Method for obtaining materials from plant cell surfaces

Abstract

The present invention relates to a method for detaching expression material from the surface or apoplast of plant cells, wherein the plant cells are treated in a liquid medium using a rotor-stator, wherein the specific heat of the rotor-stator introduced by the rotation of the rotor is at most 3 kJ per kg of the liquid medium and per g / L of the dry mass of the plant cells, and the specific heat power introduced into the medium is at most 1.5 kJ per kg of the liquid medium per minute and per g / L of the dry mass of the plant cells.

Description

Field of the Invention

[0001] The present invention relates to the isolation of proteins from cells. Background of the Invention

[0003] Methods for isolating proteins from cells or cell complexes include osmotic lysis, enzymatic or chemical lysis, sonication, and mechanical digestion. For mechanical digestion, cells are typically pulverized using a homogenizer or blender.

[0004] Short treatment times can also be used in order to break up the cell complexes and keep the cells in suspension, whereby only partial lysis of the total cell mass occurs (Orellana-Escobedo et al., Plant Cell Rep. 2015, 34(3):425-33).

[0005] If isolating proteins from individual organelles or cellular compartments, the organelles are typically isolated prior to digestion and then the isolates are used for digestion.

[0006] Witzel et al., Plant Methods 2011, 7:48; Leary et al., J Vis Exp. 2014;(94): 52113; and Córdoba-Pedregosa et al., Plant Physiology 1996, 112(3):1119-1125 describe methods for extracting proteins from the apoplast of plant cells. The space outside the protoplast is called the apoplast. It consists of the cell wall and the intercellular space. The methods described in those publications include osmotic extraction with different osmotic solutions (e.g., salts) and centrifugation. However, a disadvantage of this method is that in this extraction, only material that can be obtained by osmosis can be extracted.

[0007] US 2015 / 0140644 A1 and AU 2017 202473 B2 describe methods for obtaining proteins from the apoplast using the steps of cell wall lysis, incubation, and extraction, wherein enzymatic or chemical modification of the proteins is possible.

[0008] It was an object of the present invention to provide improved possibilities for isolating or extracting apoplast material, in particular material secreted into the apoplast.

[0009] Summary of the Invention

[0010] The present invention relates to a method for detaching expression material from the surface or from the apoplast of plant cells, wherein the plant cells are treated in a liquid medium using a rotor-stator, wherein the specific heat of the rotor-stator introduced by the rotation of the rotor is at most 3 kJ per kg of liquid medium and per g / L of plant cell dry mass, and the specific heat power introduced into the medium is at most 1.5 kJ per kg of liquid medium per minute and per g / L of plant cell dry mass.

[0011] Likewise, in another aspect, the present invention relates to a method for detaching expression material from the surface or apoplast of one or more plant cells, wherein the one or more plant cells are treated in a liquid medium with a rotor-stator, wherein the heat introduced into the rotor-stator by the rotation of the rotor is a maximum of 30 kJ per kg of liquid medium and the thermal power introduced into the medium is a maximum of 1.5 kJ per kg of liquid medium and per minute.

[0012] The parameters of these two aspects can be combined, especially since only different reference values ​​are involved, both cases being consistent with the spirit of the invention.All detailed invention descriptions and preferred embodiments described herein relate to all aspects of the invention. Detailed Description of the Invention

[0014] The present invention relates to a gentle rotor-stator treatment of plants or plant cells that, in contrast to conventional homogenization, separates material from the apoplast of the cells or plants. In this case, the protoplasts should remain largely intact to prevent contamination of the expression material to be isolated with cellular components from within the protoplast cells. Therefore, according to the present invention, the intensity and duration of the rotor-stator treatment are limited. A rotor-stator treatment with low energy input (determined as specific heat or thermal power) allows satisfactory separation of the desired expression material. Excellent results were observed when the rotor-stator specific heat introduced by the rotor rotation was a maximum of 3 kJ per kg of liquid medium and per g / L of plant cell dry mass, and when the specific heat power introduced into the medium by the rotor rotation was a maximum of 1.5 kJ per kg of liquid medium per minute and per g / L of plant cell dry mass. Equally good results were achieved when the rotor-stator heat introduced by the rotor rotation was a maximum of 30 kJ per kg of liquid medium and a thermal power introduced into the medium was a maximum of 1.5 kJ per kg of liquid medium and per minute. According to the invention, in this case the plants or plant cells are not homogenized, but are merely treated in such a way that the expression material is detached from the surface or from the apoplast.

