a polynucleotide encoding the AAV-binding protein, prepared by the method described above, can be done using methods commonly used by those skilled in the art. Specifically, transformation can be performed using methods described in known literature such as Molecular Cloning (Cold Spring Harbor Laboratory), 256, 1992. By screening the transformants obtained by the method described above using an appropriate method, transformants (genetically modified E. coli) capable of expressing the AAV-binding protein can be obtained.
[0036] To prepare the expression vector from the genetically modified E. coli, the transformant can be cultured and the resulting culture can be prepared using an alkaline extraction method or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (manufactured by Qiagen).
[0037] The transformant (genetically modified Escherichia coli) capable of expressing the AAV-binding protein used in the manufacturing method of this disclosure can be cultured in a medium suitable for culturing the target host (Escherichia coli). An example of a preferred medium is LB (Luria-Bertani) medium supplemented with the necessary nutrients. In order to selectively grow the transformant depending on whether or not the expression vector has been introduced, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the culture medium. For example, if the vector contains a kanamycin resistance gene, kanamycin should be added to the culture medium.
[0038] In addition to carbon, nitrogen, and inorganic salt sources, the culture medium may also contain appropriate nutrients, and optionally, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. The culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C, but should be selected according to the characteristics of the protein to be expressed. The pH of the culture medium is pH 6.8 to 7.4, preferably around pH 7.0.
[0039] Furthermore, if the expression vector contains an inducible promoter, it is preferable to induce the AAV-binding protein under conditions that allow for good expression. IPTG (Isopropyl-β-D-thiogalactopyranoside) can be used as an example of an inducer. By measuring the turbidity of the culture medium (absorbance at 600 nm) and adding an appropriate amount of IPTG when it is approximately between 0.5 and 1.0, and then continuing the culture, the expression of the AAV-binding protein can be induced. The concentration of IPTG added can be appropriately selected from the range of 0.005 mmol / L to 1.0 mmol / L, but a range of 0.01 mmol / L to 0.5 mmol / L is preferred. Various conditions for IPTG induction can be carried out under conditions that are well known in the art.
[0040] If proper folding does not occur in the transformant during culture, the expressed protein is often contained in an insoluble fraction such as inclusion bodies. The manufacturing method of this disclosure is characterized by solubilizing the AAV-binding protein contained in the insoluble fraction with a denaturing agent, and then performing refolding by gradually reducing the concentration of the denaturing agent in the solution of the solubilized AAV-binding protein through multiple dialysis operations.
[0041] The insoluble fraction may be recovered by centrifuging the culture after incubation and collecting the precipitate. Alternatively, the insoluble fraction may be recovered by centrifuging the culture after incubation to collect the bacterial cells, then crushing the cells by adding an enzyme treatment agent or surfactant, and then centrifuging again to collect the precipitate. The insoluble fraction obtained by the above methods may be washed with ethanol, acetone, water, etc.
[0042] The denaturing agent used to solubilize AAV-binding proteins contained in the insoluble fraction can be appropriately selected from among the denaturing agents commonly used for protein solubilization. Examples include guanidine salts, urea, and arginine salts. The solvent for the denaturing agent is not particularly limited as long as the denaturing agent dissolves in it, but may include, for example, water, acetate buffer, phosphate buffer, MES (2-Morpholinoethanesulfonic acid) buffer, HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, borate buffer, etc. If the denaturing agent is a guanidine salt, its concentration should preferably be 5 mol / L or higher.
[0043] In the solubilization process, the denaturing agent can be applied to the AAV-binding protein, but it can also be done, for example, by adding a solution containing the denaturing agent to the insoluble fraction and incubating it for a certain period of time. The solution containing the denaturing agent may also contain reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol in addition to the denaturing agent. Since reducing proteins that have formed disulfide bonds in the wrong positions during culture cleaves the disulfide bonds once they have been formed, it is effective to further include a reducing agent in the solution containing the denaturing agent.
