Manufacturing process of membrane-penetrating proteins

Abstract

A process for producing a transmembrane protein is disclosed, comprising the steps of: expressing a transmembrane protein in a host organism present in an aqueous medium; releasing the transmembrane protein from the host organism; adding a detergent solution to dissolve the transmembrane protein; recovering a liquid fraction of the dissolved transmembrane protein; treating the liquid fraction by chromatography to bind or retain the transmembrane protein on a stationary phase; and eluting the stationary phase with an elution buffer to produce the transmembrane protein. This process can produce a relatively large amount of transmembrane protein in an efficient manner without compromising the quality of the final product.

Description

Technology Field

[0001] The present invention relates to a process for manufacturing transmembrane proteins. The process according to the present invention is suitable for industrial applications of manufacturing large quantities of transmembrane proteins. Accordingly, the present invention aims to apply unit operations suitable for at least pilot plant manufacturing. In particular, the present invention aims to avoid ultracentrifugation. Background Technology

[0002] Approximately one-third of the genes in the human genome encode membrane proteins. Membrane proteins play a role in many important cellular activities, including energy conversion, cell signaling, cell-cell interactions, cell adhesion, cell migration, protein transport, viral fusion, neural synaptic activity, and the transport of ions and metabolites. Transmembrane proteins are embedded in the lipid bilayer of the cell membrane and consist of a hydrophobic portion and a hydrophilic portion.

[0003] Recently, transmembrane proteins have been successfully incorporated into thin-film composites grafted onto porous structural supports. Furthermore, transmembrane proteins represent important pharmacological targets and interesting research topics in relation to cell biology and protein biochemistry. Therefore, there is a need for pilot plants or large-scale manufacturing of transmembrane proteins.

[0004] WO2017137361(A1) discloses self-assembled nanostructures formed between aquaporin water channels (AQPs) and membrane-penetrating proteins such as polyalkyleneimines (PAIs). The self-assembled nanostructures are subsequently incorporated into a thin film composite (TFC) membrane grafted onto a porous support membrane. The porous support membrane may be a hollow fiber, flat sheet, spiral-wound membrane, etc., for reverse or forward osmosis.

[0005] WO2013 / 043118 discloses a thin film composite (TFC) membrane in which aquaporin water channels (AQP) are incorporated into the active layer of the membrane. Furthermore, the said patent discloses a method for manufacturing the thin film composite membrane and the use of the thin film composite membrane in filtration processes such as nanofiltration and osmotic filtration. The TFC membrane comprises lipid-AQP / copolymer-AQP vesicles incorporated into the TFC active layer. WO2010 / 146365 describes the preparation of a TFC-aquaporin-Z (AqpZ) filtration membrane using an amphiphilic triblock copolymer as a vesicle-forming material for incorporating immobilized AQP.

[0006] WO2014 / 108827 discloses a hollow fiber (HF) module having a fiber modified into a thin film composite (TFC) layer containing an aquaporin water channel incorporated into a vesicle before the aquaporin water channel is incorporated into the TFC layer.

[0007] Industrial methods for isolating large quantities of transmembrane proteins from cells are generally not available to those skilled in the art. Prior art has focused on producing relatively small amounts of transmembrane proteins for research purposes. Consequently, relatively cumbersome and labor-intensive manufacturing methods were permitted. However, with the recent increase in demand for transmembrane proteins for industrial membranes used in reverse and forward osmosis, the need for more efficient manufacturing methods has become evident. The object of the present invention is to provide a process for manufacturing transmembrane proteins that can produce relatively large quantities of transmembrane proteins in an efficient manner without compromising the quality of the final product. The problem to be solved

[0008] The object of the present invention is to provide a process for manufacturing transmembrane proteins to produce a relatively large amount of transmembrane proteins in an efficient manner without compromising the quality of the final product. means of solving the problem

[0009] The present invention relates to a process for manufacturing a membrane-penetrating protein and includes the following steps.

[0010] a. A step of expressing a membrane-penetrating protein in a host organism present in an aqueous medium;

[0011] b. A step of releasing the above-mentioned membrane-penetrating protein from a host organism;

[0012] c. A step of adding a detergent solution to dissolve the above-mentioned membrane-penetrating protein;

[0013] d. A step of recovering the liquid fraction of the solubilized membrane-penetrating protein as the supernatant by centrifugation;

[0014] e. applying the above liquid fraction to chromatography to bind or retain the membrane-penetrating protein on the stationary phase; and

[0015] f. A step of eluting the stationary phase with an elution buffer to prepare the above-mentioned membrane-penetrating protein, comprising,

[0016] Centrifugation in step d is performed at 500g to 30,000g.

[0017] This process has the advantage of being able to process large quantities of aqueous media containing host organisms. Therefore, this process can process volumes of 50L or more, such as application unit operations of 100L or more suitable for pilot plants or full-scale manufacturing. In addition, this process is scalable and can be easily applied to large quantities of aqueous media containing host organisms.

[0018] Surprisingly, the inventors discovered that transmembrane proteins expressed by the host organism dissolve to a higher degree than other proteins when a detergent solution is added. It is estimated that more than 60% of the proteins in the vesicles formed by the detergent solution are transmembrane proteins of interest. Additionally, the size of the vesicles formed by the detergent was observed to be about 0.5 µm, which is larger than usual, suggesting that transmembrane proteins form a more stable superform with the detergent than naturally occurring phosphorescent lipids.