[0015] Using the method according to the present invention, expression material is obtained from the cell surface or the apoplast (the entire cell wall and intercellular space). This material typically reaches these locations via the secretory pathway and is typically also located in the culture medium of the plant cells. However, during the process of the present invention, it was discovered that a large amount of secreted material adheres to surfaces, particularly the cell wall or the apoplast. This adhered material is obtained according to the present invention, and an increase in yield can be achieved using the method according to the present invention. Compared to methods without rotor-stator processing (i.e., isolation of secreted expression material), an approximately ten-fold increase in yield was observed.

[0016] The treatment intensity, treatment duration and treatment capacity of the rotor-stator are selected within the maximum parameters according to the invention to obtain a sufficient recovery of the expressed material (desired product). However, the treatment intensity, treatment duration and treatment capacity, which represent the energy introduced and are quantified as heat or specific heat, are limited because product contamination occurs at higher energy inputs.

[0017] Preferably, the specific heat of the rotor-stator introduced by the rotation of the rotor is at most 3 kJ per kg of liquid medium and per g / L of plant cell dry mass (abbreviated as 3 kJ / kg / (g / L) or 3 kJ / kg / g / L). Particularly preferably, the specific heat can be kept low to further reduce possible residual contamination. Therefore, preferably, the specific heat of the rotor-stator introduced is at most 2.75 kJ / kg / (g / L), or more preferably at most 2.5 kJ / kg / (g / L), at most 2.25 kJ / kg / (g / L), at most 2 kJ / kg / (g / L), at most 1.75 kJ / kg / (g / L), at most 1.5 kJ / kg / (g / L), at most 1.25 kJ / kg / (g / L), or at most 1 kJ / kg / (g / L).

[0018] Independent of the amount of plant material, the invention also provides an optional parameter for the amount of heat introduced. In some embodiments, this is relevant because the rotor-stator transfers energy to the cell medium independently of the plant cells; this can manifest as heating. Preferably, the amount of heat introduced by the rotor-stator through the rotation of the rotor is a maximum of 30 kJ per kg of liquid medium (abbreviated to kJ / kg), preferably a maximum of 25 kJ / kg, a maximum of 20 kJ / kg, a maximum of 15 kJ / kg, or a maximum of 10 kJ / kg.

[0019] This introduced specific heat or the amount of heat introduced can be adjusted, for example, by a time-limited treatment duration and / or treatment intensity (the same applies to the parameters of specific heat power or heat power):

[0020] In the method according to the invention, the rotor-stator is operated at low intensity, for example, at low speed. The amount of heat (or thermal output) introduced by a specific method at a specific intensity under selected parameters can be measured in comparative experiments, for example, by increasing the temperature of water or another medium with a known heat capacity. When determining the heat output of the method, other effects that influence the temperature, in particular temperature losses, should be excluded or taken into account in the calculation in order to determine the heat output or thermal output of the rotor-stator itself; preferably, the heat output or thermal output is determined in a Dewar flask.

[0021] Independent of the instrument, the specific heat power introduced or the heat power introduced is considered relevant (as above, "specific" means based on the amount of plant material optionally involved). Preferably, the specific heat power introduced into the medium is a maximum of 1.5 kJ per kg of liquid medium per minute and per g / L of plant cell dry mass (abbreviated as kJ / kg / min / (g / L) or kJ / kg / min / g / L). Particularly preferably, the specific heat output is a maximum of 1.25 kJ / kg / min / (g / L), a maximum of 1 kJ / kg / min / (g / L), a maximum of 0.8 kJ / kg / min / (g / L), a maximum of 0.6 kJ / kg / min / (g / L), a maximum of 0.5 kJ / kg / min / (g / L), a maximum of 0.4 kJ / kg / min / (g / L), a maximum of 0.3 kJ / kg / min / (g / L), a maximum of 0.2 kJ / kg / min / (g / L), a maximum of 0.15 kJ / kg / min / (g / L), a maximum of 0.125 kJ / kg / min / (g / L), or a maximum of 0.1 kJ / kg / min / (g / L). Similarly, the heat output introduced into the medium is a maximum of 1.5 kJ per kg of liquid medium and per minute (abbreviated to kJ / kg / min). Preferably, the heating power is at most 1.25 kJ / kg / min, at most 1 kJ / kg / min, at most 0.8 kJ / kg / min, at most 0.6 kJ / kg / min, at most 0.5 kJ / kg / min, or at most 0.4 kJ / kg / min.

