Method for extracting target proteins from prokaryotic cells

Abstract

The present invention addresses the problem of, in a method for extracting a target protein from prokaryotic cells expressing the target protein, preventing the contamination of the target protein with foreign substances and increasing the recovery rate of the target protein.　The method for extracting a target protein from prokaryotic cells that is disclosed in the present description is characterized by comprising: a step for mixing a culture solution containing prokaryotic cells that express the target protein with a carboxylic acid at a final concentration of 0.5-15% (v / v) inclusive to prepare a mixed liquid; and a step for extracting the target protein from the prokaryotic cells in the mixed liquid.

Description

Technical Field

[0001] This specification discloses a method for extracting a target protein from prokaryotic cells expressing the target protein.

Background Art

[0002] When expressing a protein using Escherichia coli as a host cell by genetic recombination technology, since the cytoplasm of Escherichia coli is a reducing environment, disulfide bonds involved in protein folding are difficult to form, and there may occur problems such as the formation of inclusion bodies and insolubilization, or the protein being obtained as an inactive protein. As one of the solutions, a method of secreting the expressed protein from the cytoplasm to the outside of the inner membrane and accumulating it in the periplasm is known. The periplasm is the space between the inner membrane and the outer membrane of Escherichia coli cells, and since it is a chemically oxidative environment, formation of disulfide bonds and thereby functionally accurate folding of the protein easily occur (Non-Patent Document 1).

[0003] When a protein expressed in the cytoplasm of an Escherichia coli host cell has a signal sequence that promotes transport from the cytoplasm to the periplasm, the protein can be secreted into the periplasm.

[0004] On the other hand, as documents that disclose a method for extracting a heterologous protein from recombinant microbial cells expressing the heterologous protein, the following Patent Documents 1 to 4 can be cited.

[0005] Patent Document 1 describes a method for extracting a soluble protein from a microbial population, which includes contacting a microbial population expressing the soluble protein with a solution containing 25% (v / v) to 40% (v / v) carboxylic acid in an amount effective for extracting the soluble protein from the microbial population. Patent Document 1 describes contacting the solution with wet cells obtained by recovering the cultured cells.

[0006] Patent Document 2 describes a method for isolating periplasmic polypeptides from prokaryotic cells, which includes the step of incubating prokaryotic cells in a solution with a pH of about 7 to about 10 containing predetermined concentrations of Tris-HCl and EDTA at about 15 minutes to about 6 hours at about 25°C.

[0007] Patent Document 3 describes a method for extracting periplasmic proteins, which includes suspending a cell pellet obtained from a culture of a prokaryotic microorganism expressing periplasmic proteins in an arginine-containing buffer and recovering the proteins in the supernatant.

[0008] Patent Document 4 describes a method for recovering periplasmic proteins from Gram-negative bacteria by osmotic shock, characterized by adding a polymer flocculant to the suspension of the Gram-negative bacteria. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Patent No. 6694381 [Patent Document 2] Special Publication No. 2015-528477 [Patent Document 3] Japanese Patent Application Publication No. 08-242879 [Patent Document 4] Japanese Patent Application Publication No. 07-184680 [Non-patent literature]

[0010] [Non-Patent Document 1] JHChoi and SYLee, Appl Microbiol Biotechnol (2004) 64:625-635 [Overview of the project] [Problems that the invention aims to solve]

[0011] Methods for extracting target proteins from the periplasm of prokaryotic cells containing the target protein in the periplasm have been conventionally studied, as described in the above-mentioned Patent Documents 2-4. However, these methods extract a large amount of impurities originating from the cytoplasm of the host prokaryotic cell along with the target protein, making subsequent purification of the target protein difficult.

[0012] Patent Document 1, which discloses a method for extracting soluble proteins from a microbial population, states that soluble proteins are soluble in the cytoplasm or periplasm, but does not describe experimental results of actually extracting soluble proteins from the periplasm. The present inventors, following Patent Document 1, contacted prokaryotic cells containing the target protein in the periplasm with a solution containing 25% (v / v) to 40% (v / v) carboxylic acid (acetic acid). They found that the extracted target protein was contaminated with many impurities and that the recovery rate of the target protein was low.