[0019] The aqueous medium containing the host organism can be used directly in the process. However, to obtain a cleaner product and avoid handling an excess amount of aqueous medium, the aqueous medium containing the host organism in step a is generally filtered before the release of membrane-penetrating proteins according to step b.

[0020] Generally, an aqueous medium containing a host organism is upconcentrated by filtering through a filter in which the pores are small enough to allow cell debris and the medium to pass through, while the cells are retained. In a suitable embodiment, the aqueous medium containing the host organism of step a is filtered through a microfiltration membrane having a pore diameter of 0.5 micrometers or less. Preferably, the pore diameter is 0.1 μm or less.

[0021] After a filtration step appropriately performed by microfiltration, the host organism can be separated from the remaining aqueous medium. Although various separation processes are possible, it is generally preferable to separate the host organism after filtration by centrifugation of the aqueous medium containing the host organism. The centrifugation process is generally performed by gravity to form a pellet within a suitable time. Therefore, centrifugation is typically performed at 500g or more, e.g., 1000g or more, preferably 2000g or more. The density of the pellet must not be too high to prevent difficulties in subsequent steps. Accordingly, centrifugation is generally not performed at 30,000g or more, e.g., 20,000g or more, suitably at 15,000g or more, preferably 8,000g or more.

[0022] The cells can be harvested as a pellet and the supernatant can be discarded. It is preferable to wash the isolated host organism with isotonic saline to dissolve contaminating salts and then centrifuge as described above to separate the washed host organism. The used washing solution that appears in the supernatant can be discarded.

[0023] The pellet containing the washed cells can be stored by freezing at -20°C or used directly in the next step. Optionally, a dilution buffer is added before step b. The dilution buffer may contain a protease inhibitor to prevent the degradation of membrane-penetrating proteins, a pH regulator such as TRIS and phosphate to maintain the pH value within a desired range, and / or an ion scavenger such as EDTA.

[0024] Transmembrane proteins can be released from a host organism in various ways, preferably by chemical or mechanical lysis of cells. When chemical lysis of cells is appropriately performed, a water-soluble lysis solution is added to release transmembrane proteins from the host organism. In a preferred embodiment of the present invention, the water-soluble lysis buffer is a detergent solution that simultaneously lyses transmembrane proteins.

[0025] In order for cell lysis and solubilization of membrane-penetrating proteins to occur, host cells are generally subjected to the action of a detergent during stirring. Generally, host cells are allowed to react with the detergent for at least 1 hour, e.g. 2 hours, preferably at least 6 hours.

[0026] When mechanical lysis of cells is generally used, the release of transmembrane proteins from host cells is performed by a homogenizer. For the homogenization of milk, it is appropriate to use the same type of homogenizer used in the dairy industry. A suitable example is the Stansted 7575 homogenizer. After processing in the homogenizer, the cells open up, and transmembrane proteins are released.

[0027] A cationic coagulant may be added after the release of membrane-penetrating proteins. Cationic coagulants are believed to interact particularly with negatively charged cell debris to form flocs. In a preferred embodiment of the present invention, the cationic coagulant is a polyamine compound such as Superfloc C581.

[0028] Floc formation generally does not occur immediately. Therefore, it is desirable for the cationic coagulant to react with the cell portion of the resuspended solution to form flocs during low-speed stirring. Generally, the cationic coagulant and the cell debris are allowed to interact between 10 minutes and 2 hours while stirring at room temperature.

[0029] When chemical lysis of the cells is used, the liquid fraction of step e is recovered as the supernatant from centrifugation of the suspension containing the flocs. As the membrane-penetrating proteins are dissolved by the detergent solution, they appear in the supernatant.

[0030] When mechanical lysis of cells is used, the liquid fraction of step e is recovered from the resuspension of the solid fraction, and said solid fraction is resuspended in a detergent solution to dissolve the transmembrane proteins. In one embodiment of the present invention, the solid fraction is formed due to the fact that cell fragments containing transmembrane proteins interact with the coagulant and form flocs that can be separated from the liquid by centrifugation. In another embodiment of the present invention, it was surprisingly discovered that it is possible to harvest transmembrane proteins from the pellet when mechanical lysis of cells is used without using a coagulant. The pellet obtained by centrifugation can be resuspended in a detergent solution to solubilize the transmembrane proteins. In this step, the liquid fraction of step e is harvested as a supernatant by centrifugation.

[0031] The centrifugation process is generally performed by gravity to form pellets within a suitable time. Therefore, centrifugation is typically performed at 500g or more, e.g., 1000g or more, preferably 2000g or more. The density of the pellets must not be too high to prevent difficulties in subsequent steps. Accordingly, centrifugation is generally not performed at 30,000g or more, e.g., 20,000g or more, suitably 15,000g or more, preferably 8,000g or more. When applying gravity of less than 30,000g, purifiers commonly used in dairy farming, such as spore removal centrifuges from GEA, Tetra Pak, Alfa Laval, and SPX Flow Separation Technology, can be used. Purifiers useful for the present invention may also be referred to as Bactofuge by some manufacturers. Thus, the present invention can omit the application of ultracentrifuges capable of only small batch centrifugation and processing.

