Method for producing proteins using genetically modified E. coli

Abstract

To provide a method that optimizes a medium for use in culturing recombinant Escherichia coli, capable of expressing recombinant protein, and efficiently produces the recombinant protein when using the Escherichia coli for the protein synthesis.SOLUTION: The foregoing problem is solved by measuring a sodium concentration in recombinant Escherichia coli, and culturing the Escherichia coli in a medium to which sodium has been added to have its concentration equal to the measured sodium concentration.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing a recombinant protein using genetically modified Escherichia coli capable of expressing the recombinant protein. In particular, this invention relates to a method for efficiently producing the protein by optimizing the culture medium used for culturing the Escherichia coli. [Background technology]

[0002] The production of recombinant proteins using genetically modified E. coli has been reported in many cases, including in Patent Document 1. E. coli is known to contain various inorganic elements in addition to carbon and nitrogen. Therefore, in order to efficiently express recombinant proteins from genetically modified E. coli, it is necessary to add not only carbon and nitrogen sources, but also inorganic elements such as inorganic salts, to the culture medium used for culturing the E. coli. Furthermore, it is important that the composition of the culture medium is suitable for genetically modified E. coli.

[0003] As an example of optimizing the addition of inorganic elements to the culture medium, Patent Document 2 describes using E. coli strain W3110 (ATCC 27325), which is capable of expressing recombinant proteins, with 2.8 × 10⁶ magnesium ions. -2 The document discloses an example of achieving high-volume expression of the recombinant protein by culturing it in a medium containing a concentration of mol / L (final concentration per unit of medium) or higher. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2000-501936 [Patent Document 2] Japanese Patent Publication No. 2013-085531 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a method for efficiently producing a recombinant protein using genetically modified Escherichia coli capable of expressing the recombinant protein, by optimizing the culture medium used for culturing the Escherichia coli. [Means for solving the problem]

[0006] As a result of diligent research to solve the aforementioned problems, the inventors of the present invention have found that recombinant proteins can be efficiently produced by determining the concentration of a specific inorganic element added to the culture medium based on the concentration of that specific inorganic element contained in Escherichia coli capable of expressing recombinant proteins, and have completed the present invention.

[0007] In other words, the first aspect of the present invention is A method for producing recombinant protein, comprising the steps of culturing genetically modified Escherichia coli capable of expressing recombinant protein, and recovering the recombinant protein expressed by the Escherichia coli contained in the obtained culture, The manufacturing method is characterized in that the culture step is carried out in a manner that includes at least the steps of measuring the sodium concentration contained in the E. coli and culturing the E. coli in a culture medium to which sodium has been added to achieve a concentration equivalent to the measured sodium concentration.

[0008] A second aspect of the present invention is the manufacturing method described in the first aspect, wherein the culture step further includes a step of adding an inducer when the concentration of genetically modified Escherichia coli cells reaches a certain concentration.

[0009] Furthermore, a third aspect of the present invention is the manufacturing method described in the second aspect, wherein the constant concentration of isopropyl-β-thiogalactopyranoside is 70 or more and 120 or less in absorbance at 600 nm, and the final concentration after the addition of the inducer is 0.04 mmol / L or more and 4 mmol / L or less.

[0010] Furthermore, a fourth aspect of the present invention is: The manufacturing method according to any one of the first to third embodiments, wherein the process of culturing genetically modified Escherichia coli is carried out by fed-batch culture, in which a fed-batch solution containing a carbon source and a nitrogen source is added during the culture process, and the carbon source and nitrogen source contained in the culture medium at the start of the culture and in the fed-batch solution are, respectively, in the manner described below; [Initial culture medium] Carbon source: glucose of 20 g / L or less, Nitrogen source: yeast extract of 80 g / L or less [Fluid-added solution] Carbon source: glucose between 300 g / L and 900 g / L, Nitrogen source: yeast extract between 100 g / L and 500 g / L.

[0011] Furthermore, a fifth aspect of the present invention is a manufacturing method according to any of the first to fourth aspects, wherein the recombinant protein is an adeno-associated virus (AAV) binding protein.