[0013] Therefore, this specification discloses a method for extracting a target protein from prokaryotic cells expressing the target protein, which enables the suppression of contamination of the target protein with impurities and the improvement of the recovery rate of the target protein. [Means for solving the problem]

[0014] This specification discloses the following methods as means described above.

[0015] (1) A step of preparing a mixture by mixing a carboxylic acid with a final concentration of 0.5% (v / v) or more and 15% (v / v) or less with a culture medium containing prokaryotic cells expressing the target protein, and The process of extracting target proteins from prokaryotic cells in a mixture. A method for extracting a target protein from prokaryotic cells, characterized by including [a specific element]. (2) The method according to (1), wherein the prokaryotic cell can express a nucleic acid containing a nucleotide sequence encoding an amino acid sequence of a fusion polypeptide of a signal peptide that promotes secretion into the periplasm and a target protein. (3) The method according to (1) or (2), wherein the prokaryotic cell is a gram-negative bacterium cell. (4) The method according to any one of (1) to (3), wherein the prokaryotic cell is an Escherichia coli (E. coli) cell. (5) The method according to any one of (1) to (4), wherein the carboxylic acid is one or more selected from formic acid, acetic acid, propionic acid, malonic acid, citric acid and lactic acid. (6) The method according to any one of (1) to (5), wherein the target protein is a minimized antibody. (7) A method for extracting a target protein from a prokaryotic cell, comprising adding a carboxylic acid of 0.5% (v / v) or more and 15% (v / v) or less to a culture solution containing a prokaryotic cell expressing the target protein to extract the target protein. Here, the method of (7) may have any further features described in (2) to (6). This specification incorporates the disclosure of Japanese Patent Application No. 2021-038314, which is the basis of the priority of this application.

Effects of the Invention

[0016] According to the method for extracting a target protein from a prokaryotic cell disclosed in this specification, it is possible to suppress the contamination of impurities into the target protein and increase the recovery rate of the target protein.

Modes for Carrying Out the Invention

[0017] <Prokaryotic cells expressing the target protein> The prokaryotic cell may be any prokaryotic cell that serves as a host for expressing the target protein, preferably a bacterium, particularly preferably a cell of a bacterium having a periplasm such as a gram-negative bacterium.

[0018] The periplasm refers to the space between the inner membrane (cytoplasmic membrane) surrounding the cytoplasm of prokaryotic cells and the outer membrane. Preferred examples of Gram-negative bacterial cells having a periplasm include cells of bacteria belonging to the genus Escherichia, and particularly preferably, Escherichia coli cells.

[0019] As used herein, the target protein refers to a protein to be produced in prokaryotic cells. The prokaryotic cells can expressibly hold a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of the target protein.

[0020] Prokaryotic cells may express the target protein as a fusion polypeptide in which one or both of the N-terminal side and the C-terminal side thereof are further linked to other polypeptides. Examples of other polypeptides include, but are not limited to, signal peptides, tag peptides, etc. Specific examples of signal peptides include signal peptides that promote secretion into the periplasm, which will be described later. Examples of tag peptides include tag peptides (histidine tags) composed of a plurality (for example, 6 to 10) of histidine residues and FLAG tag peptides.

[0021] Preferably, prokaryotic cells can expressibly hold a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of a fusion polypeptide of a signal peptide that promotes secretion into the periplasm and the target protein. In the prokaryotic cells according to this embodiment, the fusion polypeptide expressed in the cytoplasm passes through the inner membrane and is secreted into the periplasm, and can accumulate in the periplasm as the target protein from which the signal peptide has been removed.

[0022] Signal peptides that promote secretion into the periplasm include, for example, the signal peptides described in Non-Patent Document 1, and specific examples include pelB signal peptide (SEQ ID NO: 5), heat-stable enterotoxin II (STII) signal peptide (SEQ ID NO: 6), outer membrane protein A (OmpA) signal peptide (SEQ ID NO: 7), and maltose outer membrane porin (LamB) signal peptide (SEQ ID NO: 8).

[0023] The target protein is not particularly limited and can be selected according to the purpose. The target protein is preferably a heterologous protein. For example, antibodies or low-molecular-weight antibodies, particularly low-molecular-weight antibodies, can be exemplified as preferred target proteins.