[0022] The more intense or longer the treatment, the greater the amount of material obtained. Preferably, the heat introduced by the rotation of the rotor (rotor-stator) is at least 1 kJ per kg of liquid medium, particularly preferably at least 2 kJ / kg, and / or the specific heat of the rotor-stator is at least 0.1 kJ per kg of liquid medium and per g / L of plant cell dry mass, particularly preferably at least 0.2 kJ / kg / (g / L).

[0023] Preferably, the heat power introduced into the medium by the rotation of the rotor is at least 0.2 kJ per kg of liquid medium and per minute, preferably at least 0.4 kJ / kg / min, and / or the specific heat power introduced into the medium is at least 0.02 kJ per kg of liquid medium per minute and per g / L of dry mass of plant cells, preferably at least 0.04 kJ / kg / min / (g / L).

[0024] The expression material preferably comprises a protein. Proteins, particularly recombinantly expressed proteins, can be targeted to the secretory pathway using corresponding signal sequences and thus directed to accumulation on the surface or in the apoplast. Preferably, the expression material is in the apoplast of a plant cell and can be obtained from the apoplast using the methods of the present invention. Likewise preferably, the expression material is a secretory material, preferably a protein secreted through the cell membrane or cell wall.

[0025] According to the present invention, a rotor-stator is used to process plant cells in a liquid medium to obtain expression material.

[0026] The rotor-stator comprises at least one rotor which exerts shear forces on the plant cells by its rotational movement. By means of these shear forces, the surfaces and cell walls of the plant cells or apoplasts are loosened, mechanically influenced or abraded or parts of the surface are removed.

[0027] The rotor rotates relative to the stator. The stator can be a counterpart to the housing jacket or the rotor. The rotor can have cutting or shearing elements with edges or shearing surfaces. A common design has a comb-like structure, in which a plurality of shearing projections (e.g., teeth or tines), typically arranged parallel to the axis of rotation, exert the shearing or cutting action. In this context, "plurality" can include, for example, 2, 3, 4, 5, 6, 7, 8, or more shearing projections.

[0028] The stator can be arranged as a counterpart to the rotor and in particular as its cutting or shearing element. Optionally, similar to the rotor, the stator can have its own cutting or shearing element, for example also in the form of a comb. Such a configuration is known in rod homogenizers, as described in DE 10 2005 031 459 A1.

[0029] In other embodiments, the stator may be a housing structure, such as is common for through-flow homogenizers.An example of a through-flow rotor-stator is described in WO 2009 / 062610 A1.

[0030] Examples of rotor-stators are rod homogenizers or shear pumps. Shear pumps are particularly used in flow-through systems.

[0031] Preferably, there is a gap between the rotor and the stator, for example the size of at least one plant cell or larger, so that at least plant cells in the form of protoplasts can pass between the rotor and the stator. Suitable gap sizes are 50 μm, 70 μm, 80 μm, 100 μm, 150 μm or larger and any range in between these distances, preferably up to a maximum of 500 μm, or up to 300 μm, or up to 200 μm.

[0032] As noted, the intensity is kept low to protect the plant cells in protoplast form and to prevent or reduce contamination of the secreted expression material to be harvested with cell contents. To this end, in conventional rotor-stator models, the rotational speed is reduced, for example in embodiments where the rotor is operated at a maximum of 15,000 revolutions per minute, preferably 1,000 to 15,000 revolutions per minute. Possible rotational speeds are 3,000 to 14,000, 4,000 to 13,000, 5,000 to 12,000, or 6,000 to 11,000 revolutions per minute.

[0033] The plant cells can be present in a container into which the rotor-stator is introduced. To this end, the rotor-stator can be introduced into a container containing the medium. This "intermittent" configuration (for discontinuous processes) is particularly used in rod homogenization. For larger scales, the use of a through-flow rotor-stator is preferred. According to this embodiment, the rotor-stator can have an interior space with at least one inlet and outlet, through which the liquid medium is continuously conveyed. An example of this is a shear pump for a continuous process.