[0012] Furthermore, a sixth aspect of the present invention is the production method according to the fifth aspect, 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 312th serine to the 500th aspartic acid of the amino acid sequence described in SEQ ID NO: 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 312th serine to the 500th aspartic acid 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 amino acid residues from the 312th to the 500th, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein it has 70% or more homology to the amino acid sequence consisting of the amino acid residues from the 312th to the 500th, and has AAV binding activity. [Effects of the Invention]

[0013] The present invention relates to a method for producing recombinant protein, comprising the steps of culturing genetically modified Escherichia coli capable of expressing recombinant protein and recovering the recombinant protein expressed by the Escherichia coli contained in the obtained culture, characterized in that the culturing step is carried out in a manner that includes at least the steps of measuring the sodium concentration contained in the Escherichia coli and culturing the Escherichia coli in a medium to which sodium has been added to a concentration equivalent to the measured sodium concentration. The production method of the present invention significantly increases the amount of recombinant protein produced per culture medium compared to other methods. Therefore, the present invention can be said to be useful for the industrial production of recombinant protein. [Modes for carrying out the invention]

[0014] The present invention will be described in detail below. The present invention's production method involves culturing genetically modified Escherichia coli capable of expressing recombinant proteins. To obtain the recombinant Escherichia coli, for example, a gene encoding a recombinant protein (hereinafter also simply referred to as the "recombinant protein gene") can be inserted into a suitable expression vector, and the Escherichia coli can be transformed using the inserted vector.

[0015] Insertion of recombinant protein genes into expression vectors can be achieved by genetic engineering at an appropriate location within the vector. The appropriate location can be freely determined as long as it does not disrupt the replication function, desired antibiotic markers, or regions involved in transduction of the expression vector. When inserting into an expression vector, an oligonucleotide with promoter function may also be added. Examples of promoters that function in E. coli include the lac promoter, trc promoter, or T7 promoter. The expression vector to be inserted is not particularly limited as long as it is stable and replicable within the transformed E. coli. Specific examples of expression vectors include the pTrc, pUC, pBR, pET, and broad-host-range plasmid vectors, which are known to be stable and replicable within E. coli.

[0016] Preferred examples of Escherichia coli used for transformation include Escherichia coli JM109 strain (ATCC 53323), Escherichia coli MV1184 strain (ATCC 47108), Escherichia coli GM31 strain (NBRP ME7741), Escherichia coli HB101 strain (ATCC 33694), Escherichia coli JM101 strain (NBRP ME9043), Escherichia coli W3110 strain (ATCC 27325), and Escherichia coli BL21(DE3) strain (NBRC 108896). Among them, Escherichia coli W3110 strain is more preferred. In addition, for the above-mentioned Escherichia coli, Escherichia coli mutant strains obtained by mutation treatment using conventionally known means such as chemical substances such as nitrosoguanidine and ethyl methanesulfonate, ultraviolet rays, and radiation may also be used.

[0017] Transformation of Escherichia coli using the above expression vector may be carried out by a known method (for example, the methods described in Method in Enzymology, 216, 469-631, 1992, Academic Press and Method in Enzymology, 204, 305-636, 1991, Academic Press).

[0018] The present invention is characterized in that a transformant (genetically engineered Escherichia coli) capable of expressing a recombinant protein obtained by the above-mentioned method is cultured by a method including at least the steps shown in <1> and <2> below; <1> A step of measuring the sodium concentration contained in the transformant <2> A step of culturing the transformant in a medium supplemented with sodium so as to have a concentration equivalent to the measured sodium concentration.

[0019] The aforementioned <1> In the process, the sodium concentration contained in the transformant (genetically modified E. coli) can be measured using known inorganic element analysis methods, such as ICP emission spectrometry, ICP mass spectrometry, atomic absorption spectrometry, X-ray fluorescence analysis, ion chromatography, capillary electrophoresis, titration, colorimetric analysis, electrode method, and precipitation gravimetric analysis. There are no particular limitations on the method for preparing the transformant for sodium concentration measurement; it is sufficient to use a culture medium capable of growing E. coli, such as LB (Luria-Bertani) medium or 2YT medium, and culture it appropriately under conditions such as temperature and aeration stirring that allow E. coli to grow, and then perform pretreatment such as cell recovery / washing, drying, and extraction using a method suitable for the sodium concentration analysis method to be performed. It is preferable to prepare the transformant using an efficient culture method that has high productivity in producing the recombinant protein to be expressed, as this allows for more efficient production of recombinant protein in the present invention.

[0020] The aforementioned <2> In the process described above, <1> Recombinant proteins are expressed by culturing the E. coli in a medium to which sodium has been added to achieve a concentration equivalent to that of the genetically modified E. coli measured in the previous step. <1> As a measurement value in the process, for example, the above <2> The sodium concentration measurement value in the genetically modified Escherichia coli obtained in a culture (pre-culture) performed immediately before the main culture step may be used, or the sodium concentration measurement value in the Escherichia coli cultured at a different time than the pre-culture and the main culture may be reused.

[0021] The aforementioned <2> The process is preferable in that it can be judged quantitatively, for example, by using the degree of surplus (denoted as "S") defined by the following formula (1) as an indicator (Japanese Patent Publication No. 2007-143492).

