Production process of a membrane protein

Description

1 / 27 PROCESS FOR PRODUCING A MEMBRANE PROTEIN Divided from BR 11 2020 020379 3, deposited on 05.04.2019 TECHNICAL FIELD

[001] The present invention relates to a process for producing a membrane protein. The process according to the invention is suitable for industrial application where a large quantity of membrane proteins must be produced. Thus, the process aims at applying unit operations suitable for at least a pilot plant production. Notably, the present invention aims to avoid an ultracentrifugation apparatus. BACKGROUND

[002] 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 trafficking, viral fusion, neural synaptic activities, and the transport of ions and metabolites. Membrane proteins are embedded in the lipid bilayer of the cell membrane and are comprised of hydrophobic and hydrophilic portions.

[003] Recently, membrane proteins have been successfully integrated into a thin-film composite layer grafted onto a porous structural support layer. Furthermore, membrane proteins represent important pharmaceutical targets and interesting subjects of study with regard to cell biology and protein biochemistry. There is, therefore, a need for pilot-scale or large-scale production of membrane proteins.

[004] WO2017137361 (A1) reveals self-assembled nanostructures formed Petition 870260039804, dated 04 / 29 / 2026, page 14 / 49 2 / 27 between transmembrane proteins, such as aquaporin water channels (AQPs) and polyalkyleneimines (PAIs). The self-assembled nanostructures are subsequently incorporated into thin-film composite (TFC) membranes grafted onto a porous support membrane. The porous support membrane can be a hollow fiber, a flat sheet, a spirally wound membrane, etc., for reverse osmosis or direct osmosis.

[005] WO2013 / 043118 discloses thin-film composite (TFC) membranes in which aquaporin water channels (AQPs) are incorporated into the active layer of the membrane. Furthermore, it discloses a method for producing thin-film composite membranes and their uses in filtration processes such as nanofiltration and osmotic filtration processes. The TFC membranes comprise lipid-AQP / AQP-copolymer vesicles that are incorporated into the active layer of the TFC. WO2010 / 146365 describes the preparation of TFC-aquaporin-Z (AqpZ) filtration membranes that use an amphiphilic triblock copolymer as a vesicle-forming substance to incorporate immobilized AQPs.

[006] WO2014 / 108827 discloses a hollow fiber (HF) module having fibers modified with a thin film composite (TFC) layer comprising aquaporin water channels wherein the aquaporin water channels are incorporated into vesicles prior to incorporation into the TFC layer.

[007] Industrial methods for isolating large quantities of membrane proteins from cells are generally not available to those skilled in the art. In the state of the art, the focus has been on producing relatively small quantities of membrane proteins for research purposes. Therefore, a relatively complicated and laborious production method has been tolerated. However, with the recent higher demands for membrane proteins in industrial membranes for reverse osmosis and direct osmosis, Petition 870260039804, dated 04 / 29 / 2026, page 15 / 49 3 / 27 It became clear that a more efficient production method was needed. The objective of the present invention is to provide a process for the production of membrane proteins, where the process can produce relatively large quantities of membrane proteins in an efficient manner without compromising the quality of the final product. SUMMARY OF THE INVENTION

[008] The present invention relates to a process for producing a membrane protein, comprising the steps of: a. To express a membrane protein in a host organism present in an aqueous medium, b. To release the membrane protein from the host organism, c. Add a detergent solution to solubilize the membrane protein. d. Recover a liquid fraction of the solubilized membrane protein as the supernatant by centrifugation. e. Subject the liquid fraction to chromatography to bind or retain the membrane protein in a stationary phase, and f. Elute the stationary phase with an elution buffer to produce the membrane protein, where centrifugation in step d is performed at 500 g to 30,000 g.

[009] The present process has the advantage of being able to handle a large quantity of aqueous medium containing the host organism. Thus, the process is capable of handling quantities of 50 L or more, such as 100 L or more, by applying unit operations suitable for pilot plant or large-scale production. Furthermore, the process is scalable and can be easily adapted to large quantities of aqueous medium containing the host organism. Petition 870260039804, dated 04 / 29 / 2026, page 16 / 49 4 / 27

[010] Surprisingly, the inventors discovered that membrane proteins expressed by the host organism become solubilized to a greater extent than other proteins when a detergent solution is added. It is estimated that at least about 60% of the proteins in the vesicles formed by the detergent solution are the membrane proteins of interest. Furthermore, the detergent-formed vesicles were observed to be larger than normal, about 0.5 µm, suggesting that the membrane protein forms a more stable superform with the detergent than the phospholipids used by nature.

[011] The aqueous medium containing the host organism can be used directly in the process. However, to obtain a cleaner product and to avoid handling excessive amounts of aqueous medium, the aqueous medium comprising the host organism from step a is usually filtered before the release of the membrane protein according to step b.

[012] Generally, the aqueous medium comprising the host organism is concentrated by filtration through a filter having pores small enough to allow the passage of cellular debris and the medium, while the cells are retained. In a suitable embodiment, the aqueous medium comprising the host organism from 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.