[0044] The manufacturing method of the disclosed invention is characterized in that, when removing the denaturing agent contained in the solution of the AAV-binding protein solubilized by the method described above, the denaturing agent is removed by gradually reducing its concentration through multiple dialysis operations (step dialysis method), rather than by diluting it with an excess amount of buffer that does not contain the denaturing agent (dilution method). By removing the denaturing agent through step dialysis, the proportion of monomers of the AAV-binding protein is increased, in other words, the monodispersity of the AAV-binding protein in the solution is improved.
[0045] The number of steps in which the denaturing agent concentration is reduced by the stepwise dialysis method, and whether or not the reduction in the denaturing agent concentration is kept constant at each step, should be determined appropriately, taking into consideration the amino acid sequence of the AAV-binding protein to be refolded and the higher-order structure it should ideally adopt. However, if the number of steps is too large, the refolding operation time will be prolonged, which may lead to chemical modification of the protein or, conversely, promote aggregation, so it is best to keep the number of steps to the minimum necessary. Therefore, "multiple dialysis operations" in this specification may refer to one or more dialysis operations, and may be 1 to 10 times, 1 to 5 times, 1 to 3 times, or 1 to 2 times. In the refolding process, dialysis should be performed with a solution containing a lower concentration of denaturing agent than the concentration used for solubilization, but dialysis may be performed multiple times with a solution containing the same concentration of denaturing agent, or with a solution containing a lower concentration of denaturing agent than the solution used in the immediately preceding dialysis operation.
[0046] For example, if the denaturing agent used for solubilization is a guanidine salt of 5 mol / L or more, the solution of AAV-binding protein solubilized with the denaturing agent can be dialyzed with a buffer containing a guanidine salt of 0.5 mol / L to 2.0 mol / L (first dialysis step) to reduce the guanidine salt concentration in the solution. Then, the solution of AAV-binding protein after the first dialysis step can be dialyzed with a buffer that does not contain guanidine salt to remove the guanidine salt contained in the solution, thereby improving the proportion of monomers of the AAV-binding protein, or in other words, improving the monodispersity of the AAV-binding protein in the solution.
[0047] Here, the solution on the side of the dialysis membrane where AAV-binding proteins are absent may be called the extradialysis solution, and the solution on the side of the dialysis membrane where AAV-binding proteins are present may be called the intradialysis solution. In this specification, dialysis with a buffer containing guanidine salt in a concentration of 0.5 mol / L to 2.0 mol / L may be interpreted as dialysis using a buffer containing guanidine salt in a concentration of 0.5 mol / L to 2.0 mol / L as the extradialysis solution. Stepped dialysis may be rephrased as a method of gradually lowering the concentration of the denaturing agent in the intradialysis solution.
[0048] In the refolding process, oxidizing agents such as cystine or oxidized glutathione may be added to the non-dialysis solution. This is to oxidize the thiol groups generated by the cleavage of disulfide bonds during the reduction treatment with the above-mentioned oxidizing agents, thereby rearranging them into correct disulfide bonds.
[0049] The pore size of the dialysis membrane used in stepwise dialysis can be set appropriately, as long as it is small enough to prevent the passage of AAV-binding proteins and large enough to allow the passage of denaturants.
[0050] The method disclosed herein involves culturing genetically modified Escherichia coli containing a polynucleotide encoding an AAV-binding protein, and expressing the AAV-binding protein. A step of solubilizing the AAV-binding protein with a denaturing agent, The process includes a step of refolding the solubilized AAV-binding protein, but may also include other steps. For example, a purification step of the AAV-binding protein by ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., may be included between the solubilization step and the refolding step. For example, if the AAV-binding protein has a polyhistidine tag, a purification step by Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography may be included, and a certain amount of imidazole may be added to the solution containing the denaturant used in the solubilization step.
[0051] The solution containing the AAV-binding protein after the aforementioned refolding can be purified using, for example, liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography, and by combining these chromatography methods, the AAV-binding protein can be prepared in high purity.