[0024] "Antibody" is a general term that focuses on the function of immunoglobulins. All immunoglobulins basically have the same molecular structure, with a "Y"-shaped quadruple chain structure (two light chains and two heavy chain polypeptide chains) as their basic structure.

[0025] The lower half of the "Y" shape of an antibody, corresponding to the vertical bar, is called the Fc region, and the upper half, corresponding to the "V" shape, is called the Fab region. The Fc region has an effector function that triggers a reaction after the antibody binds to an antigen, while the Fab region has the function of binding to the antigen. The Fab region and Fc region of the heavy chain are connected by a hinge, and papain, a proteolytic enzyme found in papaya, breaks down this hinge, cleaving it into two Fab regions (fragments) and one Fc region. The part of the Fab region closest to the tip of the "Y" shape (domain) shows a variety of changes in its amino acid sequence to enable binding to various antigens, and is therefore called the variable region (V region). The variable region of the light chain is called the VL region, and the variable region of the heavy chain is called the VH region. The Fab regions and Fc regions other than the V region are regions that change relatively little and are called the constant region (C region). The steady region of the light chain is called the CL region, and the steady region of the heavy chain is called the CH region, but the CH region is further divided into three parts: CH1 to CH3. The Fab region of the heavy chain consists of the VH region and CH1, and the Fc region of the heavy chain consists of CH2 and CH3. The hinge region is located between CH1 and CH2.

[0026] Among antibodies derived from camelids and fish such as sharks, there are heavy-chain antibodies that consist only of heavy chains without light chains. Heavy-chain antibodies derived from camelids are distinguished from normal IgG antibodies (IgG1) that have light chains and are called IgG2 and IgG3. On the other hand, heavy-chain antibodies derived from fish are called IgNARs (new antigen receptors).

[0027] In this specification, a low-molecular-weight antibody is an antibody fragment in which a portion of a full-length antibody (whole antibody, such as whole IgG) is missing, and is not particularly limited as long as it has the ability to bind to an antigen.

[0028] In this specification, it is preferable that the low-molecular-weight antibody does not have a CH2 domain and a CH3 domain.

[0029] In this specification, it is preferable that the low-molecular-weight antibody includes either or both of the heavy chain variable region (VH) and the light chain variable region (VL). The amino acid sequence of VH or VL may include additions, deletions, and / or substitutions. Furthermore, a portion of either or both of VH and VL may be deleted, as long as they bind to the antigen.

[0030] There are no particular restrictions on the low molecular weight antibodies, and they can be appropriately selected depending on the purpose. Examples include those containing the variable region (VHH) of a heavy chain antibody derived from a camelid animal, the variable region (V-NAR) of a heavy chain antibody derived from a fish, Fab, Fab', F(ab')2, single-chain antibody (scFv), diabody, triabody, and minibody. Among these, those containing the variable region (VHH) of a heavy chain antibody derived from a camelid animal, or a single-chain antibody (scFv), are preferred in terms of stability and production efficiency.

[0031] The aforementioned low-molecular-weight antibody may be one in which two or more antibody fragments are linked. Examples of low-molecular-weight antibodies in which two or more antibody fragments are linked include monovalent or bivalent single-chain Fv (scFv, sc(Fv)2), scFv dimer (Diabody), in which the variable region of the antibody is linked with a linker such as a peptide linker, and tandem VHH in which two or more VHHs are linked with a linker such as a peptide linker.

[0032] Prokaryotic cells may retain nucleic acids containing a base sequence encoding the amino acid sequence of a target protein (or the fusion protein) either in an expression vector or as part of their genomic DNA.

[0033] Plasmid vectors containing the nucleic acids mentioned above can be used as expression vectors. It is preferable that the expression vector is capable of autonomous replication within prokaryotic cells. The expression vector preferably contains DNA comprising a base sequence encoding the amino acid sequence of a target protein (or the fusion protein) and a promoter functionally linked to a position where the DNA can be transcribed. Preferably, the expression vector is recombinant DNA capable of autonomous replication within prokaryotic cells and comprising a base sequence composed of a promoter, a ribosome-binding sequence, a base sequence encoding the amino acid sequence of a target protein (or the fusion protein), and a transcription termination sequence.