[013] After the filtration step, properly performed by microfiltration, the host organism can be isolated from the rest of the aqueous medium. Although several separation processes are possible, it is generally preferable that the host organism be isolated after centrifugation filtration of the aqueous medium comprising the host organism. The centrifugation process is generally carried out with a g-force that, within Petition 870260039804, dated 04 / 29 / 2026, page 17 / 49 After 5 / 27 of a suitable period of time, a pellet is formed. Therefore, centrifugation is generally carried out at 500 g or more, such as 1000 g or more, and preferably 2000 g or more. The pellet density should not be too high to avoid difficulties in subsequent steps. Therefore, centrifugation is generally not carried out above 30,000 g, such as not above 20,000 g, appropriately not above 15,000 g, and preferably not above 8,000 g.

[014] The cells are harvested as pellets and the supernatant can be discarded. It is preferable that the isolated host organism be washed with an isotonic saline solution to dissolve contaminating salts and subsequently centrifuged as described above to isolate the washed host organism. The used washing solution that appears in the supernatant can be discarded.

[015] The pellet containing the washed cells can be stored by freezing at -20 °C or used directly in the next step. Suitablely, a dilution buffer is added before step b. The dilution buffer may contain protease inhibitors to prevent membrane protein degradation, pH regulators such as TRIS and phosphate to maintain a pH value within a desired range, and / or ion scavengers such as EDTA.

[016] Membrane protein can be released from the host organism in several ways, preferably by chemical or mechanical lysis of the cells. When chemical lysis of the cells is properly performed, an aqueous lysis solution is added to release the membrane protein from the host organism. In a preferred aspect of the invention, the aqueous lysis buffer is a detergent solution, which simultaneously solubilizes the membrane protein. Petition 870260039804, dated 04 / 29 / 2026, page 18 / 49 6 / 27

[017] For cell lysis and membrane protein solubilization to occur, host cells can generally be subjected to the action of detergent during agitation. Host cells can generally react with detergent for at least one hour, as well as for 2 hours, and preferably for at least 6 hours.

[018] When mechanical cell lysis is used, the release of the host cell membrane protein is usually carried out by a homogenizer. It is suitable to use a homogenizer of the same type used in the dairy industry for milk homogenization. A suitable example includes the Stansted 7575 homogenizer. After treatment in the homogenizer, the cells rupture, releasing the membrane protein.

[019] After the release of the membrane protein, a cationic flocculant can be added. The cationic flocculant is believed to interact with, inter alia, negatively charged cellular debris, forming flocs. In a preferred aspect of the invention, the cationic flocculant is a polyamine compound, such as Superfloc C581.

[020] The formation of flocs does not usually occur instantaneously. It is therefore preferable that the cationic flocculant be able to react with the cell parts of the resuspension during gentle agitation to form flocs. Usually, the cationic flocculant and cell debris are allowed to interact for 10 minutes to 2 hours while being agitated at room temperature.

[021] When chemical lysis of cells is used, the liquid fraction of the step is recovered as the supernatant from a centrifugation of the suspension containing the flocs. The membrane protein appears in the supernatant as the membrane protein is solubilized by the detergent solution.

[022] When mechanical cell lysis is used, the liquid fraction of the step is recovered from a resuspension of a solid fraction, in which the fraction Petition 870260039804, dated 04 / 29 / 2026, page 19 / 49 7 / 27 The solid fraction is resuspended in a detergent solution to solubilize the membrane protein. In one embodiment of the invention, the solid fraction is formed because the cell fragments containing the membrane protein interact with the flocculant and form flocs, which can be separated from the liquid by centrifugation. In another embodiment of the invention, it was surprisingly found that it was possible to harvest the membrane protein from the pellet when using mechanical lysis of the cells, without the use of a flocculant. The pellet obtained by centrifugation can be resuspended in a detergent solution to solubilize the membrane protein. In this step, the liquid fraction from step e is harvested as the supernatant by centrifugation.

[023] The centrifugation process is usually carried out with a g-force that, within a suitable period of time, forms a pellet. Therefore, centrifugation is usually carried out at 500 g or more, such as 1000 g or more, and preferably 2000 g or more. The pellet density should not be too high to avoid difficulties in subsequent steps. Therefore, centrifugation is usually not carried out above 30,000 g, such as not above 20,000 g, suitably not above 10,000 g and preferably not above 8,000 g. Applying a g-force below 30,000 g makes it possible to use a clarifier commonly used in dairies, such as spore removal centrifuges from GEA, Tetra Pak, Alfa Laval and SPX Flow Seital Separation Technology. The clarifiers useful in the present invention may also be referred to as Bactofuges by some manufacturers.Thus, the present invention can omit the application of ultracentrifuges, which are only capable of batch centrifugation and processing small quantities.

[024] The detergents used in the present invention include alkyl maltopyranosides, such as n-dodecyl-D-maltopyranoside (DDM), n-decyl-D-maltopyranoside (DM), or 5-cyclohexylpentyl β-D-maltoside. Petition 870260039804, dated 04 / 29 / 2026, p. 20 / 49 8 / 27 (Cymal-5); alkyl glycopyranosides, such as n-octyl-D-glucopyranoside (OG); amine oxides, such as n-lauryldimethylamine N-oxide (LDAO); phosphocholines, such as n-dodecylphosphocholine (FC-12), n-tetradecylphosphocholine (FC14), or n-hexadecylphosphocholine (FC-16); or polyoxyethylene glycols. In a suitable embodiment of the invention, the detergent is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octylglucoside (OG), dodecyl maltoside (DDM), or combinations thereof. LDAO is a preferred detergent because large, stable vesicles are produced, suggesting that the detergent is capable of displacing naturally occurring phospholipids.