[0022] Degree of excess S = (M m -M x ) / M x (1) In formula (1), The excess degree S is the excess degree of sodium in the culture medium. M mrepresents the sodium content in the medium (unit: mg / L (medium)), M x represents the sodium content per medium in the Escherichia coli when the density of recombinant Escherichia coli in the medium is X (unit: g / L (medium)), respectively. Note that M x is the sodium content M c (unit: mg / g (cells)), and it may be calculated from M c by X. The density X of recombinant Escherichia coli may be set to any value, or it may be set based on the amount of the Escherichia coli actually cultured and recovered. Considering the uptake efficiency of inorganic elements into the Escherichia coli and the utilization efficiency within the Escherichia coli, it is preferable to set the density X of recombinant Escherichia coli within the range of 70% to 300% of the wet cell mass recovered.

[0023] In the present invention, the lower the absolute value of the surplus represented by formula (1), the more preferable it is in that the deviation of the sodium concentration between the Escherichia coli and the medium is small. However, for example, when adding components containing a plurality of inorganic elements such as peptones and extracts described later to the medium, it is not necessary to strictly match the sodium concentration contained in the Escherichia coli and the sodium concentration added to the medium, and an equivalent concentration may be used. As the "equivalent concentration" in this specification, the absolute value of the surplus S i is less than 1.0, preferably less than 0.5, and more preferably less than 0.2, can be exemplified.

[0024] In the present invention, the culture medium used for culturing genetically modified E. coli is acceptable as long as the sodium concentration is equivalent to that of the genetically modified E. coli, and the E. coli can grow and recombinant proteins can be expressed. Examples of carbon sources include glucose, fructose, maltose, sucrose, raw sugar, and molasses, with glucose being preferred. Yeast extract (e.g., CAS number: 8013-01-2) is preferred as the nitrogen source, but polypeptone, casein and its metabolites, corn steep liquor, soy protein, meat extract, fish extract, etc. may also be used as nitrogen sources. Examples of inorganic salts include phosphates such as diammonium hydrogen phosphate, ammonium dihydrogen phosphate, 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, iron ammonium 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.

[0025] In the present invention, there are no particular limitations on the method of culturing genetically modified Escherichia coli. It may be cultured by batch culture, semi-batch culture (also called fed-batch culture), or perfusion culture, or a combination thereof. However, if nutrients such as carbon sources and nitrogen sources are added to the culture medium all at once at the start of cultivation, the growth of Escherichia coli and the expression of recombinant proteins by the Escherichia coli may be inhibited, and by-products such as organic acids may also be produced, which may adversely affect the efficiency of protein expression and the quality of the obtained protein. For this reason, it is preferable to culture genetically modified Escherichia coli using fed-batch culture, in which the amount of nutrients added at the start of cultivation is kept to a minimum, and nutrients are supplied as appropriate (fed-batch) during cultivation.

[0026] In the present invention, when culturing genetically modified 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 if the carbon source is glucose, and 80 g / L or less if the nitrogen source is yeast extract. It is preferable to use high-concentration solutions for the carbon source and nitrogen source added in the fed-batch culture because this suppresses the increase in the volume of the culture medium. Specifically, it is preferable to use 300 g / L to 900 g / L if the carbon source is glucose, and 100 g / L to 500 g / L if the nitrogen source is yeast extract. Furthermore, the aforementioned inorganic salts may be added under conditions where the sodium concentration in the Escherichia coli and the sodium concentration in the culture medium are equivalent.

[0027] When culturing genetically modified Escherichia coli using the fed-batch culture method, the carbon source and nitrogen source must be added while maintaining predetermined low concentrations of the carbon source and nitrogen source in the culture medium. In this specification, "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 the carbon source, culturing is performed with a carbon source concentration exceeding 5 g / L, which may suppress the growth of E. coli and the expression of recombinant proteins due to the accumulation of organic acids, which are by-products. Therefore, the 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. There are no particular limitations on the method for monitoring carbon source depletion; for example, it can be monitored by a decrease in respiratory activity. A decrease in respiratory activity may manifest as, for example, an increase in dissolved oxygen (DO) concentration 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 favorable indicator for monitoring carbon source depletion because its response is rapid, as microbial respiratory activity decreases and then rapidly increases when carbon sources are depleted.

[0028] In this invention, the culture conditions for genetically modified Escherichia coli are not particularly limited as long as the E. coli can grow and express AAV-binding protein. However, the culture temperature is preferably between 15°C and 50°C, and particularly preferably between 20°C and 33°C. The pH is preferably between 6 and 8. The culture time can be set arbitrarily, but is usually set between several hours and 100 hours.