[0034] Preferably, the stator defines a 10 cm 3 (0.01 L) to 1 m 3 (1000 L). Preferred volumes are 0.1 L to 800 L, or 0.5 L to 600 L, or 1 L to 400 L, or 2 L to 200 L. Volumes of up to 100 L are preferred, particularly preferably 0.65 L to 50 L, for example 1 L to 40 L. These volumes are particularly suitable for processing plant cell culture media.

[0035] Preferably, the amount of liquid medium treated is at most 50,000 kg, preferably 0.5 g to 50,000 kg, for example 1 g to 25,000, 2 g to 10,000 kg, 5 g to 5,000 kg, 10 g to 2,500 kg, 20 g to 1,000 kg, 30 g to 500 kg, 50 g to 250 kg, 100 g to 100 kg, 200 g to 50 kg, 500 g to 250 kg, 1 kg to 100 kg, 2 kg to 50 kg or 4 kg to 20 kg. Such amounts are preferably used per run in a discontinuous process.

[0036] Preferably, the plant cells are present in the liquid medium at a concentration of 0.2 g / L to 60 g / L (mass of plant cells as dry mass). Preferred concentrations of plant cells (always based on dry mass) are 0.5 g / L to 50 g / L, 1 g / L to 40 g / L, 2 g / L to 30 g / L, 4 g / L to 20 g / L, particularly preferably about 10 g / L, for example 5 g / L to 15 g / L. These plant cell concentrations can be processed particularly effectively with the rotor-stator.

[0037] Preferably, the plant cells are treated with a rotor-stator for 2 to 150 minutes. In a continuous process, these times refer to the average treatment time of the plant cells. Preferred times are particularly 3 to 120 minutes, 5 to 100 minutes, 8 to 80 minutes, 10 to 60 minutes, or particularly preferably 12 to 40 minutes. In large culture volumes, longer rotor-stator treatments can be performed. Preferably, another possible time is 1 to 24 hours, preferably 2 to 20 hours, 3 to 16 hours, 4 to 12 hours. Therefore, all preferred treatment times are within the range of 3 to 24 hours, and relate to each range between the mentioned treatment times or even longer.

[0038] Preferably, the plant cells can be cultured in suspension culture. In the method according to the invention, the suspension can be processed directly as a liquid medium. Alternatively, it is also possible to first isolate the moss, for example, from a solid culture, a liquid culture or a suspension culture, and then suspend it in an aqueous medium under conditions suitable for the rotor-stator process. Plants particularly suitable for use in the method according to the invention are non-woody plants. Preferred plants are algae and mosses, in particular bryophytes. Preferably, the bryophyte or cell is a moss, preferably Physcomitrella patens ( P. patens The bryophyte may be any of the bryophytes, but is preferably selected from mosses, liverworts or hornworts, particularly preferably selected from the class Mosses or Physcomitrella ( Physcomitrella), Cucurbitaceae ( Funaria ), Sphagnum ( Sphagnum ), Ceratocystis ( Ceratodon ), Marchantia ( Marchantia ) and Sphaerocarpos. Physcomitrella patens is particularly preferred. Most preferably, the method according to the invention is performed using cells from plant tissue, such as protonema from Physcomitrella patens. Preferred algae are selected from green algae, for example from the order Chlorellales ( Chlorellales ), preferably selected from the family Chlorellaceae, more preferably selected from the genus Protothecoides ( Auxenochlorella ) or Chlorella ( Chlorella ), especially Chlorella vulgaris ( Chlorella vulgaris ), and selected from the order Volvocales, preferably from the family Haematococcaceae, more preferably from the genus Haematococcus ( Haematococcus ), especially Haematococcus pluvialis ( Haematococcus pluvialis ), and selected from the order Eustigmatales, preferably Loboceae, Chlorobothryaceae, Pseudocharaciopsidaceae and Eustigmataceae. Further preferred plants are tobacco, beans or lentils. Preferably, the plant is an aquatic plant, for example from the genus Lemna ( Lemna )、Spiderwort( Spirodela ), Spirodela ( Landoltia ), Wolffia ( Wolffia ) or Wolffia ( Wolffiella ).

[0039] Plant cells form the subject of the present invention. As used herein, "plant cell" may refer to an isolated cell, an individual cell, but also to a cell in or from a plant tissue, preferably a tissue selected from the group consisting of callus, protonema, phloem, xylem, mesophyll, stem, leaf, thallus, chloroplast, rhizoid or gametophore, or a cell in a plant organism.