[025] The liquid fraction is subjected to a chromatography process in which the membrane protein is bound to or retained by means of a stationary phase. The type of chromatography can be selected as affinity chromatography, ion exchange chromatography, size exclusion chromatography, displacement chromatography, liquid chromatography, high-performance liquid chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, etc. Usually, the chromatography process is preparative chromatography, as opposed to analytical chromatography, to achieve a purification of the membrane protein.

[026] In a currently preferred embodiment, the chromatography method is selected as affinity chromatography, according to which a first part of an affinity pair is associated with the membrane protein and the second part of the affinity pair is associated with the stationary phase. Examples of a stationary phase include beads and column material. In a preferred embodiment of the invention, the stationary phase associated with a second part of the affinity pair is present in a column. Usually, the parts of the affinity pairs are associated with the bonds Petition 870260039804, dated 04 / 29 / 2026, page 21 / 49 9 / 27 covalent bonds to the membrane protein or to the stationary phase. However, other types of association are also possible, such as hybridization, affinity binding through antibody-antigen interaction, etc.

[027] A number of affinity pairs applicable in the present invention are known to those skilled in the art and include a biotin-streptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an aptamer affinity pair, a capture protein pair, an IgG FC receptor pair, a metal-chelating lipid pair, a metal-chelating lipid (by HIS) labeled protein pair, or a combination thereof. In a currently preferred embodiment, the affinity pair is a metal-chelating lipid (by HIS) labeled protein pair.

[028] The metal is usually immobilized on the column material in a technology referred to as immobilized metal affinity chromatography (IMAC). The metal is usually selected as Cu(II) or Ni(II), preferably Ni(II). Using a Ni-NTA resin, His-labeled proteins can be purified automatically or manually. A suitable stationary phase is “Capto Chelating” from GE Healthcare or alternatively the HisTrap Gel filtration material (Ni Sepharose 6 Fast Flow) from GE Healthcare.

[029] The first part of the affinity pair, being a histidine marker, is usually linked to the C-terminal of the membrane protein. Whereas the histidine marker usually comprises 6 consecutive histidine amino acids, it is preferred in the present invention that the histidine marker comprises 8 or more histidine molecules. The high number of histidine amino acids in the histidine marker makes it possible to effectively separate specific and non-specific binding proteins.

[030] After the addition of the liquid fraction containing the membrane protein Petition 870260039804, dated 04 / 29 / 2026, page 22 / 49 10 / 27 for the column, the liquid can penetrate the resin, by gravity or pressure. Appropriately, the elution buffer comprises imidazole. Imidazole has the ability to interact with the His-label binding to the metal ion immobilized on the resin. At a certain concentration of imidazole, the His-labeled membrane protein will be released.

[031] To separate the non-specific binding protein from the membrane protein of interest, the column, generally before elution with the elution buffer, is washed with a wash buffer comprising 40% or less of the imidazole concentration in the elution buffer. The imidazole concentration in the elution buffer is generally in the range of 200mM to 2000mM imidazole. In preferred embodiments of the invention, the imidazole concentration in the elution buffer is 400 mM or more. To obtain a more effective elution of His-labeled membrane protein, the imidazole concentration in the buffers is generally 600mM or 800mM or more.

[032] For most applications, the His-labeled tag generally has a negligible influence on the structure, function, or immunogenicity of the protein. However, it may be desirable for certain applications to remove the His-labeled tag after it has served its function of binding the membrane molecule to the column. The His-labeled tag can be removed by introducing a cleavable linkage between the membrane protein and the His-labeled tag. Suitable cleavable linkages include pH-sensitive linkers, disulfide linkers, protease-sensitive linkers, and beta-glucuronide linkers.

[033] In their natural environment, membrane proteins span the entire lipid bimembrane, that is, from the interior of the cell to the extracellular space. Many transmembrane proteins function as entry points for specific substances, thus allowing the exchange of these substances between the interior of the cell and the extracellular fluid. A characteristic Petition 870260039804, dated 04 / 29 / 2026, page 23 / 49 11 / 27 A striking feature of transmembrane proteins is the presence of a hydrophobic area, which ensures the integration of the transmembrane protein into the membrane. In addition, the transmembrane protein has hydrophilic segments on both sides of the hollow fibrous area, where these hydrophilic segments are directed towards the interior of the cell and the extracellular fluid, respectively.

[034] While it is believed that any membrane protein can be produced according to the present invention, it is generally desirable to use the process of producing membrane proteins that transport ions (ion channels) and water (aquaporin water channels). Ion channels include chloride channels and metal ion transporters. Certain chloride channels, in addition to the chloride ion, also conduct HCO3-, I-, SCN-, and NO3-. Metal ion transporters include magnesium transporters, potassium ion channels, sodium ion channels, calcium channels, proton channels, etc. In a particular embodiment of the invention, the membrane protein is an outer membrane protein (OmpA).