[0029] When genetically modified Escherichia coli contains an expression vector into which an inducible promoter and a polynucleotide encoding a recombinant protein (recombinant protein gene) are inserted, it is preferable to further include in the culture step of the present invention a step of adding an inducer when the cell concentration of the Escherichia coli reaches a certain concentration, because this improves the amount of recombinant protein expressed per Escherichia coli. In the present invention, the inducer refers to a gene expression inducer that induces the expression of a gene inserted into the expression vector. An example of such an inducer is isopropyl-β-thiogalactopyranoside (IPTG). In particular, when the cell concentration at which the inducer is added is such that the absorbance at 600 nm (OD) is high. 600 ) is more preferable if the inhibitor is isopropyl-β-thiogalactopyranoside (IPTG) with a concentration of 70 to 120 and / or a final concentration of 0.04 mmol / L to 4 mmol / L (preferably 0.08 mmol / L to 0.4 mmol / L), as this particularly improves the recombinant protein expression level per E. coli.

[0030] To recover the recombinant protein expressed by the method described above, the protein can be isolated and purified from the culture using a method suitable for the protein's expression mode in genetically modified E. coli. For example, if the protein is expressed in the culture supernatant, the bacterial cells can be separated by centrifugation, and the recombinant protein can be purified from the resulting culture supernatant. Alternatively, if the protein is expressed intracellularly (including in the periplasm), the bacterial cells can be collected by centrifugation, then the cells can be disrupted by adding an enzyme treatment agent or surfactant, and the recombinant protein can be extracted and purified.

[0031] To purify the recovered recombinant protein, methods known in the art can be used, one example being separation / purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc. By combining these chromatography methods, the protein can be prepared to a high degree of purity.

[0032] The recombinant proteins that can be produced by the method of the present invention are not particularly limited, and as an example, insulin Phosphorus, interferon, interleukin, antibodies, erythropoietin, growth hormone, and the proteins of those receptors, as well as receptors for bacteria and viruses, and DNA polymers Enzymes such as lases, RNA polymerase, reverse transcriptase, and ribonuclease (RNase) Examples include enzymes used in the field of genetic engineering. Hereafter, recombinant enzymes produced by the method of the present invention will be described. A preferred example of a protein is adeno-associated virus (AAV)-binding protein, which will be described in detail.

[0033] AAV-binding proteins are not particularly limited as long as they are polypeptides capable of binding to AAV. Examples include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVRs).

[0034] A preferred embodiment when the AAV-binding protein is AAVR is a polypeptide shown in any of the following (i) to (iii). (i) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in SEQ ID NO: 1, (ii) A polypeptide having an amino acid sequence that includes at least the amino acid residues from the 312th serine to the 500th aspartic acid 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 amino acid residues from the 312th to the 500th, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein it has 70% or more homology to the amino acid sequence consisting of the amino acid residues from the 312th to the 500th, and has AAV binding activity.

[0035] The amino acid sequence described in Sequence ID No. 1 is the amino acid sequence of KIAA0319L (official database: UniProt, accession number: Q8IZA0), which is one form of AAVR. The amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) in the amino acid sequence described in Sequence ID No. 1 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

[0036] The polypeptide described in any of (i) to (iii) above only needs to include at least the regions corresponding to PKD1 and PKD2 of KIAA0319L described above, for example, It may include all or part of the regions corresponding to other extracellular domains on the C-terminal side of PKD2 (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)), or all or part of the regions corresponding to signal sequences such as the MANSC (Motif At N terminus with Seven Cysteines) domain and cysteine-rich regions on the N-terminal side of PKD1, or all or part of the transmembrane and intracellular regions on the N-terminal and / or C-terminal sides of the extracellular region.

[0037] Examples of the above (ii) include polypeptides containing at least the amino acid sequence described in Sequence ID No. 2, and AAV-binding proteins disclosed in WO2021 / 106882. Examples of substitutions, deletions, insertions, or additions described in (ii) include amino acid residue substitutions disclosed in WO2021 / 106882.

[0038] In (ii) above, "one or several" means one or more amino acid substitutions, one or more amino acid residues, depending on the position and type of amino acid substitution in the three-dimensional structure of AAVR. For example, it means one or more of the following: one or more and 50, one or more and 30, one or more and 20, one or more and 10, one or more and 9, one or more and 8, one or more and 7, one or more and 6, one or more and 5, one or more and 4, one or more and 3, one or more and 2, or one. The substitution of "one or several" amino acid residues may occur at positions other than those disclosed in WO2021 / 106882, for example, as long as it has AAV binding activity.

[0039] 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. It is generally known to those skilled in the art that conservative substitutions maintain the function of the protein between the substituted and unsubstituted portions. Examples of conservative substitutions include substitutions between glycine and alanine, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). In addition, 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 AAVR or differences in species.