[0032] The detergents used in the present invention include alkyl maltopyranosides such as n-dodecyl-β-D-maltopyranoside (DDM), n-decyl-β-D-maltopyranoside (DM), or 5-cyclohexylpentyl β-D-maltoside (Cymal-5); alkyl glucopyranosides such as n-octyl-β-D-glucopyranoside (OG); amine oxides such as n-lauryl dimethylamine N-oxide (LDAO); phosphocholines such as n-dodecyl phosphocholine (FC-12), n-tetradecyl phosphocholine (FC-14), or n-hexadecyl phosphocholine (FC-16); or polyoxyethylene glycol, etc. In a suitable embodiment of the present invention, the detergent is selected from the group consisting of lauryl dimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM), or combinations thereof. LDAO is a preferred detergent because it generates large and stable vesicles, which suggests that the detergent can replace naturally occurring phospholipids.

[0033] The liquid fraction undergoes a chromatography process in which the membrane-penetrating protein binds to or is retained on the stationary phase. The type of chromatography can be selected from affinity chromatography, ion exchange chromatography, size exclusion chromatography, displacement chromatography, liquid chromatography, high-performance liquid chromatography, reverse-phase chromatography, hydrophobic interaction chromatography, etc. Generally, the chromatography process is a preparatory chromatography, as opposed to analytical chromatography, which forms the purification of membrane-penetrating proteins.

[0034] In a currently preferred embodiment, the chromatographic method is selected as affinity chromatography, so that the first part of the affinity pair is associated with a transmembrane protein and the second part of the affinity pair is associated with a stationary phase. Examples of stationary phases include beads and column materials. In a preferred embodiment of the present invention, the stationary phase associated with the second part of the affinity pair is present in a column. Generally, a part of the affinity pair is associated with a covalent bond to a transmembrane protein or a stationary phase. However, other types of bonding are also possible, such as affinity bonding through hybridization or antibody-antigen interactions.

[0035] A number of affinity pairs applicable in the present invention are known to those skilled in the art and include biotin-streptavidin pairs, antibody-antigen pairs, antibody-hapten pairs, aptamer affinity pairs, capture protein pairs, Fc receptor-IgG pairs, metal chelate lipid pairs, metal chelate lipid histidine (HIS) tag protein pairs, or combinations thereof. In a currently preferred embodiment, the affinity pair is a metal chelate lipid-histidine (HIS)-tag protein pair.

[0036] The metal is generally immobilized on the column material in a technique called immobilized metal affinity chromatography (IMAC). The metal is generally selected as Cu(II) or Ni(II), preferably Ni(II). His-tagged proteins can be purified automatically or manually using Ni-NTA resin. Suitable stationary phases are GE Healthcare's "Capto Chelating" or GE Healthcare's HisTrap Gel filtration material (Ni Sepharose 6 Fast Flow).

[0037] The first portion of the affinity pair, which is a histidine tag, is typically attached to the C-terminus of a transmembrane protein. While a histidine tag typically contains six consecutive histidine amino acids, in the present invention, it is preferable that the histidine tag contains eight or more histidine molecules. A large number of histidine amino acids in the histidine tag enables effective separation of specific binding proteins and non-specific binding proteins.

[0038] After adding a liquid fraction containing membrane-penetrating proteins to the column, the liquid is allowed to penetrate the resin by gravity or pressure. Suitably, the elution buffer contains imidazole. Imidazole has the ability to interact with the binding of His tags to metal ions immobilized on the resin. At a specific concentration of imidazole, histidine-tagged membrane-penetrating proteins are released.

[0039] To separate non-specifically bound transmembrane proteins from transmembrane proteins of interest, the column is generally washed with a washing buffer containing 40% or less of the imidazole concentration in the elution buffer before elution with the elution buffer. The imidazole concentration in the elution buffer is generally in the range of 200 mM to 2000 mM imidazole. In a preferred embodiment of the present invention, the imidazole concentration in the elution buffer is 400 mM or higher. To obtain more effective elution of His-tagged transmembrane proteins, the imidazole concentration in the buffer is generally 600 mM or 800 mM or higher.

[0040] In most applications, the impact of His tags on protein structure, function, and immunogenicity is generally negligible. However, in certain applications, it may be desirable to remove His tags after they have provided the function of binding membrane molecules to the column. His tags can be removed by introducing a cleavable link between the membrane-transmitting protein and the His tag. Suitable cleavable links include pH-sensitive linkers, disulfide linkers, protease-sensitive linkers, and beta-glucuronide linkers.

[0041] Transmembrane proteins extend across the entire double lipid membrane in the natural environment, that is, from the inside of the cell to the extracellular space. Most transmembrane proteins function as gateways for specific substances, allowing for their exchange between the intracellular and extracellular fluids. A characteristic feature of transmembrane proteins is the presence of hydrophobic regions that ensure their integration into the membrane. Transmembrane proteins also possess hydrophilic segments on either side of hollow fiber regions, with these hydrophilic segments facing the intracellular and extracellular fluids, respectively.

[0042] While it is believed that any transmembrane protein can be produced according to the present invention, it is generally preferred to use this process to produce transmembrane proteins that transport ions (ion channels) and water (aquaporin water channels). Ion channels include chloride channels and metal ion transporters. In addition to chloride ions, certain chloride channels are HCO3 - , I - , SCN - and NO3 - It conducts. Metal ion transporters include magnesium transporters, potassium ion channels, sodium ion channels, calcium channels, proton channels, etc. In a specific embodiment of the present invention, the membrane-penetrating protein is outer membrane protein A (OmpA).