[035] In a preferred embodiment of the invention, the membrane protein is an aquaporin water channel. Aquaporin water channels facilitate the transport of water into and out of a cell. In an industrial membrane, aquaporin water channels ensure the flow of water by osmosis, while other components in the solution are rejected. The membrane protein, as an aquaporin water channel, can emanate from various sources, including prokaryotic and eukaryotic organisms. A prokaryotic source for aquaporin includes E. coli, Kyrpidia spormannii, Methanothermobacter sp., Novibacillus thermophilus, Saccharomyces cerevisiae, and Halomonas sp. A eukaryotic source for aquaporin includes Oryza sativa japonica (Japanese rice), Eucalyptus grandis, Solanum tuberosum (Potato). Petition 870260039804, dated 04 / 29 / 2026, p. 24 / 49 12 / 27 Danish), and Milnesium tardigradum (water bear).

[036] The nucleic acid sequence of the membrane protein from the source organism is generally codon-optimized using the Geneart (a subsidiary of Thermo Fisher Scientific) service to enhance expression in the host organism. The resulting gene is appropriately synthesized with the addition of C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks. The synthetic gene fragment is restriction-digested and ligated into a vector fragment. The resulting ligation mixture is preferentially transformed into an organism, such as Escherichia coli DH10B. Antibiotic-resistant transformants are appropriately selected in a medium containing the antibiotic. Transformants are confirmed by sequencing genetic constructs. The isolated vector DNA is subsequently transferred to the production host.The production host can be selected from a number of suitable eukaryotic or prokaryotic organisms, such as Escherichia coli and Saccharomyces cerevisiae.

[037] The host organism is usually manipulated to express a native membrane protein in a higher than normal amount or to express the non-native membrane protein. One way to express the membrane protein may be by transforming the host organism with a vector comprising DNA encoding the membrane protein, as discussed above. Another possibility for achieving overexpression of the membrane protein could be by upregulating the expression of a native membrane protein, for example, by attenuating a repressor or inserting a suitable promoter region. An additional possibility for achieving expression of a non-native protein would be to transfect a host organism with a virus or bacteriophage containing an acid Petition 870260039804, dated 04 / 29 / 2026, p. 25 / 49 13 / 27 nucleic acid encoding membrane protein. EXAMPLES Example 1

[038] An E. coli BL21 strain is prepared comprising a vector producing aquaporin proteins linked to a C-terminal His-linked marker. The His-linked marker contains ten consecutive histidine molecules linked to the primary sequence of the aquaporin membrane protein.

[039] The E. coli strain is fed in a standard medium to obtain a total fermentation broth of 150 µL. E. coli cells were harvested by filtering the fermentation broth through a microfiltration membrane with a pore diameter of 0.05 µm. The filtrate containing the E. coli cells is reduced to approximately 50 µL and subsequently centrifuged at 5300g for 20 minutes. Thus, the E. coli cells are concentrated upwards by microfiltration and the remaining medium is subsequently removed as the supernatant by centrifugation.

[040] The pellets obtained by centrifugation are collected and added at a concentration of 1:1 by volume with 0.9% sodium chloride to wash cells and dissolve contaminating salts. Subsequently, the washing solution is removed in a centrifuge operating at 5300g for 20 minutes. The supernatant is discarded and the washed cells are collected as pellets. The pellets can be stored by freezing at -20 °C or used directly in the next step.

[041] The pellets comprising the E. coli cells were solubilized in approximately 47 L of TRIS buffer used as a binding buffer. After stirring for about one hour, 6.4 L of detergent (5% LDAO) was added for solubilization to a final concentration of 0.6%. The mixture was incubated overnight at room temperature with gentle stirring. Cell lysis Petition 870260039804, dated 04 / 29 / 2026, page 26 / 49 14 / 27 occurred by resuspending the cells in a buffer containing a detergent. Through cell lysis, membrane proteins are released from the inner cell membranes and solubilized by the detergent.

[042] To remove negatively charged cellular material, a polyamine (Superfloc C581) was added in a volume of 427 mL. The mixture was incubated for 30 minutes with agitation at room temperature to coagulate negatively charged molecules such as cellular debris, DNA, RNA, and some proteins. The membrane protein solubilized by the detergent remains in the aqueous phase. The mixture was centrifuged at a maximum speed of 5300 g for 15 minutes. The pellet containing the solubilized cellular debris, DNA, RNA, and proteins is discarded, and the supernatant is collected. The supernatant is 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.

[043] A column containing the affinity resin “Capto Chelating” from GE Healthcare is provided. The resin is loaded with Ni2+ which binds to the His10 marker. The column was loaded with the diluted supernatant and washed with 10 column volumes of 200 mM imidazole wash buffer to remove non-specific binding components. Subsequently, 2.5 column volumes of 1000 mM aqueous imidazole elution buffer were used to release the membrane protein from the column. The protein eluted from the column was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which showed only a single band indicating a purity above 80%. Example 2

[044] An E. coli BL21 strain is prepared comprising a vector producing aquaporin proteins bound to a C-terminus His marker. The Petition 870260039804, dated 04 / 29 / 2026, page 27 / 49 The 15 / 27 His marker contains ten consecutive histidine molecules bound to the primary sequence of the aquaporin membrane protein.

[045] The E. coli strain is fed in a standard medium to obtain a total fermentation broth of 250 μL. E. coli cells were harvested by filtering the fermentation broth through a PES flat sheet membrane with a pore diameter of 0.05 μm. The filtrate containing the E. coli cells is reduced to approximately 50 μL and subsequently centrifuged at 5300g in a 16 μL Sorvall centrifuge for 20 minutes. Thus, the E. coli cells are concentrated upwards by microfiltration and the remaining medium is subsequently removed as the supernatant by centrifugation. The pellets can be stored by freezing at -20 °C or used directly in the next step.