[0040] The homology of amino acid sequences in (iii) above is sufficient if it is 70% or more, but it may have a higher homology (for example, 80% or more, 85% or more, 90% or more, or 95% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homologous amino acid sequence" means homology to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, 31(3), Yodosha). The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0041] Recombinant proteins produced by the method of the present invention may have oligopeptides added to their N-terminus or C-terminus that are useful for accelerating analysis and purification from solutions containing contaminants, and for stabilizing the protein. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and C-myc tags.

[0042] Furthermore, a signal peptide may be added to the N-terminus of the recombinant protein produced by the method of the present invention to promote efficient expression in the host. Examples of signal peptides that stimulate protein secretion into the periplasm include PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Publication No. 2011-097898). [Examples]

[0043] The present invention will be described in more detail below using examples and comparative examples in which adeno-associated virus (AAV) binding proteins are recombinant proteins, but the present invention is not limited to these examples.

[0044] Comparative Example 1: Production of AAV-binding protein from genetically modified E. coli (Part 1, standard medium without control of inorganic elements) (1) Genetically modified E. coli strain W3110 was transformed with an expression vector containing a polynucleotide encoding the AAV-binding protein AVR10s consisting of the amino acid sequence described in SEQ ID NO: 2 (SEQ ID NO: 3) and an inducible promoter, and the resulting recombinant E. coli capable of expressing the AAV-binding protein was inoculated into 2×YT medium (tryptone: 16 g / L, yeast extract (CAS number: 8013-01-2): 10 g / L, NaCl: 5 g / L, kanamycin sulfate: 50 mg / L) and pre-cultured at 30°C for 16 hours. AVR10s (SEQ ID NO: 2) is a polypeptide in which the amino acid substitutions shown in (i) to (x) below have occurred in the amino acid residues from the 312th serine (Ser) to the 500th aspartic acid (Asp) of KIAA0319L (UniProt accession number: Q8IZA0, SEQ ID NO: 1), which correspond to the extracellular domains 1 and 2 (PKD1 and PKD2) of KIAA0319L (hereinafter also referred to as the wild-type AAV-binding protein). Compared to the wild-type AAV-binding protein, it is an AAV-binding protein with improved stability to alkali (WO2021 / 106882). (i) The valine at position 317 in SEQ ID NO: 1 (position 6 in SEQ ID NO: 2) is replaced with aspartic acid. (ii) The tyrosine at position 342 in SEQ ID NO: 1 (position 31 in SEQ ID NO: 2) is replaced with serine. (iii) Lysine at position 362 in SEQ ID NO: 1 (position 51 in SEQ ID NO: 2) is replaced with glutamic acid. (iv) Lysine at position 371 in SEQ ID NO: 1 (position 60 in SEQ ID NO: 2) is replaced with asparagine. (v) The valine at position 381 in sequence number 1 (position 70 in sequence number 2) is replaced with alanine. (vi) Isoleucine at position 382 in sequence number 1 (position 71 in sequence number 2) is replaced with valine. (vii) The glycine at position 390 in sequence number 1 (position 79 in sequence number 2) is replaced with serine. (viii) Lysine at position 399 in SEQ ID NO: 1 (position 88 in SEQ ID NO: 2) is replaced with glutamic acid. (ix) The 476th serine in SEQ ID NO: 1 (165th in SEQ ID NO: 2) is replaced with arginine. (x) The asparagine at position 487 in SEQ ID NO: 1 (position 176 in SEQ ID NO: 2) is replaced with aspartic acid. (2) After preparing 1.2 L of standard culture medium with the composition shown in Table 1, 36 mL of the pre-culture solution from (1) was added to perform the main culture. An Able BMS-03PI culture apparatus was used, with a stirring 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. The pH fluctuation during culture was controlled within the above range by adding 14% (w / v) aqueous ammonia or 50% (w / v) phosphoric acid.

[0045] [Table 1] (3) When the DO (dissolved oxygen concentration) measured by the DO electrode attached to the BMS-03PI exceeded 40% saturation, the fed-batch pump was activated, and the operation of supplying fed-batch medium with the composition shown in Table 2 was continued until the end of the culture period until the DO returned to below 40% saturation.

[0046] [Table 2] (4) Between 19 and 21 hours after the start of culture, the culture temperature was changed to 25°C and the stirring speed to 600 rpm, and isopropyl-β-thiogalactopyranoside (IPTG) was added to induce the expression of AAV-binding proteins. Between 19 and 21 hours after the start of culture, the absorbance of the culture medium at 600 nm (OD600) was in the range of 70 to 120, and the final concentration of added IPTG was 0.10 mmol / L. (5) 48 hours after the start of cultivation, the cultivation was terminated and the cultured bacterial cells were recovered by centrifugation of the culture medium. (6) The obtained wet bacterial cells were extracted using a commercially available extraction reagent (BugBuster, Merck) according to the standard protocol attached to the reagent, and the supernatant (cell-free extract) was obtained by centrifugation. (7) The obtained cell-free extracts were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) alongside AAV-binding protein standards of known concentration. (8) Using image analysis software (ImageQuant TL 10.0, Cytiva), the concentration of the band corresponding to AAV-binding protein was quantified, and by comparing it with the band concentration in the standard sample, the amount of AAV-binding protein contained in the cell-free extract was quantified, and the production amount per culture medium was calculated.