[0040] In the method according to the invention, the medium preferably has a physiological pH, in particular in order to protect the protoplasts (cell components in the cell wall, in particular from the cell membrane) as described above, and to prevent contamination of the expression material in the apoplast / on the cell surface. The pH of the liquid medium is preferably 3.5-8.5, particularly preferably 4-8, or 4.5-7, or 5-6.5, in particular 5.5-6, or a combination of these values, such as a pH of 5-8.

[0041] Also for the protection of the protoplasts, the osmotic pressure of the liquid medium is preferably physiological, in particular in order to avoid swelling stress, for example when the osmotic pressure is too low. Optionally, an upper limit for the osmotic pressure can also be provided to avoid osmotic contraction stress. Preferably, the medium has an osmotic pressure of at least 0.1 osmol / L, or preferably at least 0.150 osmol / L. The osmotic pressure can be adjusted by dissolving substances such as salts or other medium components such as sugars or sugar alcohols. Preferably, an alkali metal salt such as Na + and / or K + Preferably, a halide ion such as Cl is provided - or F - or I - , phosphate or acetate as anions. Other possible buffer components include Tris (tris (hydroxymethyl) aminomethane).

[0042] In addition, in order to further protect protoplasts, surface-active polymers can be provided in the liquid medium for rotor-stator processing.Surface-active polymers are for example described in WO 2013 / 156504 A1, and preferably encompass uncharged polymers, for example emulsifiers, such as polyalkylene glycols, particularly polyethylene glycol. Especially, the polymer is a nonionic water-soluble surface-active polymer. Preferably, it does not denature the protein. Example is selected from following polymers or copolymers: polyethers such as polyalkylene glycols, polysorbates or polyvinyl pyrrolidone, polyvinyl alcohol, water-soluble cellulose derivatives such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose or hydroxyethyl cellulose, vinyl pyrrolidone vinyl acetate copolymer (copolyvidone), polyvinyl acetate, partially hydrolyzed polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol copolymer, and their mixtures. Further possibilities are polysorbates, for example polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, preferably polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, Tween® 80), polyoxyethylene (40) stearate. The surface-active polymer is preferably present in the medium in a concentration of at least 0.05% by weight, particularly preferably at least 0.08%, at least 0.1% or at least 1.5% (all % data are in % by weight). The molecular weight of the surface-active polymer, such as PEG, is preferably at least 500 Da, particularly preferably at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, at least 6,000 Da, at least 8,000 Da, at least 10,000 Da, at least 20,000 Da, or at least 30,000 Da. Particularly preferably, the molecular weight is from 500 Da to 2,000,000 Da, preferably from 1,000 Da to 200,000 Da or from 1,200 Da to 80,000 Da.

[0043] The liquid medium is preferably aqueous, in particular water or a water mixture compatible with the cells. In particular, it can be a culture medium for plant cells (and with plant cells), as long as the plant has not been separated from it beforehand.

[0044] Preferably, the expression material has been expressed before the plant cell is treated with the rotor-stator so that it accumulates on the surface of the plant cell or in the apoplast. Here, the plant cell can be cultured and / or grown, for example, under plant growth conditions (nutrient medium, illumination) in a medium, which is generally known (see, for example, Frank et al. Plant Biol 7, (2005): 220–227). Expression or cultivation is preferably carried out for 13 minutes to 1 month (30 days) or longer, for example 2 months (60 days), for example 1 hour to 22 days, or 5 hours to 15 days, for example 10 hours to 7 days or 20 hours to 3 days. In the continuous cell culture in which cells are regularly removed in order to obtain a product (expression material), these time ranges or the shortest time can correspond to the average period of the cultured cells.

[0045] Other methods of damaging or cracking or homogenizing the protoplasts should be avoided. The cell wall is preferably not cracked, in particular not enzymatically and / or chemically and / or osmotically and / or ultrasonically. Preferably, the cell wall should remain untouched or intact except for the rotor-stator treatment according to the invention. In particular, the cell membrane (protoplast) should remain intact, wherein in the method according to the invention, the viability of the cells does not play any special role, but contamination of the liquid medium by components of the cell interior, in particular the cytoplasm, should be avoided.

[0046] The present invention will now be further described by means of the following figures and examples, but the invention is not limited to these embodiments.