[046] Pellets containing E. coli cells were resuspended in approximately 50 L of buffer (aqueous solution of the protease inhibitor PMSF and EDTA) and homogenized at 1000 bar (1.0 x 10⁸ Pa) in a Stansted nm-GEN 7575 homogenizer. To isolate the cellular material of interest, a polyamine (Superfloc C581) was added at a concentration of 12 mL / L. The temperature was maintained around 10-15 °C. The mixture was incubated for 30 minutes with stirring at room temperature. The mixture was centrifuged at a maximum speed of 5,300 g for 30 minutes. The pellet containing the membrane protein was discarded, and the supernatant was removed.

[047] The pellet was resuspended in a 0.9% sodium chloride solution to obtain a total protein concentration of approximately 50 mg / mL. Solubilization of the membrane protein was performed by adding 28 L of TRIS binding buffer and 4.5 liters of 5% LDAO to 5 L of the resuspended pellet material. At room temperature and with gentle stirring, the mixture was left to incubate for 2 to 24 hours. Petition 870260039804, dated 04 / 29 / 2026, page 28 / 49 16 / 27

[048] After the solubilization process, the mixture was centrifuged in 2 L containers at 5300g for 90 minutes. The supernatant was recovered and the LDAO concentration was adjusted to 0.2% by adding dilution buffer.

[049] A column containing the affinity resin “Capto Chelating” from GE Healthcare is provided. The resin is loaded with Ni2+ which binds to the His10 marker. The column was equilibrated by loading the binding buffer with 0.2% LDAO. Subsequently, the column was loaded with the diluted supernatant and washed with 10 column volumes of 200 mM imidazole wash buffer to remove non-specific binding components. Subsequently, 2.5 column volumes of 1000 mM aqueous imidazole elution buffer are used to release the membrane protein from the column. The protein eluted from the column was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which showed only a single band indicating a purity above 95%. Example 3

[050] An E. coli BL21 strain is prepared comprising a vector producing aquaporin proteins bound to a C-terminus His marker. The His marker contains ten consecutive histidine molecules bound to the primary sequence of the aquaporin membrane protein.

[051] The E. coli strain is fed in a standard medium to obtain a total fermentation broth of 250 L. The fermentation batch had an OD600 13 when collected, and was induced for 42.5 hours. The material was homogenized twice at 100 MPa in a Stansted nm-GEN 7575 homogenizer. 10 mL were taken from the lysed material and added to a 15 mL falcon tube.

[052] Centrifugation was then performed for 1 hour with 5300 geo sediment was separated from the supernatant. The sediment was then resuspended in 10 mL of 0.9% NaCl. The BCA assay was used to determine the Petition 870260039804, dated 04 / 29 / 2026, page 29 / 49 17 / 27 concentration (mg / mL of total protein) of the resuspended pellets and the supernatants separated after the first rotation.

[053] The resuspended pellets and separated supernatants were finally solubilized (1 mL) for 2 hours in 0.6% LDAO (120 pL of 5% Carbosynth LDAO stock was used for 1 mL of material) and centrifuged again at 5300 G for 15 minutes. Once again, the supernatants were separated from the pellets. The pellets were resuspended (to 1 mL again) in w / w LDAO binding buffer.

[054] All samples were analyzed on SDS gel, which showed that approximately 60% of the proteins in the supernatant were aquaporin membrane protein. Example 4

[055] After purification of Aquaporin Z in example 2, the size distribution of the protein solubilized in LDAO was measured.

[056] The comparison was performed between the same elution buffer, with or without protein, to evaluate whether the particle size distribution was influenced by the buffer components (including detergent) or by the membrane protein.

[057] Aquaporin Z protein was eluted from the IMAC column using elution buffer with 1000 mM imidazole and 0.2% w / v LDAO. The purified protein was measured for its protein concentration by amino acid analysis. 6.44 mg / mL of clear protein solubilized in elution buffer were loaded into 3 separate cuvettes (Sarstedt, 4 mL, PMMA, ca. no. 67,755, Nümbrecht, Germany) and analyzed for size distribution using the Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern UK) with Malvern Zetasizer v.7.02 software. The results presented in Table 1 are the averages of the samples performed in triplicate. Petition 870260039804, dated 04 / 29 / 2026, pp. 30 / 49 18 / 27 Table 1: Sample Concentration of AqpZ (mg / mL) Size, diameter (d.nm) Population intensity (%) Pop. 1 Pop. 2 Pop. 3 Purified AqpZ in elution buffer 6.44 315.6 ± 108 ± 17.1 ± 116.8 nm 32.3 nm 2.5 nm (avg.) 45.9% 35.5% 14.9% Elution buffer 0 0.85 ± 8.6 ± 1.2 103 ± (avg.) 0.16 nm nm 5.5 nm 56.8% 41.1% 2.1%

[058] The elution buffer sample (without protein) contains a population distribution with 97.9% of particles having a size of 8.6 nm or smaller, indicating that there were no large detergent micelles in the solution that could be retained by microfiltration. 81.4% of the particles in the sample with protein showed a size of 108 nm or larger, thus indicating that the presence of protein and detergent together forms large soluble particles that are retained during microfiltration. Interestingly, the experiment showed that large, stable particles composed of LDAO micelles and membrane protein are produced. Example 5 Expression of aquaporin labeled from Oryza sativa japonica (Japanese rice) in Escherichia coli and its purification using IMAC.