[0052] The AAV-binding proteins produced by the method of this disclosure can be used, for example, for the purification or analysis of AAVs. When used for this purpose, there is no particular limitation on the AAVs to which the proteins bind; they may be naturally occurring AAVs or artificially produced AAVs. Examples of naturally occurring AAVs include serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), and serotype 13 (AAV13). Examples of artificially produced AAVs include AAVrh8, AAVrh10, and chimeric AAVs that possess two or more characteristics (cell tropism or infectivity) from these serotypes. [Examples]
[0053] The present disclosure will be described in further detail below using examples and comparative examples, but the present disclosure is not limited to these examples.
[0054] Example 1: Purification of GPRwild (1) A polynucleotide encoding an AAV-binding protein (named GPR wild) consisting of the amino acid sequence described in Sequence ID No. 2 was inserted between the cspA 5'UTR (untranslated region) sequence and the cspA 3'UTR sequence of pCold III (Takara Bio Inc.) using the Gibson Assembly method to create an expression plasmid (named pCold-GPR wild). In Sequence ID No. 2, the first methionine (M) to the fifth valine (V) is the translation amplification sequence (TEE), the sixth arginine (R) to the 235th lysine (K) is the N-terminal extramembrane region of the AAV-binding protein GPR108 (UniProt No. Q9NPR9) (i.e., the amino acid residues from the 33rd arginine (R) to the 262nd lysine (K) in Sequence ID No. 1), the histidine (H) from the 236th to the 241st histidine (H) is the histidine tag, and the cysteine (C) from the 242nd cysteine (C) to the 248th glycine (G) is the immobilization tag.
[0055] (2) The transformants obtained by transforming E. coli strain BL21(DE3) with pCold-GPRwild prepared in (1) were inoculated into 3 mL of 2×YT medium (1.6%(w / v)Tryptone, 1%(w / v)Yeast Extract, 0.5%(w / v)Sodium Chloride) containing 50 μg / mL ampicillin, and pre-cultured by aerobic shaking at 37°C overnight.
[0056] (3) 2 mL of the pre-culture solution from (2) was inoculated into a 5 L baffled flask containing 1 L of 2 × YT medium with 50 μg / mL ampicillin, and the culture was performed aerobically with shaking at 37°C.
[0057] (4) Three hours after the start of culture, the samples were cooled on ice, and IPTG (Isopropyl β-D-thiogalactopyranoside) was added to each sample to a final concentration of 0.1 mmol / L. The samples were then incubated with shaking at 15°C for 24 hours.
[0058] (5) After the culturing was complete, the cultured cells (transformed organisms) were collected by centrifuging the culture solution at 4°C and 8000 rpm for 20 minutes.
[0059] (6) To 1 g of the bacterial cells recovered in (5), 10 times the amount of solubilization buffer (20 mmol / L Tris hydrochloride buffer (pH 7.4) containing 150 mmol / L sodium chloride, 20 mmol / L imizopropyl ionazole, 0.01% (w / v) Tween 20 (trade name), 0.5% (w / v) Triton X-100 (trade name), 2 mmol / L magnesium chloride, 2.5 units / mL DNase I, and 0.2 mg / mL lysozyme) was added to suspend the bacterial cells, and the mixture was further stirred at room temperature for 1 hour to solubilize the cells. The solubilized bacterial solution after stirring was centrifuged at 15000 × g for 30 minutes at 4°C, and the precipitate was collected after removing the supernatant.
[0060] (7) The precipitate recovered in (6) was suspended in acetone and centrifuged at 15,000 g for 30 minutes at 4°C, and the supernatant was removed. The precipitate was suspended in ultrapure water and centrifuged at 15,000 g for 30 minutes at 4°C, and the supernatant was removed to recover the inclusion bodies.
[0061] (8) To 1 g of the inclusion body obtained in (7), 10 times the volume of denaturation buffer A (20 mmol / L Tris hydrochloride buffer (pH 7.4) containing 150 mmol / L sodium chloride, 20 mmol / L imizopropyl iodazole, and 6 mol / L guanidine hydrochloride) was added, and the inclusion body was solubilized by stirring overnight at 4°C. After stirring, the solubilized solution was centrifuged at 15000 × g for 30 minutes at 4°C, and the supernatant was collected.