[0034] Suitable plasmid vectors include pET-28b (available from Merck), pQEK1, pCA24N (DNA RESEARCH, 12, 191-299 (2005)), pACYC177, pACYC184 (available from Nippon Gene Co., Ltd.), pQE30, pQE60, pQE70, pQE80 and pQE9 (available from QIAGEN); pTipQC1 (available from QIAGEN or Hokkaido System Science Co., Ltd.), pTipRT2 (available from Hokkaido System Science Co., Ltd.); pBS vector, Phagescript vector, Bluescript vector, pNH8A, and pNH16. Examples include A, pNH18A and pNH46A (available from Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540 and pRIT5 (available from Addgene); pRSF (available from MERCK); and pAC (available from Nippon Gene Co., Ltd.), pUCN18 (can be prepared by modifying pUC18 (available from Takara Bio Inc.)), pSTV28 (available from Takara Bio Inc.), pUCNT (International Publication No. 94 / 03613), etc.

[0035] An inducible promoter may be used as the promoter included in the expression vector. Alternatively, an inducible promoter may be created by functionally ligating a promoter with an operator sequence.

[0036] Examples of inducible promoters include isopropyl-β-thiogalactopyranoside (IPTG) inducible promoters, photoinducible promoters that induce gene expression under light irradiation, araBAD promoters (arabinose inducible), rhaBAD promoters (rhamnose inducible), tet promoters (drug inducible), penP promoters (drug inducible), cspA promoters (temperature-inducible promoters that respond to low temperatures), and promoters containing tetO or lacO operators as operator sequences. IPTG inducible promoters, araBAD promoters, rhaBAD promoters, tet promoters, penP promoters, cspA promoters, or promoters containing tetO or lacO operators as operator sequences are preferred.

[0037] When introducing the expression vector containing the nucleic acid into prokaryotic cells, it is preferable that the copy number of the expression vector in the cell is preferably 2 or more, more preferably 3 or more, more preferably 5 or more, more preferably 10 or more, more preferably 15 or more, and more preferably 20 or more.

[0038] The method for introducing expression vectors into prokaryotic cells is not particularly limited, but can be carried out by transformation methods such as the competent cell method, electroporation, calcium chloride method, electrical pulse method, protoplast method, or particle gun method.

[0039] Homologous recombination can be used in a prokaryotic cell that retains nucleic acids containing a base sequence encoding the amino acid sequence of a target protein (or the fusion protein) as part of its genomic DNA.

[0040] Prokaryotic cells expressing target proteins can be cultured in a suitable culture medium. The medium can be either synthetic or natural, as long as it contains nutrients necessary for the proliferation of prokaryotic cells and the expression of target proteins, such as carbon sources, nitrogen sources, inorganic salts, vitamins, and yeast extracts.

[0041] Any carbon source that can be utilized by the prokaryotic cells can be used, and examples include carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid.

[0042] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium chloride and ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone.

[0043] Examples of inorganic salts include trisodium phosphate, monosodium hydrogen phosphate, magnesium sulfate, iron(II) sulfate, manganese(II) chloride, sodium chloride, and potassium carbonate.

[0044] Examples of vitamins include biotin and thiamine. Furthermore, substances required for the growth of the prokaryotic cells (for example, required amino acids in the case of amino acid-requiring strains) can be added as needed.

[0045] In culturing the aforementioned prokaryotic cells, a culture medium containing glucose, yeast extract, trisodium phosphate, monosodium hydrogen phosphate, ammonium chloride, magnesium sulfate, iron(II) sulfate, and manganese(II) chloride is preferably used. The pH of the medium is preferably adjusted to 6-8.

[0046] In culturing the prokaryotic cells, the culture conditions are not particularly limited, but preferably include shaking culture and stirring culture. It is also preferable to culture while aerating with air. The culture temperature is 20 to 50°C, preferably 25 to 40°C, and more preferably 25 to 35°C. The culture time is 3 hours to 5 days, preferably 5 hours to 4 days.

[0047] <Method for extracting target proteins from prokaryotic cells expressing target proteins> The method disclosed herein for extracting a target protein from prokaryotic cells expressing the target protein is: A step of preparing a mixture by mixing a carboxylic acid with a final concentration of 0.5% (v / v) or more and 15% (v / v) or less with a culture medium containing prokaryotic cells expressing a target protein, and The process of extracting target proteins from prokaryotic cells in a mixture. It is characterized by including.