[059] The aquaporin-encoding gene from Oryza sativa japonica (UNIPROT: A3C132) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized with the addition of ten C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with NPetition flanks 870260039804, 29 / 04 / 2026, p. 31 / 49 19 / 27 terminals and C-terminals, respectively (Gene ID: aquaporin_Oryza_sativa_Japonica). The synthetic gene fragment was digested with NdeI / XhoI restriction enzymes and ligated to the digested NdeI / XhoI fragment and purified pUP1909 vector. The resulting ligation mixture was transformed into Escherichia coli DH10B, and kanamycin-resistant transformants were selected on LB agar plates with kanamycin. Transformants were confirmed by sequencing genetic constructs. The isolated vector DNA was subsequently transferred to the production host, Escherichia coli BL21.

[060] In order to express aquaporin heterologously in E. coli, the production host was grown in minimal medium consisting of 30 g / L glycerol, 6 g / L (NH4)2HPO4, 3 g / L KH2PO4, 5 g / L NaCl, 0.25 g / L MgSO4^7H?O, 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 CoCb6H2O, 1.5 g / L MnCb^HzO, 0.4 g / L CuCb-ZHzO, 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, 0.25 g / L MgSO4^7H2O was added.

[061] E. coli was cultivated in 3L Applikon Bioreactors with ez-Control in a batch fermentation process. Protein production was induced by the addition of IPTG added at a final concentration of 0.5 mM at an optical density (OD 600 nm) of approximately 30. The culture was induced for approximately 24 hours and bacterial cells were collected by centrifugation at 5300 g for 20 min.

[062] The pellets containing E. coli cells were resuspended in buffer (aqueous solution of the protease inhibitor PMSF and EDTA) and homogenized at 1000 bar (1.0 x 108 Pa) in a Stansted nmGEN 7575 homogenizer. The temperature was maintained around 10-15 °C. The mixture was Petition 870260039804, dated 04 / 29 / 2026, pp. 32 / 49 20 / 27 centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contains the membrane protein and the supernatant is discarded.

[063] The pellet was resuspended in a 0.9% sodium chloride solution to obtain a total protein concentration of approximately 50 mg / mL. Solubilization of the membrane protein was performed by adding 28 L of TRIS binding buffer and 4.5 liters of 5% LDAO to 5 L of the resuspended pellet material. At room temperature and with gentle stirring, the mixture was left to incubate for 2 to 24 hours.

[064] After the solubilization process, the mixture was centrifuged in 2 L containers at 5300g for 90 minutes. The supernatant was recovered and the LDAO concentration was adjusted to 0.2% by adding dilution buffer.

[065] After solubilization and clarification, the protein was captured using IMAC and eluted in elution buffer containing 1000 nM imidazole and 0.2% w / v LDAO. Elution fractions were analyzed by SDS Page and revealed only a single main band that migrated at 27 kDa, which corresponds to the size of the aquaporin from Japanese rice. Furthermore, the result was confirmed by comparison to a negative control purification from E. coli transformed with an empty vector. The negative control resulted in no purified protein. Western blot analysis with antibodies (TaKaRa Bio) specific for the histidine marker resulted, as expected, in a clear signal from the purified protein and no signal from the negative control, confirming the origin of the purified protein as a histidine-labeled membrane protein. Example 6 Expression of labeled aquaporin from Eucalyptus grandis in Escherichia coli and its purification using IMAC.

[066] The gene encoding aquaporin from Eucalyptus grandis Petition 870260039804, dated 04 / 29 / 2026, pp. 33 / 49 21 / 27 (UNIPROT: A0A059C9Z4) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance the expression of E. coli. The resulting gene was synthesized with the addition of ten codons encoding C-terminal histidine, along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_Eucalyptus_grandis). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Example 7 Expression of labeled aquaporin from Solanum tuberosum (Danish potato) in Escherichia coli and its purification using IMAC.

[067] The aquaporin-encoding gene from Solanum tuberosum (UNIPROT: Q38HT6) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized by adding ten C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_Solanum_tuberosum). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Example 8 Expression of aquaporin labeled from Milnesium tardigradum (water bear) in Escherichia coli and purified using IMAC.

[068] The gene encoding aquaporin from Milnesium tardigradum (UNIPROT: G5CTG2) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized with the addition of ten codons encoding Petition 870260039804, dated 04 / 29 / 2026, pp. 34 / 49 22 / 27 C-terminal histidine, along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_Milnesium_tardigradum). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Example 9 Expression of aquaporin labeled from Halomonas sp. in Escherichia coli and its purification using IMAC.

[069] The aquaporin-encoding gene from Halomonas sp. (UNIPROT: A0A2N0G6U6) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized by adding ten C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_Halomonas_sp). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Example 10 Expression of labeled aquaporin from Kyrpidia spormannii in Escherichia coli and its purification using IMAC.

[070] The aquaporin-encoding gene from Kyrpidia sp. (UNIPROT: A0A2K8N5Z5) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized by adding ten C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_Kyrpidia_spormannii). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Petition 870260039804, dated 04 / 29 / 2026, pp. 35 / 49 23 / 27 Example 11 Expression of aquaporin labeled from Methanothermobacter sp. in Escherichia coli and its purification using IMAC.