[0047] Attached photos:

[0048] Figure 1: Determination of energy input in water using a T25 Ultraturrax rod homogenizer (IKA / Staufen). (A) Temperature profile in 1.5 L of water at 10,000 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg].

[0049] Figure 2: Determination of energy input in water using an FSP712VC-2.2kW-FU shear pump homogenizer (Fristram / Hamburg). (A) Temperature profile in 50 L of water at 2,800 rpm. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg].

[0050] Figure 3: Percent release of biomass-bound product (moss-aGal) during treatment using a shear pump (dashed line) and a T25 Ultraturrax rod (solid line). The shear pump treatment of the reactor culture was performed in a volume of 50 L. The reactor culture was treated with a T25 Turrax rod in a volume of 0.65 L.

[0051] Figure 4: Specific energy input by a T25 Ultraturrax rod (A) and a shear pump (B), both for 10 g / L biomass dry mass. Specific energy input using a T25 Ultraturrax rod at 19,000 rpm and 1 g / L biomass dry mass according to a comparative example (C).

[0052] Figure 5 : Percent release of biomass-bound products (moss-aGal) during treatment using a shear pump (dashed line) and a T25 Ultraturrax rod (solid line). The shear pump treatment of the reactor culture was carried out in a volume of 50 L. The reactor culture was treated with a T25 Turrax rod in a volume of 0.65 L. Biomass: 9.2 g / L (shear pump), 8.2 g / L (T25).

[0053] Figure 6 Western blot analysis of product release (moss-aGal) compared to intracellular marker protein release (Rubisco, large subunit). For intracellular proteins in saline medium (e.g., 20 mM Tris, 100 mM NaCl, pH 7), energy input of up to 32.9 kJ / kg could be detected with minimal amounts. Under osmotic stress conditions (demineralized water), approximately 10 kJ / kg could be detected. The amount of target protein (moss-aGal) increased in saline medium and under osmotic stress conditions in response to energy input.

[0054] Figure 7 Microscopic analysis of the T25 process in demineralized water. Up to an energy input of 16.5 kJ / kg, moss cells remained intact. At an energy input of 32.9 kJ / kg, cell integrity was maintained, but significantly more particles were present, indicating the onset of cellular digestion.

[0055] Figure 8 Microscopic analysis of T25 processes in saline buffer (20 mM Tris, 100 mM NaCl, pH 7). Moss cells remained intact up to an energy input of 16.5 kJ / kg. At an energy input of 32.9 kJ / kg, cellular integrity was maintained, but significantly more particles were present, indicating the onset of cell digestion.

[0056] Figure 9: Microscopic analysis of the shear pumping process in saline medium (reactor culture). Up to an energy input of 18.41 kJ / kg, moss cells remained intact. From an energy input of 27.20 kJ / kg, cell integrity remained, but significantly more particles were present, indicating the onset of cell digestion.

[0057] Figure 10 Microscopic analysis of the shear pumping process in demineralized water. Up to an energy input of 9.62 kJ / kg, moss cells remained intact. From an energy input of 18.41 kJ / kg, cell integrity remained, but significantly more particles were present, indicating the onset of cellular digestion.

[0058] Figure 11 Microscopic analysis of moss cells during sonication, as a comparative image of cell digestion. After a short period of sonication (1 minute), the cells have already lost their integrity. After 3 minutes, only cell fragments and empty cell debris are visible (100% in a 50 mL sample).

[0059] Figure 12: Comparative example: Determination of energy input in water using a T25 homogenizer (IKA / Staufen) at high energy and 19,000 rpm. (A) Temperature profile in 1 L of water. (B) Calculated energy input [kJ]. (C) Calculated energy input [kJ / kg].

[0060] Figure 13 Comparative Example: At 19,000 rpm, product release was significantly faster, especially up to an energy input of 7 kJ / kg. From an energy input of 84 kJ / kg, product loss occurred due to the high temperature and shear stress.

[0061] Example:

[0062] Example 1: Determination of the energy input with the aid of a Turrax rod and a shear pump

[0063] Temperature was used as a measurable variable as a measure of the energy input into the aqueous medium. 1.5 L of H2O were dispersed in a Dewar flask at 10,000 rpm for 30 minutes using a T25-S25N-18G Turrax rod (IKA Staufen), and the temperature profile was measured. The room temperature during the experiment was 20.5 to 20.7°C. The energy input data were calculated using the specific heat capacity of H2O (4,190 Jkg -1 K -1) Calculations were performed similarly to the energy input data from the literature (Orellana-Escobedo et al., Plant cell Rep. 2015, 34(3), 425–433) at 19,000 rpm.