[0048] This method can suppress contamination of the extracted target protein with impurities and increase the recovery rate of the target protein. This method is particularly useful for extracting target proteins present in the periplasm.

[0049] When the carboxylic acid mixture concentration is less than 0.5% (v / v) or greater than 15% (v / v), there is a tendency for the recovery rate of the target protein to decrease or for the amount of impurities mixed into the extracted target protein to increase. The carboxylic acid mixture concentration is more preferably 3% (v / v) or more and 10% (v / v) or less.

[0050] In this specification, the mixed concentration of carboxylic acid in a culture medium containing prokaryotic cells expressing a target protein refers to the ratio of the volume of the mixed carboxylic acid to the total volume of the culture medium after the entire amount of carboxylic acid has been mixed (i.e., the final concentration). Here, the volume of carboxylic acid refers to the volume of the liquid carboxylic acid at room temperature and pressure when using carboxylic acids that are liquid at room temperature and pressure, such as formic acid, acetic acid, propionic acid, and lactic acid, and to the volume of the saturated aqueous solution of the carboxylic acid at 25°C when using carboxylic acids that are solid at room temperature and pressure, such as malonic acid and citric acid.

[0051] In this specification, when multiple carboxylic acids are mixed, the mixed concentration of the carboxylic acids in a culture medium containing prokaryotic cells expressing a target protein refers to the sum of the individual mixed concentrations of the multiple carboxylic acids.

[0052] The mixing concentration of carboxylic acid in the culture medium containing prokaryotic cells expressing the target protein is preferably 0.5% (v / v) or higher, more preferably 1% (v / v) or higher, more preferably 3% (v / v) or higher, and particularly preferably 5% (v / v) or higher. When the mixing concentration of carboxylic acid is within this range, the contamination of the target protein with impurities can be particularly effectively suppressed, and the recovery rate of the target protein can be further increased.

[0053] The mixed concentration of the carboxylic acid is particularly preferably 1%(v / v) to 15%(v / v), more preferably 3%(v / v) to 15%(v / v), more preferably 5%(v / v) to 15%(v / v), and more preferably 5%(v / v) to 10%(v / v).

[0054] The type of carboxylic acid is not particularly limited. The carboxylic acid may also be a hydroxy acid. Preferred examples of carboxylic acids include one or more selected from formic acid, acetic acid, propionic acid, malonic acid, citric acid, and lactic acid. More preferably, one or more selected from formic acid, acetic acid, and propionic acid, and even more preferably, one or more carboxylic acids selected from formic acid and acetic acid.

[0055] "Culture medium containing prokaryotic cells expressing a target protein" refers to a suspension in which prokaryotic cells are suspended in a culture medium, obtained by culturing prokaryotic cells expressing a target protein in a culture medium and expressing the target protein within the prokaryotic cells. Preferably, such a culture medium is one obtained by culturing prokaryotic cells expressing a target protein in the above-mentioned culture medium. In the method according to this disclosure, the target protein expressed in the prokaryotic cells can be extracted into a liquid fraction by mixing a predetermined amount of carboxylic acid with the culture medium before separating the prokaryotic cells from the culture medium after culturing. Preferably, the culture medium is a suspension containing prokaryotic cells at a wet weight of 50 to 200 g / L.

[0056] The target protein is extracted from the prokaryotic cells in a mixture formed by mixing the aforementioned amount of carboxylic acid with a culture medium containing prokaryotic cells expressing the target protein. The extraction time is not particularly limited, but is preferably 0.5 hours or more, more preferably 1 hour or more, preferably 10 hours or less, more preferably 8 hours or less, and more preferably 5 hours or less. The extraction temperature is not particularly limited, but is preferably in the range of 15°C to 30°C, more preferably 20°C to 30°C. During extraction, the mixture may be left to stand, or it may be stirred temporarily or continuously.

[0057] After extraction of the target protein, cells can be removed by appropriate solid-liquid separation methods such as centrifugation and filtration, and the liquid fraction from which the target protein has been eluted can be recovered. The target protein can then be further purified and recovered from the liquid fraction. [Examples]

[0058] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Detailed methods of recombinant DNA technology used in the following examples are described in the following books: Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Green Publishing Associates and Willey-Interscience).