[071] The aquaporin-encoding gene from Methanothermobacter sp. (UNIPROT: A0A223ZCQ2) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized by adding ten C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_ Methanothermobacter sp. The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Example 12 Expression of labeled aquaporin from Novibacillus thermophilus in Escherichia coli and its purification using IMAC.

[072] The aquaporin-encoding gene from Novibacillus thermophilus (UNIPROT: A0A1U9K5R2) was the codon optimized using the Geneart service (a subsidiary of Thermo Fisher Scientific) to enhance E. coli expression. The resulting gene was synthesized by adding ten C-terminal histidine-encoding codons along NdeI / XhoI restriction sites with N-terminal and C-terminal flanks, respectively (Gene ID: aquaporin_ (Novibacillus thermophilus). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5. Example 13 Expression of outer membrane protein A (OmpA) from Escherichia coli in E. coli Petition 870260039804, dated 04 / 29 / 2026, pp. 36 / 49 24 / 27

[073] The expression and purification of OmpA (UNIPROT: P0A910) in E. coli was 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 approximately 30-50% by SDS-PAGE, thus clearly attesting that OmpA was successfully isolated in the membrane fraction following the standard purification process. An additional size exclusion chromatography (SEC) step could have further purified OmpA. Example 14

[074] Construction of a Saccharomyces cerevisiae strain expressing aquaporin-Z (AqpZ) from Escherichia coli fused to an N-terminal histidine-labeled yEGFP and separated by a Tobacco Etch Virus (TEV) protease cleavage site.

[075] AqpZ (UNIPROT ID: P60844) from E. coli was the codon optimized for expression in S. cerevisiae using the Geneart service for enhanced expression in S. cerevisiae. For expression in S. cerevisiae, AqpZ was fused with N-terminal yeast green fluorescent protein (yEGFP) (Brendan P. Cormack et al, Microbiology (1997), 143, 303-311) to allow visual detection and quantification of membrane protein expression. In addition, eight histidines (His8) were added to the N-terminal of yEGFP as an IMAC purification marker. His8-yEGFP and AqpZ were genetically separated by means of a TEV protease cleavage site incorporated by PCR primers during construction.

[076] The rapid and efficient construction of the plasmid encoding the His8-yEGFP-TEV-AqpZ fusion was performed by homologous in vivo recombination of overlapping regions incorporated by the primers in S. cerevisiae between a His8-yEGFP-TEV PCR fragment, a TEV-AqpZ PCR fragment, and the Petition 870260039804, dated 04 / 29 / 2026, pp. 37 / 49 25 / 27 linearized expression plasmid derived from the digestion of SalI, HindlII and BamHI of the pEMBLyex4 plasmid, as described in (Scientific Reports 7: 16899). The TEV cleavage site allows for the subsequent removal of the His8-yEGFP protein by the TEV protease.

[077] The selection of S. cerevisiae transformants was performed on minimal medium plates deficient in 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 identical to those listed in Example 15. Example 15

[078] Expression of His8-yEGFP-TEV-AqpZ with Saccharomyces cerevisiae.

[079] A single colony of transformed yeast cells was selectively propagated to saturation in approximately 5 mL of minimal glucose medium supplemented with 60 mg / L leucine and 30 mg / L lysine. 200 μL of this culture was subsequently propagated in 5 mL of minimal glucose medium supplemented with 30 mg / L lysine for selection by high plasmid copy numbers. Frozen stocks of cells with high plasmid copy numbers were prepared.

[080] 200 pL thawed from a frozen stock were added to 10 mL of minimal medium supplemented with lysine and grown to 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 minimal medium with an initial concentration of 20 g / L of glucose as a carbon source and 30 g / L of glycerol supplemented with extra amino acids. The culture was grown in a 3L Applikon® bioreactor equipped with ezControl connected to Lucullus® software running on a PC (Applikon, Petition 870260039804, dated 04 / 29 / 2026, pp. 38 / 49 26 / 27 Netherlands, and SecureCell, Switzerland).

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

[082] 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. The ASD-10 consists of 50 g / L (NH4)2SO4, 8.75 g / L KH2PO4, 1.25 g / L K2HPO4, 5 g / L MgCOrTHzO, 1 g / L NaCl, 5 mg / L H3BO3, 1 mg / L KI, 4 mg / L M mg / L ZnSO4-7H2O, 0.4 mg / L CuSO4^5H2O, 2 mg / L FeCb and 2 mg / L Na2MoO4^2H2O. The V-200 consisted of 4 mg / L of Biotin, 400 mg / L of D-pantothenic acid, 0.4 mg / L of folic acid, 2000 mg / L of myo-inositol, 80 mg / L of niacin, 40 mg / L of p-amino acid, 80 mg / L of pyridine, pyridine mg / L 40 mg / L riboflavin and 80 mg / L thiamine.When initiated, the medium containing the additional amino acids consisted of 600 mg / L of alanine, 600 mg / L of arginine, 600 mg / L of cysteine, 3000 mg / L of glutamic acid, 2000 mg / L of lysine, 600 mg / L of methionine, 1500 mg / L of phenylalanine, 600 mg / L of proline, 10000 mg / L of serine, 900 mg / L of tyrosine, 4500 mg / L of valine, 2000 mg / L of aspartic acid, 4000 mg / L of threonine, 600 mg / L of histidine, and 600 mg / L of tryptophan. Example 16 Cloning of His8-yEGFP-TEV-AQP5 from Milnesium tardigradum in Petition 870260039804, dated 04 / 29 / 2026, pp. 39 / 49 27 / 27 a Bioreactor with Saccharomyces cerevisiae