[0064] 50 L of H2O were circulated from a Nalgene container via a fabric-stabilized PVC hose at 2800 rpm using a shear pump (shear pump FSP712, Fristam Hamburg). The temperature in the Nalgene container was measured using a temperature sensor (precision thermometer, G002.1, Carl Roth). The experimental setup was carried out in a room temperature-controlled at 19°C. Heat losses to the environment were neglected in this experimental approach. The energy input data were calculated using the specific heat capacity (4,190 J • kg -1 K -1 )calculate.

[0065] Example 2: Production of moss culture

[0066] In Wave TMMoss production strains were sterilely cultured in 200 L disposable bioreactor bags (Cellbag 200, GE Healthcare) in a Rocking Motion bioreactor (Wave200, GE Healthcare) for 3-4 weeks. Culture parameters included a shaking frequency of 19 to 25 rpm, a shaking angle of 9°, a temperature of 24-26°C, and an air supply rate of 2 L / min, using 2% CO2-enriched air. Illumination was provided by four LED modules (serial numbers 120268 to 120282, Infors AG) with "warm white" LEDs mounted above the bioreactor bag. Moss cultivation was performed under 24-hour continuous illumination. SM07 (100 mM NaCl, 6.6 mM KCl, 2.0 mM MgSO4 x 7H2O, 1.8 mM KH2PO4, 20.4 mM Ca(NO3)2 x 4H2O, 0.05 mM Fe Na-EDTA, 4.9 mM MES, 0.1% (w / v) PEG4000, 100.26 µM H3BO3, 0.11 µM CoCl2 x 6H2O, 0.1 µM CuSO4 x 5H2O, 5 µM KI, 85.39 µM MnCl2 x4H2O, 1.03 µM Na2MoO4 x 2H2O, 0.11 mM NiCl2 x 6H2O, 0.04 Na2SeO3 x 5H2O, 0.039 zinc acetate x 2H2O) was used as the mineral salt medium. The pH value of 5-6 was adjusted by automatically adding 0.25 M H2SO4 and 0.25 M NaOH using a WAVEPOD 1 and Pump 20 (GE Healthcare). Recombinant α-galactosidase (aGal or α-Gal A) ("moss-aGal") was expressed as described in WO 2016 / 146760 A1.

[0067] Example 3: Release and analysis of moss-bound products

[0068] To analyze the time course of the release of moss-bound products, the cultures obtained from Example 2 were exposed to different energy inputs using T25-S25N-18G Turrax rods and a shear pump FSP 712. The released products were analyzed by means of moss-aGal ELISA (Biogenes / Germany). PL Determination of product concentration (c PLThe extent of cell digestion was determined by microscopic image analysis of moss cells (microscope: Axiovert 200 oper Stemi SV11, equipped with AxioCam camera, AxioSoft software, and KL 1500 LCD cold light source (Carl Zeiss). Comparison with microscopic images of total digestion was possible by microscopic analysis of 50 mL of ultrasonic digestion at 100% power (probe: UW2070, Bandelin; amplifier: HD 2070, Bandelin) for 20 minutes. At the molecular level, qualitative analysis was performed by Western blotting on the released products and the intracellular marker protein Rubisco. The primary antibodies used were anti-aGal (H00002717-D01P, abnova) and anti-Rubisco (AS03037, Agrisera), and anti-rabbit HRP (abcam, AS03037) was used as a secondary antibody.

[0069] In order to analyze the released (c PL ) and releasable moss-bound products (c PX ) ratio between untreated cultures and cells were digested using a ball mill (steel balls: RB-3 / G20W, Schleer; ball mill: MM300, Retsch). After separation of cell debris, product concentrations were determined using ELISA (Biogenes). In this regard, the protein aGal (α-galactosidase, also known as "moss-aGal") expressed in the apoplast was assayed.

[0070] Example 4: Results and Discussion

[0071] The energy input (caloric value, kJ / kg, kg based on liquid medium) of two different homogenizers (T25-S25N-18G Turrax rod and FSP 712 shear pump) in culture medium (kg) was determined by temperature measurement (Figures 1-5). The homogenizers were set to a low speed, resulting in a low heating output (caloric value, kJ / kg / min). This allowed the total heat input to be determined over a specific time period (0-60 minutes). The energy input in the water was calculated based on the measured temperature using the specific heat coefficient of H2O [4.182 kJ / kg*K]. The heat (Figures 1-3) or the specific heat relative to the dry mass of the plant part (Figures 4-5) was calculated.