[0043] In a preferred embodiment of the present invention, the transmembrane protein is an aquaporin water channel. Aquaporin water channels facilitate the transport of water into and out of the cell. In industrial membranes, aquaporin water channels ensure the flow of water by osmosis while rejecting other components in the solution. Transmembrane proteins such as aquaporin water channels can be released from various sources, including prokaryotes and eukaryotes. The prokaryotic sources of aquaporins are E. coli , Kyrpidia spormannii , Methanothermobacter sp., Novibacillus thermophilus , Saccharomyces cerevisiae , and Halomonas It includes sp. Eukaryotic sources of aquaporins include Oryza sativa Japonica (Japanese rice), Eucalyptus grandis, Solanum tuberosum (Danish potatoes) and Milnesium tardigradum (Water bear) is included.

[0044] The nucleic acid sequence of the transmembrane protein in the source organism is typically codon-optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in the host organism. The generated gene is appropriately synthesized by adding histidine encoding the C-terminal codon, along with N-terminal and C-terminal lateral restriction sites. The synthesized gene fragment can be degraded with restriction enzymes and ligated into the vector fragment. The resulting ligation mixture is preferably transformed into an organism such as E. coli DH10B. Antibiotic-resistant transformants are appropriately selected in a medium containing antibiotics. The transformants were identified by sequencing of the genetic construct. The isolated vector DNA was subsequently transferred to a production host. The production host is E. coli ( Escherichia coli ) and Saccharomyces cerevisiae( Saccharomyces cerevisiae It can be selected from a number of suitable prokaryotic or eukaryotic organisms such as ).

[0045] Host organisms are generally engineered to express natural transmembrane proteins in greater amounts than usual or to express non-natural transmembrane proteins. One method of expressing transmembrane proteins may be to transform the host organism with a vector containing DNA encoding transmembrane proteins, as previously described. Another possibility for obtaining overexpression of transmembrane proteins is to upregulate the expression of natural transmembrane proteins, for example, by attenuating repressors or inserting suitable promoter regions. An additional possibility for obtaining expression of non-natural proteins is to transfect the host organism with a virus or bacteriophage containing nucleic acid encoding transmembrane proteins. Effects of the invention

[0046] As described above, according to the present invention, there is an advantage in that a relatively large amount of membrane-penetrating protein can be produced in an efficient manner without compromising the quality of the final product. Specific details for implementing the invention

[0047] Example 1

[0048] Prepare an E. coli BL21 strain containing a vector that produces an aquaporin protein linked to a His tag at the C-terminus. The His tag contains 10 consecutive histidine molecules attached to the primary sequence of the aquaporin membrane protein.

[0049] E. coli strains were cultured in standard medium to obtain a total of 150 L of fermentation liquid. E. coli cells were harvested by filtering the fermentation liquid through a microfiltration membrane with a pore diameter of 0.05 µm. The filtrate containing E. coli cells was reduced to approximately 50 L and then centrifuged at 5300 g for 20 minutes. Thus, the E. coli cells are concentrated upward by microfiltration, and the remaining medium is subsequently removed as the supernatant by centrifugation.

[0050] The pellet obtained by centrifugation is collected, and 0.9% sodium chloride is added in a 1:1 volume ratio to wash the cells and dissolve contaminating salts. Afterward, the wash solution is removed from a centrifuge operating at 5300g for 20 minutes. The supernatant is discarded, and the washed cells are collected as a pellet. The pellet can be frozen at -20°C for storage or used directly in the next step.

[0051] A pellet containing E. coli cells was dissolved in approximately 47 L of TRIS buffer, which was used as a binding buffer. After stirring for about 1 hour, 6.4 L of detergent (5% LDAO) was added for solubilization to achieve a final concentration of 0.6%. The mixture was incubated overnight at room temperature with low-speed stirring. Cell lysis occurred upon resuspension of the cells in the buffer containing the detergent. Membrane proteins are released from the inner membrane of the cell and dissolved by the detergent.

[0052] To remove negatively charged cell material, polyamine (Superfloc C581) was added in a volume of 427 mL. The mixture was incubated at room temperature with stirring for 30 minutes to coagulate cell debris, negatively charged molecules such as DNA and RNA, and a certain amount of protein. Membrane proteins dissolved by the detergent remained in the aqueous phase. The mixture was centrifuged at a maximum speed of 5300 g for 15 minutes. The pellet containing cell debris, DNA, RNA, and dissolved protein was discarded, and the supernatant was collected. The supernatant was transferred to a container containing 107 L of dilution buffer to obtain a final volume of 160 L with a final concentration of 0.2% LDAO.

[0053] A column containing GE Healthcare’s affinity resin “Capto Chelating” is provided. The resin is packed with Ni2+ that binds to the His10 tag. The diluted supernatant was loaded onto the column and then washed with 10 column volumes of wash buffer containing 200 mM imidazole to wash away non-specific binding components. Subsequently, membrane proteins were released from the column using 2.5 column volumes of elution buffer containing 1000 mM imidazole aqueous solution. The proteins eluted from the column were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which showed a single band indicating a purity of over 80%.

[0054] Example 2

[0055] Prepare an E. coli BL21 strain containing a vector that produces an aquaporin protein linked to a His tag at the C-terminus. The His tag contains 10 consecutive histidine molecules attached to the primary sequence of the aquaporin membrane protein.