[0047] The results of culture and AAV-binding protein production are shown in Table 3. The amount of AAV-binding protein per culture medium was 3.8 g / L (culture medium).

[0048] [Table 3]

[0049] Comparative example 2 Elemental analysis (1) Approximately 10 g of cultured microbial cells recovered by centrifugation in Comparative Example 1(5) were suspended in 300 mL of pure water, and the cells were recovered again by centrifugation. This process was repeated twice to wash the cells, and then the cells were dried in a dry heat dryer for two days. (2) After the dried bacterial cells were crushed in a mortar, the concentrations of inorganic elements (specifically, calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), nickel (Ni), and zinc (Zn)) in the genetically modified E. coli were measured by analyzing them using an ICP emission spectrometer (instrument name: Vista-PRO [Seiko Instruments]). (3) The yeast extract added to the culture medium was also measured in the same manner using the ICP emission spectrometer described in (2).

[0050] Table 4 shows the measurement results of the inorganic element concentrations contained in the yeast extract. Table 5 shows the inorganic element concentrations in the cultured cells and the culture medium at the end of the culture, as well as the surplus of each inorganic element calculated from these measurement results. The inorganic element concentrations in the culture medium at the end of the culture are calculated from the inorganic element concentrations in the culture medium with the composition shown in Table 1, the inorganic element concentrations in the fed-batch medium with the composition shown in Table 2, and the inorganic element concentrations in the yeast extract shown in Table 4. Inorganic elements with concentrations below 1 mg / L (specifically, cobalt, copper, molybdenum, and nickel) are excluded from the surplus calculation.

[0051] [Table 4]

[0052] [Table 5]

[0053] Table 4 shows that the concentrations of calcium, iron, magnesium, manganese, and zinc in a standard culture medium are equivalent to those in the cultured bacterial cells (absolute value of excess is less than 1.0), indicating that there are no problems with the culture mediums with the compositions described in Tables 1 and 2 for these inorganic elements. On the other hand, potassium and sodium have excess values ​​of 10 or more, indicating that in a standard culture medium with the compositions described in Tables 1 and 2, potassium and sodium are in large excess relative to the concentrations in the cultured bacterial cells.

[0054] Example 1: Production of AAV-binding protein from genetically modified E. coli (Part 1, sodium concentration control) Table 4 shows that the culture media with the compositions described in Tables 1 and 2 contained a large excess of potassium and sodium. Therefore, genetically modified E. coli was cultured in a culture medium with controlled sodium concentration to bring the sodium concentration in the medium to the same level as that in the cultured bacteria. Specifically, the AAV-binding protein was produced in the same manner as in Comparative Example 1(2), except that the culture medium used in the main culture was changed to sodium-controlled medium A as described in Table 5. The inorganic element concentrations in the culture medium at the end of the culture were calculated from the inorganic element concentrations in sodium-controlled medium A with the composition shown in Table 5, the inorganic element concentrations in the fed-batch medium with the composition shown in Table 2, and the inorganic element concentrations in the yeast extract shown in Table 4.

[0055] [Table 6]

[0056] Comparative Example 3: Production of AAV-binding protein from genetically modified E. coli (Part 1: Potassium concentration control) Table 4 shows that the culture media with the compositions described in Tables 1 and 2 contained a large excess of potassium and sodium. Therefore, genetically modified E. coli was cultured in a culture medium with controlled potassium concentration to bring the potassium concentration in the medium to the same level as that in the cultured bacteria. Specifically, the culture medium used in the main culture of Comparative Example 1(2) was changed to the potassium-controlled medium described in Table 5, and the culture medium used in the fed-batch medium of Comparative Example 1(3) was changed to the potassium-controlled medium described in Table 7. Otherwise, AAV-binding protein was produced in the same manner as in Comparative Example 1. Furthermore, the inorganic element concentrations in the culture medium at the end of the culture were calculated from the inorganic element concentrations in the potassium-controlled medium with the composition shown in Table 6, the inorganic element concentrations in the fed-batch medium (potassium-controlled medium) with the composition shown in Table 7, and the inorganic element concentrations in the yeast extract shown in Table 4.