[0059] Furthermore, in the following examples, the plasmids used for transforming E. coli were prepared by introducing the constructed vector into E. coli DH5α competent cells (Takara Bio Inc.), culturing the resulting transformants, and amplifying the resulting cells. Plasmid preparation from plasmid-carrying strains was performed using the QIAprep spin miniprep kit (QIAGEN Inc.).

[0060] We prepared E. coli strains expressing the low-molecular-weight antibodies anti-Fc VHH and Caplacizumab (tandem VHH) as target proteins, respectively.

[0061] The amino acid sequence of anti-Fc VHH is described in SEQ ID NO: 163 of EP2170960B1, and the amino acid sequence of Caplacizumab (tandem VHH) is described in SEQ ID NO: 1 of WO2009 / 115614A2. Based on this publicly available information, synthetic DNA of the gene encoding anti-Fc VHH with the pelB signal peptide (SEQ ID NO: 5) added upstream, and the gene encoding Caplacizumab with the STII signal peptide (SEQ ID NO: 6) added upstream were prepared and used in vector construction. Both the pelB signal peptide and the STII signal peptide are signal peptides that promote the transport of expressed proteins to the periplasm.

[0062] For PCR, Prime STAR Max DNA Polymerase (manufactured by Takara Bio Inc.) was used, and the reaction conditions were carried out according to the method described in the attached manual.

[0063] [Experiment 1: Construction of a target protein expression vector] Nucleic acid fragments, each with a restriction enzyme site of NdeI upstream and Bpu1102I downstream of the respective synthetic gene, were prepared by PCR using the pelB signal peptide-added anti-Fc VHH synthetic DNA as a template and primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), and by PCR using the STII signal peptide-added Caplacizumab synthetic DNA as a template and primers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4). After treatment with NdeI and Bpu1102I, these fragments were inserted into the NdeI and Bpu1102I sites of pET-28b (Merck) to prepare anti-Fc VHH expression vectors and Caplacizumab expression vectors.

[0064] [Experiment 2: Obtaining transformed E. coli] 0.1 μL of each prepared expression vector was mixed with 1 μL of the competent E. coli cell line BL21(DE3) on ice and allowed to stand for 30 minutes. The mixture was then heated at 42°C for 45 seconds and cooled on ice. After cooling, 100 μL of SOC medium (20 g / L bactotryptone (Becton Dickinson & Co. (BD)), 5 g / L bacto yeast extract (BD), 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium sulfate, 10 mM magnesium chloride, 20 mM glucose) was added and the mixture was recovered at 37°C for 1 hour. The samples were spread onto LBK selective agar plates (10 g / L polypeptone (BD), 5 g / L Bact yeast extract (BD), 10 g / L sodium chloride, 50 μg / L kanamycin, 15 g / L agarose), and strains that grew after static incubation at 37°C for 16 hours were selected to obtain anti-Fc VHH-expressing E. coli and Caplacizumab-expressing E. coli.

[0065] [Experiment 3: Culture of transformed E. coli] One of the low-molecular-weight antibody-expressing E. coli obtained in Experiment 2 was inoculated into 50 mL of TB medium (24 g / L Bacto yeast extract (BD), 12 g / L Bactotryptone (BD), 10 g / L glycerol, 9.4 g / L dipotassium hydrogen phosphate, 2.2 g / L potassium dihydrogen phosphate) in a 500 mL Sakaguchi flask, and pre-culture was performed at 30 °C for 16 hours at a shaking rate of 110 times per minute. Approximately 1.7 L of medium, from which glucose, yeast extract, and metal salts were removed from the medium with the composition shown in Table 1, was placed in a 5 L culture tank, sterilized at 121 °C for 20 minutes, and then glucose filtered through a 0.2 μm filter, yeast extract (Oriental Yeast Co., Ltd.), and metal salts as shown in Table 1 were added to prepare the culture tank for cultivation. 5 mL of the pre-culture solution was added to the culture tank, and the main culture was performed. The culture apparatus used was a Bioneer-5L manufactured by Marubishi Bioengin Co., Ltd., with an aeration rate of 2.5 L / min, a stirring speed of 400 rpm, a culture temperature of 30°C, and a pH of 6.9 to 7.1. Furthermore, pH fluctuations during culture were controlled within the aforementioned range by adding 12.5% ​​aqueous ammonia or 4N sulfuric acid.