[0056] Escherichia coli strains were cultured in standard media to obtain a total of 250 L of fermentation broth. E. coli cells were harvested by filtering the fermentation broth through a PES plate membrane filter with a pore diameter of 0.05 µm. The filtrate containing E. coli cells was reduced to approximately 50 L and then centrifuged at 5300 g for 20 minutes using a Sorvall 16 L centrifuge. Thus, the E. coli cells were upconcentrated by microfiltration, and the remaining medium was subsequently removed as the supernatant by centrifugation. The pellet can be stored frozen at -20°C or used directly in the next step.

[0057] The pellet containing E. coli cells was resuspended in approximately 50 L of buffer (aqueous solution of the protease inhibitor PMSF and EDTA) and homogenized at 1000 bar using a Stansted nm-GEN 7575 homogenizer. Polyamine (Superfloc C581) was added at a concentration of 12 ml / L to isolate the cellular material of interest. The temperature was maintained at approximately 10–15ºC. The mixture was incubated at room temperature for 30 minutes with stirring. The mixture was centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contained transmembrane proteins, and the supernatant was discarded.

[0058] The pellet was resuspended in a 0.9% sodium chloride solution to obtain a total protein concentration of approximately 50 mg / ml. 28 L of TRIS binding buffer and 4.5 L of 5% LDAO were added to 5 L of the resuspended pellet material to perform solubilization of the transmembrane protein. The mixture was incubated at room temperature for 2 to 24 hours while stirring at low speed.

[0059] After the solubilization process, the mixture was centrifuged in a 2L container at 5300g for 90 minutes. The supernatant was recovered, and a dilution buffer was added to adjust the LDAO concentration to 0.2%.

[0060] A column containing GE Healthcare’s affinity resin, “Capto Chelating,” is provided. The resin binds to Ni that binds to the His10 tag.2+ The column was loaded with binding buffer containing 0.2% LDAO and equilibrated. Subsequently, the diluted supernatant was loaded onto the column, and non-specific binding components were washed with 10 column volumes of washing buffer containing 200 mM imidazole. Next, transmembrane proteins were released from the column using 2.5 column volumes of elution buffer containing 1000 mM imidazole aqueous solution. The proteins eluted from the column were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) chromatography, which showed only single bands indicating a purity of over 95%.

[0061] Example 3

[0062] Prepare an E. coli BL21 strain containing a vector that produces an aquaporin protein linked to a His tag at the C-terminus. The His tag contains 10 consecutive histidine molecules attached to the primary sequence of the aquaporin membrane protein.

[0063] Escherichia coli strains were cultured in standard media to obtain 250 L of total fermentation broth. The fermentation batch had an OD600 of 13 at harvest and was induced for 42.5 hours. The material was homogenized twice at 100 MPa using a Stansted nm-GEN 7575 homogenizer. 10 mL of the dissolved material was taken and added to a 15 mL Falcon tube.

[0064] Then, centrifugation was performed at 5300g for 1 hour, and the pellet was separated from the supernatant. The pellet was then resuspended in 10 mL of 0.9% NaCl. BCA analysis was used to determine the concentration (mg / mL total protein) of the resuspended pellet and the separated supernatant after the first spin.

[0065] The resuspended pellet and separated supernatant were finally dissolved in 0.6% LDAO (120 µL of Carbosynth 5% LDAO stock was used for 1 mL of material) for 2 hours (1 mL) and centrifuged again at 5300 G for 15 minutes. Once again, the supernatant was separated from the pellet. The pellet was resuspended in binding buffer without LDAO (again up to 1 mL).

[0066] All samples were analyzed on an SDS-gel and showed that approximately 60% of the proteins in the supernatant were aquaporin membrane proteins.

[0067] Example 4

[0068] After purifying aquaporin Z in Example 2, the size distribution of the protein dissolved in LDAO was measured.

[0069] To evaluate whether the particle size distribution is affected by buffer components (including detergent) or membrane proteins, the same elution buffer was compared with and without protein.

[0070] Aquaporin Z protein was eluted on an IMAC column using elution buffer containing 1000 mM imidazole and 0.2% w / v LDAO. The protein concentration of the purified protein was measured via amino acid analysis. Protein in clear, soluble elution buffer at 6.44 mg / mL was extracted into three cuvettes (Sarstedt, 4 mL, PMMA, ca. no. 67.755, N The samples were placed in the Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern UK) and the size distribution was analyzed using Malvern Zetasizer software v.7.02 (Malvern Instruments Ltd., Malvern UK). The results presented in Table 1 are the average of samples from three runs.

[0071] Sample AqpZ concentration (mg / mL) Size, Diameter (d.nm) Population Strength (%) Population 1 Population 2 Population 3 Purified AqpZ in elution buffer (average) 6.44 315.6±116.8 nm45.9% 108±32.3 nm35.5% 17.1±2.5 nm14.9% elution buffer (average) 0 0.85±0.16 nm56.8% 8.6±1.2 nm41.1% 103±5.5 nm 2.1%

[0073] The elution buffer sample (protein-free) contains a population distribution in which 97.9% of particles are 8.6 nm or smaller, indicating the absence of large detergent micelles in the solution maintained by microfiltration. In the protein-containing sample, 81.4% of the particles are 108 nm or larger, indicating that the presence of both protein and detergent forms large soluble particles maintained during microfiltration. Surprisingly, the experiment shows that large, stable particles composed of LDAO micelles and transmembrane proteins are generated.