[0057] [Table 7]

[0058] Table 8 shows the inorganic element concentrations in the culture medium at the end of cultivation in Example 1 and Comparative Example 3 (for comparison, the inorganic element concentrations in the cultured cells shown in Table 4 are also included). Table 9 shows the surplus of each inorganic element calculated from the inorganic element concentrations in the culture medium and cultured cells at the end of cultivation (inorganic elements with concentrations of less than 1 mg / L (cobalt, copper, molybdenum, and nickel) are excluded from the surplus calculation).

[0059] [Table 8]

[0060] [Table 9]

[0061] Table 9 shows that the sodium surplus in the sodium-controlled medium (Example 1) was -0.01, indicating that the sodium concentration in the medium was equivalent to the sodium concentration in the cultured cells. This confirms that the AAV-binding protein production in Example 1 was achieved through a sodium-controlled culture. Similarly, the potassium surplus in the potassium-controlled medium (Comparative Example 3) was 1.03, indicating that the potassium concentration in the medium was almost equivalent to the potassium concentration in the cultured cells. This confirms that the AAV-binding protein production in Comparative Example 3 was also achieved through a potassium-controlled culture.

[0062] Table 10 shows the results of culture and AAV-binding protein production in Example 1 and Comparative Example 3. When genetically modified E. coli was cultured in a sodium-controlled medium (Example 1), the amount of AAV-binding protein produced per unit of culture medium was improved compared to when it was cultured in a medium without control of sodium and potassium (Comparative Example 1, Table 3) (Example 1: 8.8 g / L (culture medium), Comparative Example 1: 3.8 g / L (culture medium)). From these results, it can be seen that by setting the sodium concentration in the culture medium used for culturing genetically modified E. coli for the purpose of producing recombinant proteins to a concentration equivalent to the sodium concentration contained in the E. coli, the protein can be produced efficiently.

[0063] On the other hand, when genetically modified E. coli was cultured in a potassium-controlled medium (Comparative Example 3), the bacterial density after culture decreased significantly (Comparative Example 3: 146 g / L (culture medium), Comparative Example 1: 302 g / L (culture medium)) compared to when it was cultured in a medium where sodium and potassium were not controlled (Comparative Example 1, Table 3), and the amount of AAV-binding protein produced per unit of culture medium also decreased (Comparative Example 3: 2.8 g / L (culture medium), Comparative Example 1: 3.8 g / L (culture medium)). From these results, it can be seen that even if the potassium concentration in the culture medium used for culturing genetically modified E. coli for the purpose of producing recombinant protein is the same as the potassium concentration contained in the E. coli, it does not lead to the efficient production of the protein.

[0064] [Table 10]

[0065] Comparative Example 4: Production of AAV-binding protein from genetically modified E. coli (Part 2, standard medium without control of inorganic elements) AAV-binding proteins were produced in the same manner as in Comparative Example 1, except that the polynucleotide encoding the AAV-binding protein to be inserted into the expression vector was changed to a polynucleotide encoding the AAV-binding protein AVR11a (SEQ ID NO: 5), which has the amino acid sequence described in SEQ ID NO: 4. AVR11a (SEQ ID NO: 4) is a polypeptide in which the amino acid substitution shown in (xi) below has been further induced in the amino acid residues of AVR10s (SEQ ID NO: 2), and is an AAV-binding protein with improved alkali stability compared to the wild-type AAV-binding protein and AVR10s. (xi) The 330th alanine in SEQ ID NO: 1 (19th in SEQ ID NO: 2) is replaced with valine.

[0066] Example 2: Production of AAV-binding protein from genetically modified E. coli (Part 2, sodium concentration control) The AAV-binding protein was produced in the same manner as in Comparative Example 1, except that the polynucleotide encoding the AAV-binding protein to be inserted into the expression vector was changed to the polynucleotide encoding the aforementioned AAV-binding protein AVR11a (SEQ ID NO: 4) (SEQ ID NO: 5), and the culture medium used in the main culture of Comparative Example 1(2) was changed to sodium-controlled medium B with the composition described in Table 5.

[0067] Table 11 shows the inorganic element concentrations in the culture medium at the end of cultivation in Comparative Example 2 and Example 2, calculated from the inorganic element concentrations in the standard culture medium (Comparative Example 4) with the composition shown in Table 1 or the sodium-controlled culture medium B (Example 2) with the composition shown in Table 5, the inorganic element concentrations in the fed-batch medium with the composition shown in Table 2, and the inorganic element concentrations in the yeast extract shown in Table 4. (For comparison, the inorganic element concentrations in the cultured cells shown in Table 4 are also shown again.) Furthermore, Table 12 shows the surplus of each inorganic element calculated from the inorganic element concentrations in the culture medium and cultured cells at the end of cultivation (inorganic elements with concentrations of less than 1 mg / L (cobalt, copper, molybdenum, and nickel) are excluded from the surplus calculation).