[0066] [Table 1]

[0067] A dissolved oxygen (DO) electrode manufactured by Marubishi Bioengineering was used to detect when the glucose (20 g / L) added at the beginning of the culture had been consumed. When the dissolved oxygen concentration exceeded 40% saturation, a fed-boil pump was activated to supply a mixture of 423 g / L glucose and 13 g / L magnesium sulfate heptahydrate. A Peristaltic Bio Mini Pump (manufactured by Atto Co., Ltd.) was used for supply, and the pump flow rate was adjusted to maintain the dissolved oxygen concentration at 40% saturation. The fed-boil pump was started 16 hours after the start of the culture.

[0068] After 24 hours had elapsed since the start of the culture, the culture temperature was changed from 30°C to 25°C. After the culture temperature had decreased, IPTG (isopropyl-β-thiogalactopyranoside) was added to the culture medium to a final concentration of 0.8 mM to induce the expression of low molecular weight antibodies (anti-Fc VHH or Caplacizumab).

[0069] After 72 hours of incubation, the turbidity of the culture medium for anti-Fc VHH-expressing E. coli and Caplacizumab-containing E. coli was 65.9 and 66.2, respectively. The wet cell yield was 110 g and 108 g per liter of culture medium.

[0070] [Experiment 4: Method for quantifying low-molecular-weight antibodies and impurities] The quantification of both low-molecular-weight antibodies and impurities was performed using SDS-PAGE and quantified by densitometry.

[0071] In Experiment 3, a low-molecular-weight antibody extract was obtained by treating the E. coli culture medium (hereinafter sometimes referred to as "E. coli culture medium") after 72 hours of incubation with ultrasonic disruption. An SMT UH-50 ultrasonic disperser was used for ultrasonic disruption, and the mixture was treated 60 times on ice at maximum power with intervals of 1 second. The obtained solution was centrifuged (15,000 G, 5 minutes) to remove bacterial residue, and a low-molecular-weight antibody extract was obtained from the supernatant.

[0072] 5 μL of the extract was mixed with 5 μL of 2× sample buffer (0.25 M Tris-HCl (pH 6.8), 40% glycerol, 8% sodium dodecyl sulfate, 0.02% bromophenol blue). This sample was then mixed with a molecular weight marker (Precision Plus Protein). TM Blue Standards (Bio-Rad) and a self-prepared anti-Fc VHH purified standard (1 g / L) were used together with e-PAGEL gels (E-R15L, ATTO) for SDS-PAGE electrophoresis. After electrophoresis, the gels were washed with water for 15 minutes, stained for 30 minutes, and then destained with water.

[0073] As a result, bands of low-molecular-weight antibodies were confirmed at the molecular weight positions estimated from the amino acid sequence (anti-Fc VHH: 14 kDa, Caplacizumab: 28 kDa). In densitometry quantification, the concentration of each low-molecular-weight antibody was calculated using purified anti-Fc VHH as a control. On the other hand, in the quantification of impurities, the concentration of impurities was calculated using purified anti-Fc VHH as a control, by subtracting the area value of the target low-molecular-weight antibody from the total area value of the lane in question.

[0074] The concentrations of anti-Fc VHH and Caplacizumab in the E. coli culture medium, quantified using the procedure described above, were 1.66 g / L and 1.10 g / L, respectively. On the other hand, the impurity concentrations in the culture medium of anti-Fc VHH-expressing E. coli and Caplacizumab-expressing E. coli were 15.89 g / L and 14.23 g / L, respectively. Using this value as a 100% recovery rate, the recovery rate and degree of impurity reduction in the extraction methods for the target protein (anti-Fc VHH or Caplacizumab) described in Comparative Examples 1-5 and Example 1 were calculated.

[0075] <Comparative Example 1: Extraction of target protein from E. coli culture medium by heating> In accordance with the information in APJ Middelberg Biotechnology Advances 13(1995)491-551, the target protein was extracted by heating. The E. coli culture was heated in a water bath set at 55°C for 1 hour. After heating, the E. coli culture was cooled on ice, and the supernatant was collected by centrifugation (15,000G, 5 minutes).