[0074] Example 5

[0075] In E. coli Oryza sativa Japonica Histidine-tagged aquaporin expression and purification using IMAC in (Japanese rice)

[0076] Oryza sativa Japonica The gene encoding aquaporin (UNIPROT:A3C132) was codon-optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Oryza_sativa_Japonica). The synthesized gene fragment was digested with NdeI / XhoI restriction enzymes and ligated to the NdeI / XhoI-digested and purified vector pUP1909 fragment. The resulting ligation mixture was transformed into E. coli DH10B, and kanamycin-resistant transformants were selected on LB agar plates containing kanamycin. Transformants were identified by sequencing of the gene constructs. The isolated vector DNA was subsequently transferred to the production host, E. coli BL21.

[0077] To heterogenize aquaporins in E. coli, the production host was grown in a minimal medium composed of 30 g / L glycerol, 6 g / L (NH4)2HPO4, 3 g / L KH2PO4, 5 g / L NaCl, 0.25 g / L MgSO4·7H2O, 0.4 g / L Fe(III) citrate, and 1 mL / L sterile filtered trace metal solution. The trace metal solution consisted of 1 g / L EDTA, 0.8 g / L CoCl2·6H2O, 1.5 MnCl2·4H2O, 0.4 g / L CuCl2·2H2O, 0.4 g / L H3BO3, 0.8 g / L Na2MoO4·2H2O, and 1.3 g / L Zn(CH3COO)2·2H2O. After inoculation and overnight growth, an additional 0.25 g / L MgSO4·7H2O was added.

[0078] Escherichia coli was cultured in 3L Applikon Bioreactors with ez-Control in a batch fermentation process. Protein production was induced by adding IPTG at a final concentration of 0.5 mM at an optical density of approximately 30 (OD 600 nm). Culture was induced for approximately 24 hours, and bacterial cells were harvested by centrifugation at 5300 g for 20 minutes.

[0079] A pellet containing E. coli cells was resuspended in a buffer (aqueous solution of the protease inhibitor PMSF and EDTA) and homogenized at 1000 bar using a Stansted nm-GEN 7575 homogenizer. The temperature was maintained at approximately 10–15°C. The mixture was centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contained transmembrane proteins, and the supernatant was discarded.

[0080] The pellet was resuspended in a 0.9% sodium chloride solution to obtain a total protein concentration of approximately 50 mg / ml. 28 L of TRIS binding buffer and 4.5 L of 5% LDAO were added to 5 L of the resuspended pellet material to perform solubilization of the transmembrane protein. The mixture was incubated at room temperature for 2 to 24 hours while stirring at low speed.

[0081] After the solubilization process, the mixture was centrifuged in a 2L container at 5300g for 90 minutes. The supernatant was recovered, and a dilution buffer was added to adjust the LDAO concentration to 0.2%.

[0082] After solubilization and purification, the protein was captured using IMAC and eluted in an elution buffer containing 1000 mM imidazole and 0.2% w / v LDAO. The elution fraction was analyzed by SDS-PAGE, revealing only a single major band shifting at 27 kDa, corresponding to the size of aquaporins extracted from Japanese rice. Additionally, the results were verified by comparing the purified protein with a negative control from E. coli transformed with an empty vector. The negative control did not produce the purified protein. Protein detection using a histidine tag-specific antibody (TaKaRa Bio) (Western blot analysis) showed a clear signal in the purified protein as expected, while there was no signal in the negative control, confirming that the purified protein originated from a histidine tag-transmembrane protein.

[0083] Example 6

[0084] In E. coli Eucalyptus grandis Histidine-tagged aquaporin expression and purification using IMAC

[0085] Eucalyptus grandis The gene encoding aquaporin (UNIPROT:A0A059C9Z4) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Eucalyptus_grandis). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0086] Example 7

[0087] In E. coli Solanum tuberosum Histidine-tagged aquaporin expression and purification using IMAC in (Danish potato)

[0088] Solanum tuberosum The gene encoding aquaporin (UNIPROT:Q38HT6) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Solanum_grandis). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0089] Example 8

[0090] In E. coli Milnesium tardigradum Histidine-tagged aquaporin expression in (water bear) and purification using IMAC

[0091] Milnesium tardigradum The gene encoding aquaporin (UNIPROT: G5CTG2) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Milnesium_tardigradum). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0092] Example 9

[0093] In E. coli HalomonasHistidine-tagged aquaporin expression in sp. and purification using IMAC

[0094] The gene encoding an aquaporin in Halomonas sp. (UNIPROT: A0A2N0G6U6) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Halomonas_sp). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0095] Example 10

[0096] In E. coli Kyrpidia spormannii Histidine-tagged aquaporin expression and purification using IMAC

[0097] Kyrpidia The gene encoding an aquaporin in sp. (UNIPROT: A0A2K8N5Z5) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Kyrpidia_spormannii). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0098] Example 11

[0099] In E. coli Methanothermobacter Histidine-tagged aquaporin expression in sp. and purification using IMAC

[0100] Methanothermobacter The gene encoding an aquaporin in *Methanothermobacter* sp. (UNIPROT: A0A223ZCQ2) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in *E. coli*. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Methanothermobacter_sp). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0101] Example 12

[0102] In E. coli Novibacillus thermophilus Histidine-tagged aquaporin expression and purification using IMAC.