[083] The His8-yEGFP-TEV-AQP5 construct was prepared following the procedure outlined in Example 14. The AQP5 protein from M. tardigradum (UNIPROT: G5CTG2) was the codon optimized for expression in E. coli despite the need for expression in yeast. Example 17 Purification of His8-yEGFP-TEV-AqpZ and His8-yEGFP-TEV-AQP5 from S. cerevisiae

[084] The purification of heterologously expressed membrane protein from S. cerevisiae was performed as described in Example 2, except that the applied buffer volumes were scaled down to match the reduced culture volumes and that lysis was performed at 1800 bar (1.8 x 108 Pa). Protein production and purity were successfully confirmed by two-protein fusion with SDS page and Western Blot and no contaminating proteins could be detected. Petition 870260039804, dated 04 / 29 / 2026, pp. 40 / 49

Claims

1 / 4 CLAIMS 1. A process for producing a membrane protein, characterized in that it comprises the steps of: a. expressing a membrane protein in a host organism present in an aqueous medium, b. releasing the membrane protein from the host organism, c. adding a detergent solution to solubilize the membrane protein, d. recovering a liquid fraction of the solubilized membrane protein as the supernatant by centrifugation, e. subjecting the liquid fraction to chromatography to bind or retain the membrane protein in a stationary phase, and f. eluting the stationary phase with an elution buffer to produce the membrane protein, wherein the centrifugation in step d is carried out at 500 g to 30,000 g.

2. Process according to claim 1, characterized in that the aqueous medium comprising the host organism of step a is filtered before the release of the host organism according to step b.

3. Process according to claim 2, characterized in that the aqueous medium comprising the host organism of step a is filtered through a microfiltration membrane having a pore diameter of 0.5 micrometers or less.

4. Process according to claim 3, characterized in that the host organism is isolated, optionally after filtration, by centrifugation of the aqueous medium comprising the host organism.

5. Process according to any one of claims 1 to 4, characterized in that the centrifugation is carried out at 500 g 30,000 g, Petition 870260039804, dated 29 / 04 / 2026, page 41 / 49 2 / 4 preferably at 1,000 g 10,000 g.

6. A process according to any one of claims 1 to 5, characterized in that the isolated host organism is washed with an isotonic saline solution to dissolve contaminating salts and subsequently centrifuged to isolate the washed host organism.

7. Process according to any one of claims 1 to 6, characterized in that a dilution buffer is added before step b.

8. A process according to any one of claims 1 to 7, characterized in that an aqueous lysis solution is added to release the membrane protein from the host organism.

9. Process according to claim 8, characterized in that the aqueous lysis buffer is a detergent solution that simultaneously solubilizes the membrane protein.

10. Process according to any one of claims 1 to 9, characterized in that the host cells can be subjected to the action of the detergent during agitation.

11. Process according to any one of claims 1 to 7, characterized in that the release of the membrane protein from the host cells is carried out by a homogenizer.

12. Process according to any one of claims 1 to 11, characterized in that a cationic flocculant is added to the released membrane protein to form a suspension.

13. Process according to claim 12, characterized in that the cationic flocculant is a polyamine compound.

14. Process according to claim 12 or 13, characterized in that the cationic flocculant can react with the cellular parts of the resuspension during gentle agitation to form flocs.

15. Process according to any one of claims 1 to 14, characterized in that the liquid fraction of step d is recovered as the supernatant from a centrifugation of the suspension containing the flocs.

16. Process according to any one of claims 1 to 7, characterized in that the liquid fraction of step d is recovered from a resuspension of a solid fraction, wherein the solid fraction is resuspended in a detergent solution to solubilize the membrane protein.

17. Process according to claim 16, characterized in that the liquid fraction of step d is collected as the supernatant by centrifugation.

18. Process according to any one of claims 1 to 17, characterized in that the detergent is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octylglucoside (OG), dodecyl maltoside (DDM) or combinations thereof.

19. Process according to any one of claims 1 to 18, characterized in that the centrifugation is carried out at 500 g to 30,000 g, preferably at 1,000 g to 10,000 g.

20. Process according to any one of claims 1 to 19, characterized in that the stationary phase capable of binding or retaining membrane protein is present in a column.

21. A process according to any one of claims 1 to 20, characterized in that the membrane protein is associated with a first part of an affinity pair and the stationary phase is associated with a second part of the affinity pair.

22. Process according to claim 21, characterized in that the first part of the affinity pair is a histidine marker. Petition 870260039804, dated 04 / 29 / 2026, pp. 43 / 49 4 / 4 23. Process according to claim 22, characterized in that the histidine marker comprises 8 or more histidine molecules.

24. Process according to any one of claims 1 to 23, characterized in that the elution buffer comprises imidazole.

25. Process according to claim 24, characterized in that the column, before elution with the elution buffer, is washed with a washing buffer comprising 40% or less of the imidazole concentration in the elution buffer.

26. Process according to claim 24 or 25, characterized in that the imidazole concentration in the elution buffer is 400 mM or more. Petition 870260039804, dated 04 / 29 / 2026, pp. 44 / 49