[0062] (9) 5 mL of Ni-NTA agarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was packed into an open column, equilibrated with denaturation buffer A, and the supernatant obtained in (8) was applied to adsorb GPR wild onto the Ni-NTA agarose.
[0063] After washing off impurities by applying 10 CV (Column Volume) of denaturation buffer A to column (10)(9), 5 CV of denaturation buffer B (20 mmol / L Tris-HCl (pH 7.4) containing 150 mmol / L sodium chloride, 500 mmol / L imizopropyl ionazole, and 6 mol / L guanidine hydrochloride) was added, and GPR wild adsorbed onto Ni-NTA agarose was eluted (the resulting eluted fraction is also called the purified GPR wild product).
[0064] Figure 1 shows the results of confirming the purity of the obtained GPR wild purified product by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). It can be seen that the GPR wild expressed in the inclusion bodies was obtained in high purity by solubilization and Ni-NTA agarose purification.
[0065] Example 2: Refolding of GPR wilds by step dialysis method (1) Dithiothreitol (DTT) was added to the purified GPR wild product obtained in Example 1 to a final concentration of 10 mmol / L, and then sealed in dialysis membrane tubes with an exclusion limit molecular weight of 6 kDa to 8 kDa.
[0066] (2) The dialysis tube from (1) was immersed in 100 times the volume of dialysis buffer 1 (20 mmol / L Tris hydrochloride buffer (pH 8.0) containing 1 mol / L guanidine hydrochloride, 0.4 mol / L arginine hydrochloride, and 1 mmol / L dithiothreitol (DTT)) and dialyzed by stirring at 4°C overnight.
[0067] (3) The dialysis tubes from (2) were transferred to 30 times the volume of dialysis buffer 2 (20 mmol / L Tris hydrochloride buffer (pH 8.0) containing 1 mol / L guanidine hydrochloride, 0.4 mol / L arginine hydrochloride, and 1 mmol / L L(-)-cystine dihydrochloride), and then dialysis was performed by stirring at 4°C for 3 hours.
[0068] (4) The dialysis tubes from (3) were transferred to 300 times the volume of dialysis buffer 3 (20 mmol / L Tris hydrochloride buffer (pH 7.4)), and then dialysis was performed by stirring at 4°C for 4 hours.
[0069] (5) The solution in the dialysis tube used in (4) was removed, centrifuged at 4°C and 12000×g for 10 minutes, and the supernatant was collected. The supernatant was then clarified by filtering through a 0.22 μm filter.
[0070] (6) Size exclusion chromatography (SEC) column TSKgel G3000SW XL The device (manufactured by Tosoh Corporation) was connected to the HPLC system Nexera (manufactured by Shimadzu Corporation) and equilibrated with the Arg-SEC mobile phase (manufactured by Nacalai Tesque Corporation).
[0071] (7) After applying the GPR wild clarified in (5) to the column equilibrated in (6), the mobile phase was delivered at a flow rate of 1 mL / min, and the 280 nm absorbance of the components that passed through the column was monitored.
[0072] Comparative Example 1: Refolding of GPR wild by dilution method (1) DTT was added to the purified GPR wild product obtained in Example 1 to a final concentration of 10 mmol / L.
[0073] (2) A 100-fold refolding buffer (20 mmol / L Tris hydrochloride buffer (pH 8.0) containing 1 mmol / L L(-)-cystine dihydrochloride) was prepared for the solution from (1) and cooled to 4°C.
[0074] (3) While stirring the refolding buffer prepared in (2), the solution from (1) was added dropwise using a pipette.
[0075] (4) The solution after dropwise addition was clarified by filtering through a 0.22 μm filter.