[0076] <Comparative Example 2: Extraction of target protein from E. coli culture medium by chelation> One-tenth of the volume of chelate buffer (200 mM Tris / 20 mM EDTA pH 8) was added to the E. coli culture medium. After addition, the mixture was allowed to stand at room temperature for 1 hour. After standing, the supernatant was collected by centrifugation (15,000 G, 5 minutes).

[0077] <Comparative Example 3: Extraction of target protein from E. coli culture medium using chaotropic method> Half a volume of chaotropic buffer (0.8M arginine hydrochloride, pH 8) was added to the E. coli culture medium, and the mixture was allowed to stand at room temperature for 1 hour. After standing, the supernatant was collected by centrifugation (15,000G, 5 minutes).

[0078] <Comparative Example 4: Extraction of target protein from E. coli culture solution by acid treatment> 6N HCl was added to the E. coli culture medium to adjust the pH to 4.0, 3.5, 3.0, 2.5, 2.0, and 1.5, respectively. After pH adjustment, the supernatant was collected by centrifugation (15,000G, 5 minutes).

[0079] <Comparative Example 5: Extraction of target protein from E. coli culture solution by high-concentration carboxylic acid treatment> The E. coli culture was centrifuged once, and the supernatant was removed. Acetic acid solutions of various concentrations (v / v) were added to the bacterial cells and suspended. The mixture was then allowed to stand at room temperature for 1 hour. The supernatant was collected by centrifugation (15,000G, 5 minutes).

[0080] <Example 1: Extraction of target protein from E. coli culture medium using carboxylic acid> Carboxylic acids (formic acid, acetic acid, propionic acid, malonic acid, citric acid, lactic acid) were added to E. coli culture medium to reach their respective final concentrations (v / v), and the mixtures were allowed to stand at room temperature for 1 hour. The supernatant was collected by centrifugation (15,000 G, 5 minutes). However, since malonic acid and citric acid are solids at room temperature and pressure, their concentrations were calculated assuming a saturated solution as 100%.

[0081] [Conditions and results of each comparative example and example] The conditions in Experiment 4, Comparative Examples 1-5, and Example 1, along with the concentrations of the target proteins anti-Fc VHH and Caplacizumab in the culture medium, the impurity concentration, the target protein recovery rate compared to Experiment 4 (disruption), and the degree of impurity reduction are shown in the table below.

[0082] [Table 2]

[0083] [Table 3]

[0084] [Table 4]

[0085] [Table 5] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A step of preparing a mixture by mixing a carboxylic acid with a culture medium containing prokaryotic cells having a periplasm and secreting a target protein from the periplasm, such that the final concentration is 0.5% (v / v) or more and 15% (v / v) or less, and The process of extracting target proteins from prokaryotic cells in a mixture. Includes, The aforementioned final concentration is the ratio of the volume of the mixed carboxylic acid to the total volume of the culture medium after the entire amount of carboxylic acid has been mixed. Here, the volume of the carboxylic acid refers to the volume of the liquid carboxylic acid at room temperature and pressure if a liquid carboxylic acid is used at room temperature and pressure, and to the volume of the saturated aqueous solution of the carboxylic acid at 25°C if a solid carboxylic acid is used at room temperature and pressure. A method for extracting target proteins from prokaryotic cells.

2. The method according to claim 1, wherein a prokaryotic cell is capable of expressing a nucleic acid comprising a base sequence encoding the amino acid sequence of a fusion polypeptide of a signal peptide that promotes secretion into the periplasm and a target protein.

3. The method according to claim 1 or 2, wherein the prokaryotic cell is a Gram-negative bacterial cell.

4. The method according to any one of claims 1 to 3, wherein the prokaryotic cell is an Escherichia coli (E. coli) cell.

5. The method according to any one of claims 1 to 4, wherein the carboxylic acid is one or more selected from formic acid, acetic acid, propionic acid, malonic acid, citric acid, and lactic acid.

6. The method according to any one of claims 1 to 5, characterized in that the target protein is a low-molecular-weight antibody.

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