[0103] Novibacillus thermophilus The gene encoding aquaporin (UNIPROT: A0A1U9K5R2) was codon optimized using the services of Geneart (a subsidiary of Thermo Fischer Scientific) to improve expression in E. coli. The resulting gene is synthesized by adding 10 histidines encoding the C-terminal codon, along with N-terminal and C-terminal flanking NdeI / XhoI restriction sites (Genetic ID: aquaporin_Novibacillus_thermophilus). This gene was cloned and expressed as described in Example 5. The protein was successfully purified and verified as described in Example 5.

[0104] Example 13

[0105] Expression of E. coli outer membrane protein A (OmpA) in E. coli. The expression and purification of OmpA (UNIPROT: P0A910) in E. coli were performed essentially as described in Example 5, except that E. coli was transformed with an empty vector and solubilization was extended to 24 hours. The resulting solubilized protein was estimated to have a purity of about 30–50% by SDS-PAGE, thus clearly indicating that OmpA was successfully separated from the membrane fraction according to standard purification procedures. OmpA could be further purified by an additional size exclusion chromatography (SEC) step.

[0106] Example 14

[0107] Saccharomyces cerevisiae expressing aquaporin-Z (AqpZ) from Escherichia coli isolated by fusion to N-terminal histidine-tagged yEGFP and tobacco etching virus (TEV) protease cleavage site ( Saccharomyces cerevisiae ) Strain establishment.

[0108] AqpZ (UNIPROT ID: AqpZ of Escherichia coli (UNIPROT ID: P60844) is S. cerevisiae Codons were optimized for expression in S. cerevisiae using Geneart's services to improve expression. S. cerevisiae For expression, AqpZ was fused to the N-terminus of yeast-enhanced green fluorescent protein (yEGFP) (Brendan P. Cormack et al, Microbiology (1997), 143, 303–311) to enable the visual detection and quantification of the membrane-transcending protein. Additionally, eight histidines (His8) were added to the N-terminus of yEGFP as IMAC purification tags. His8-yEGFP and AqpZ were genetically isolated by TEV protease cleavage sites incorporated by PCR primers during construction.

[0109] The rapid and efficient construction of a plasmid encoding a His8-yEGFP-TEV-AqpZ fusion was achieved between the His8-yEGFP-TEV PCR fragment, the TEV-AqpZ PCR fragment, and a linearized expression plasmid derived from the SalI, HindIII, and BamHI digestion of the pEMBLyex4 plasmid as described in (Scientific Reports 7:16899). S. cerevisiae It was performed by in vivo homologous recombination of the overlapping region integrated by the primers. The TEV cleavage site enables the subsequent removal of His8-yEGFP protein by TEV protease.

[0110] S. cerevisiae Selection of transformants was performed on minimal medium plates lacking uracil but supplemented with leucine and lysine to ensure the survival of bacterial cells with correctly recombined DNA fragments. The medium composition and amino acid concentrations used in this example were the same as those listed in Example 15.

[0111] Example 15

[0112] Saccharomyces cerevisiae using Expression of His8-yEGFP-TEV-AqpZ.

[0113] Single colonies of transformed yeast cells were selectively expanded in 5 ml of glucose minimal medium supplemented with 60 mg / L leucine and 30 mg / L lysine until saturation. Subsequently, 200 μl of this culture was expanded in 5 ml of glucose minimal medium supplemented with 30 mg / L lysine for selection for high plasmid copy numbers. Frozen stocks of high plasmid copy number cells were prepared.

[0114] 200 μl of thawed frozen stock was added to 10 ml of lysine-supplemented minimum medium and grown until saturation. 1 ml of the culture was transferred to 100 ml of the same medium. After growing overnight, an aliquot corresponding to a final OD600 of 0.05 was transferred to 1.5 liters of minimum medium containing 30 g / L glycerol supplemented with extra amino acids and an initial concentration of 20 g / L glucose as a carbon source. The culture was grown in a 3 L Applikon® bioreactor equipped with ez-Control connected to a PC (Applikon / Netherlands and SecureCell / Switzerland) running Lucullus® software.

[0115] The initial phase of fermentation was carried out at 20°C in minimal medium. When the initial amount of glucose had been metabolized, glucose was supplied to the bioreactor at a final concentration of 3% w / v. The pH of the growth medium was maintained at 6.0 by the computer-controlled addition of 1M NH4OH. When the CO2 exhaust gas leveled out due to limited glucose access, the bioreactor was cooled to 15°C before inducing recombinant AQP production. Expression of the recombinant protein began upon the addition of 50 mL / L expression medium consisting of 400 mL / L ASD-10, 400 mL / L extra amino acids, 200 g / L glycerol, and 20 g / L galactose. Yeast cells were harvested after 96 hours.

[0116] The minimum medium consisted of 20 g / L glucose, 100 mL / L ASD-10, 5 mL / L V-200, 30 g / L glycerol, and 0.1 g / L Ca2Cl. ASD-10 consisted of 50 g / L (NH4)2SO4, 8.75 g / L KH2PO4, 1.25 g / L K2HPO4, 5 g / L MgSO4·7H2O, 1 g / L NaCl, 5 mg / L H3BO3, 1 mg / L KI, 4 mg / L MnSO4·1H2O, 4.2 mg / L ZnSO4·7H2O, 0.4 mg / L CuSO4·5H2O, 2 mg / L FeCl3, and 2 mg / L Na2MoO4·2H2O. V-200 was composed of 4 mg / L biotin, 400 mg / L D-pantothenic acid, 0.4 mg / L folic acid, 2000 mg / L myo-inositol, 80 mg / L niacin, 40 mg / L p-aminobenzoate, 80 mg / L pyridoxine, 40 mg / L riboflavin, and 80 mg / L thiamine. In the case mentioned, the medium contains additional amino acids consisting of 600 mg / L alanine, 600 mg / L arginine, 600 mg / L cysteine, 3000 mg / L glutamic acid, 2000 mg / L lysine, 600 mg / L methionine, 1500 mg / L phenylalanine, 600 mg / L proline, 10000 mg / L serine, 900 mg / L tyrosine, 4500 mg / L valine, 2000 mg / L aspartic acid, 4000 mg / L threonine, 600 mg / L histidine, and 600 mg / L tryptophan.