[0076] (5) An open column packed with 5 mL of Ni-NTA agarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was equilibrated with Ni buffer A (20 mmol / L Tris hydrochloride buffer (pH 7.4) containing 150 mmol / L sodium chloride, 20 mmol / L imizopropyl alcohol, and 0.01% (w / v) Tween 20 (trade name)), and then the solution clarified in (4) was applied.
[0077] (6) After washing off impurities by applying 5 CV of Ni buffer A to the column from (5), 5 CV of Ni buffer B (20 mM Tris hydrochloride buffer (pH 7.4) containing 150 mmol / L sodium chloride, 500 mmol / L imisorbazole, and 0.01% (w / v) Tween 20 (trade name)) was added to elute the GPR wild adsorbed on the column.
[0078] (7) The GPR wild solution obtained in (6) was sealed in a dialysis membrane tube with an exclusion limit molecular weight of 6 kDa to 8 kDa, then immersed in 5 L of dialysis buffer (20 mmol / L Tris hydrochloride buffer (pH 7.4)) and dialyzed by stirring overnight at 4°C.
[0079] (8) The GPR wild solution dialyzed in (7) was subjected to SEC analysis in the same manner as in Examples 2(6) to (7).
[0080] Figure 2(a) shows the SEC analysis results (chromatogram) of GPR wild refolded by the method described in Example 2, and Figure 2(b) shows the SEC analysis results (chromatogram) of GPR wild refolded by the method described in Comparative Example 1. It can be seen that when the refolding process is performed using a stepwise dialysis method, in which the concentration of the denaturing agent is gradually reduced by multiple dialysis operations on a protein-containing solution solubilized with a buffer containing a denaturing agent, a portion of the GPR wild becomes monomeric (Figure 2(a)). On the other hand, when the refolding process is performed using a dilution method, in which the denaturing agent contained in the solubilized protein-containing solution is diluted with a large excess of a diluent that does not contain the denaturing agent, thereby removing the effect of the denaturing agent all at once, it can be seen that the GPR wild exists as approximately aggregates (Figure 2(b)). Note that aggregates may also mean aggregates, and monomers may mean AAV-binding proteins that have been properly refolded.
[0081] From the above results, it was found that when refolding the insoluble AAV-binding protein after solubilization of the protein expressed in genetically modified Escherichia coli containing a polynucleotide encoding the AAV-binding protein, the proportion of AAV-binding protein existing as monomers is improved when performed by the aforementioned stepwise dialysis method compared to the aforementioned dilution method. In other words, the monodispersibility of the AAV-binding protein in solution is improved. That is, according to the method of this disclosure, the AAV-binding protein that has been properly refolded can be efficiently recovered.
Claims
1. A step of culturing genetically modified Escherichia coli containing a polynucleotide encoding an adeno-associated virus (AAV) binding protein, and expressing the AAV binding protein. A step of solubilizing the AAV-binding protein with a denaturing agent, and a step of refolding the solubilized AAV-binding protein, A method for producing AAV-binding proteins, The aforementioned refolding process, The manufacturing method, comprising the step of gradually reducing the concentration of the denaturant contained in the solution of the solubilized AAV-binding protein by performing multiple dialysis operations.
2. The method for producing the product according to claim 1, wherein the AAV-binding protein is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid sequence includes substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions within the 33rd to 262nd amino acid residues, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 33rd arginine to the 262nd lysine of the amino acid sequence described in Sequence ID No. 1, provided that it has 70% or more identity with the amino acid sequence consisting of the 33rd to the 262nd amino acid residues, and has AAV binding activity.
3. The denaturing agent used in the solubilization step is a buffer solution containing 5 mol / L or more of a guanidine salt, The aforementioned refolding process, The first dialysis step involves dialyzing the solution of the solubilized AAV-binding protein with a buffer containing 0.5 mol / L to 2.0 mol / L of guanidine salt. The method for producing AAV-binding protein according to claim 1 or 2, further comprising the step of dialyzing the solution of AAV-binding protein after the first dialysis step with a buffer that does not contain guanidine salt.