[0068] [Table 11]

[0069] [Table 12]

[0070] Table 12 shows that the sodium surplus in sodium-controlled medium B (Example 2) was -0.82, and since the absolute value of the surplus is less than 1, it can be said that the sodium concentration in the medium is equivalent to the sodium concentration in the cultured bacteria. This confirms that the AAV-binding protein production in Example 2 was achieved through sodium-controlled culture. Table 13 shows the results of culture and AAV-binding protein production in Comparative Example 4 and Example 2. When genetically modified E. coli was cultured in sodium-controlled medium B (Example 2), the amount of AAV-binding protein produced per unit of culture medium was improved compared to when cultured in a medium where sodium and potassium were not controlled (Comparative Example 4) (Example 2: 6.9 g / L (culture medium), Comparative Example 4: 3.2 g / L (culture medium)). From these results, it can be seen that even if the recombinant protein to be produced is changed from AVR10s (SEQ ID NO: 2) to AVR11a (SEQ ID NO: 4), the protein can be efficiently produced by making the sodium concentration in the medium used for culturing genetically modified E. coli capable of expressing the protein equivalent to the sodium concentration in the E. coli.

[0071] [Table 13]

Claims

1. A method for producing recombinant protein, comprising the steps of culturing genetically modified Escherichia coli capable of expressing recombinant protein, and recovering the recombinant protein expressed by the Escherichia coli contained in the obtained culture, The culture step includes at least the steps of: performing a first batch culture of the genetically modified Escherichia coli using the culture medium and a fed-batch medium; measuring the sodium concentration contained in the Escherichia coli at the end of the first batch culture; and performing a second batch culture of a genetically modified Escherichia coli separate from the one used in the first batch culture using a sodium-controlled medium and a fed-batch medium. At the start of the first batch culture, the culture medium contained 18 g / L of disodium hydrogen phosphate dodecahydrate, 6 g / L of trisodium phosphate dodecahydrate, 40 g / L of yeast extract, 1 g / L of ammonium chloride, 10 g / L of D(+)-glucose, 1 g / L of magnesium sulfate heptahydrate, 10 mg / L of iron(II) sulfate heptahydrate, and 5 mg / L of manganese chloride tetrahydrate. The fed-batch culture medium in the first and second fed-batch cultures contains 425 g / L of D(+)-glucose, 142 g / L of yeast extract, and 12 g / L of magnesium sulfate heptahydrate. At the end of the first culture, 693 g of fed-batch medium was added to 1.2 L of the culture medium. The sodium-controlled medium at the start of the second batch culture contains 6.6 to 13.2 g / L of diammonium hydrogen phosphate, 40 g / L of yeast extract, 0 to 0.5 g / L of sodium chloride, 10 g / L of D(+)-glucose, 1 g / L of magnesium sulfate heptahydrate, 10 mg / L of iron(II) sulfate heptahydrate, and 5 mg / L of manganese chloride tetrahydrate. At the end of the second batch culture, 669g to 820g of the batch medium was added to 1.2L of the sodium-controlled medium. The absolute value of the sodium surplus S in the culture medium at the end of the second batch culture is less than 1.

0. Here, the degree of surplus S is defined as follows: Surplus degree S=(M m - M x ) / M x M m: Sodium content in the culture medium at the end of the second batch culture (unit: mg / L (culture medium)) M x: The amount of sodium present in the culture medium per unit (in mg / L (culture medium)) when the genetically modified E. coli density in the culture medium measured at the end of the first-line culture is X (in g / L (culture medium)), method.

2. The method according to claim 1, further comprising the step of adding isopropyl-β-thiogalactopyranoside at a final concentration of 0.04 mmol / L to 4 mmol / L when the cell concentration of genetically modified Escherichia coli reaches an absorbance of 70 to 120 at 600 nm.

3. The method for producing a product according to claim 1 or 2, wherein the recombinant protein is an adeno-associated virus (AAV) binding protein.

4. The method for producing the product according to claim 3, wherein the AAV-binding protein is a polypeptide selected from any of (i) to (iv) below; (i) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid 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 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, wherein the amino acid residues from the 312th to the 500th include substitutions, deletions, insertions, or additions of 1 to 10 amino acid residues, and which has AAV binding activity. (iii) A polypeptide comprising at least the amino acid residues from the 312th serine to the 500th aspartic acid of the amino acid sequence described in Sequence ID No. 1, provided that it has 90% or more identity with the amino acid sequence consisting of the 312th to the 500th amino acid residues, and has AAV binding activity. (iv) A polypeptide comprising at least the amino acid sequence described in SEQ ID NO: 2.

Images