[0117] Example 16

[0118] Saccharomyces cerevisiae In a bioreactor using Milnesium tardigradum's His8-yEGFP-TEV-AQP5 Cloning

[0119] The His8-yEGFP-TEV-AQP5 construct was prepared according to the procedure summarized in Example 14. M. tardigradum The AQP5 protein of (UNIPROT: G5CTG2) was codon-optimized for expression in E. coli despite the need for expression in yeast.

[0120] Example 17

[0121] S. cerevisiae Purification of His8-yEGFP-TEV-AqpZ and His8-yEGFP-TEV-AQP5 from

[0122] S. cerevisiae The purification of the heterologously expressed transmembrane protein was carried out as described in Example 2, except that the volume of the applied buffer was reduced to match the reduced culture volume and the lysis was performed at 1800 bar. Protein production and purity were successfully confirmed for both fusion proteins by SDS-PAGE and Western Blot, and no contaminating proteins were detected.

Claims

Claim 1 In a process for manufacturing a transmembrane protein on a test production or industrial scale, the method comprises: a. expressing said transmembrane protein in a host organism present in an aqueous medium; b. i) releasing said transmembrane protein by mechanically lysing the cells of said host organism to form lysed cell fragments containing said transmembrane protein from said host organism; ii) separating said lysed cell fragments containing said transmembrane protein into a solid fraction by centrifugation performed at 500 g to 30,000 g; c. adding a detergent solution to the resuspended solid fraction obtained in step b to lyse said transmembrane protein; d. recovering the liquid fraction of said solubilized transmembrane protein obtained in step c as a supernatant by centrifugation; e. applying said liquid fraction to chromatography to bind or retain said transmembrane protein on a stationary phase; and f. A process for manufacturing a membrane-penetrating protein, comprising the step of eluting the membrane-penetrating protein from the stationary phase to manufacture the membrane-penetrating protein, wherein the centrifugation of step d is performed at 500g to 30,000g, but does not include ultracentrifugation of the liquid fraction obtained in step d. Claim 2 A process for producing a membrane-penetrating protein according to claim 1, wherein the aqueous medium containing the host organism of step a is filtered through a microfiltration membrane having a pore diameter of 0.5 micrometers or less before the release of the membrane-penetrating protein from the host organism according to step b. Claim 3 delete Claim 4 A process for producing a membrane-penetrating protein according to claim 1, wherein the host organism is separated by centrifugation of the aqueous medium containing the host organism after the aqueous medium containing the host organism is filtered through a microfiltration membrane. Claim 5 delete Claim 6 A process for manufacturing a membrane-penetrating protein according to claim 1, wherein the separated host organism is washed with isotonic saline to dissolve contaminating salts, and then centrifuged to separate the washed host organism. Claim 7 In claim 1, the dilution buffer is added prior to step b, in the process for manufacturing a membrane-penetrating protein. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 A process for manufacturing a membrane-penetrating protein, wherein, in any one of claims 1, 2, 4, 6, or 7, the release of the membrane-penetrating protein from the host organism in step b. is performed by a homogenizer. Claim 12 A process for producing a membrane-penetrating protein according to claim 1, wherein a cationic coagulant is added to the lysed cell fragment obtained in step bi) to form a suspension. Claim 13 In paragraph 12, the above cationic coagulant is a polyamine compound, in the process of manufacturing a membrane-penetrating protein. Claim 14 A process for manufacturing a membrane-penetrating protein according to claim 12, wherein the cationic coagulant reacts with the cell portion of the suspension during stirring to form a floc of the lysed cell fragment comprising the membrane-penetrating protein and the solid fraction of step b ii), and the solid fraction of step b ii) is formed by centrifuging the floc of the suspension. Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 A process for manufacturing a membrane-penetrating protein according to claim 1, wherein the detergent added in step c is selected from the group consisting of lauryl dimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM), or a combination thereof. Claim 19 delete Claim 20 A process for manufacturing a membrane-penetrating protein, wherein the stationary phase capable of binding to or retaining the membrane-penetrating protein is present in a column, according to claim 1. Claim 21 A process for manufacturing a membrane-penetrating protein according to claim 1, wherein the chromatography is affinity chromatography, and the membrane-penetrating protein is bound to a first portion of an affinity pair and the stationary phase is bound to a second portion of an affinity pair. Claim 22 A process for manufacturing a membrane-penetrating protein, wherein, in claim 21, the first portion of the affinity pair is a histidine tag, and the histidine tag comprises eight or more histidine molecules. Claim 23 delete Claim 24 A process for manufacturing a transmembrane protein according to claim 1, wherein an elution buffer containing imidazole is used to elute the transmembrane protein. Claim 25 delete Claim 26 A process for manufacturing a membrane-penetrating protein according to claim 24, wherein the concentration of imidazole in the elution buffer is 400 mM or more.