[0072] Figure 3 and 5 Product release of the desired protein (recombinant aGal from moss, "moss-aGal") deposited on the surface or in the apoplast is shown. Release also increased with increasing treatment.

[0073] In a comparative experiment, the Turrax rod was operated at a higher thermal power, more precisely at 19,000 rpm ( Figure 4C , 12-13). The thermal power is about 100 times that of the protective treatment at 10,000 rpm (compare Figure 4A and 4C Operation at higher thermal powers leads to rapid product release, but also to cell (protoplast) disruption, resulting in contamination of the extracellular product with components from the intracellular space. These effects are already evident at 1 minute.

[0074] Figure 6 The quality of the product release of the extracellular protein (moss aGal) and the intracellular protein Rubisco is shown. Rubisco is present in plant cells at high concentrations and is therefore considered to be a highly sensitive marker for the overflow of cell contents. In experiments with physiological saline solutions, an increase in the release of Rubisco was shown at higher energy (heat) inputs exceeding 32.9 kJ / kg. In comparative experiments using softened water (VE water), contamination of Rubisco occurred earlier, starting at approximately 10 kJ / kg, because in addition to the shear stress of the homogenizer, osmosis also occurred. Different amounts of heat (energy input) were controlled by treatment time.

[0075] Microscopic analysis of the treated cell complexes mirrored these results. Figure 7 Shows the results after treatment with an Ultraturrax rotor-stator in demineralized water; Figure 8 Results are shown after treatment with an Ultraturrax rotor-stator under physiological conditions (20 mM Tris, 100 mM NaCl, pH=7). Figure 9 The cell complexes are shown after treatment with a shear pump under physiological conditions. As the treatment time (heat) increases, the number of particles formed, likely from disrupted cells, increases. Starting at approximately 30 kJ / kg, increased cell digestion is observed. For comparison, Figure 10 An experiment using a shear pump in demineralized water is shown. Here, comparable particles already appear at approximately 20 kJ / kg.

[0076] Through with Figure 7-10 Compare, Figure 11 Cell digestion by ultrasound is shown. After 1 minute, the cells have already lost their integrity.

[0077] This suggests that, on the one hand, the energy input per unit time (rotor-stator rotation intensity; thermal output) should be limited, while on the other hand, the absolute energy input (heat) in the experiments here is controlled by the treatment duration. This parameter can be considered on its own or based on the biomass (biomass dry mass, TBM). Useful figures for a gentle method that removes as many adsorbed or apoplast-bound products as possible while keeping the protoplasts largely intact are a maximum of 3 kJ / kg per g / L dry mass and a maximum of 1.5 kJ / kg / min per g / L dry mass, or a maximum of 30 kJ / kg and a maximum of 1.5 kJ / kg / min. At these low thermal outputs, possible treatment times range from 2 to 150 minutes—depending on the intensity of the rotation, generally longer than the brief but intense treatments commonly used to date.

Claims

1. A method for detaching expression material from the surface or apoplast of plant cells, wherein the plant cells are treated in a liquid medium with a rotor-stator, wherein the specific heat of the rotor-stator introduced by the rotation of the rotor is at most 3 kJ per kg of the liquid medium and per g / L of the dry mass of the plant cells, and the specific heat power introduced into the medium is at most 1.5 kJ per kg of the liquid medium per minute and per g / L of the dry mass of the plant cells, wherein the rotor-stator is a rod homogenizer or a shear pump, and / or wherein the stator has a comb-like structure, wherein the plant cell is a moss cell, The expression material is a protein secreted through the cell membrane.

2. The method according to claim 1, wherein The expression material is in the apoplast of the plant cell.

3. The method according to claim 1 or 2, wherein: The heat of the rotor-stator introduced by the rotation of the rotor is at least 1 kJ per kg of the liquid medium, and / or the specific heat of the rotor-stator is at least 0.1 kJ per kg of the liquid medium and per g / L of the dry mass of the plant cells.

4. The method according to any one of claims 1 to 2, characterized in that The heat power introduced into the medium by the rotation of the rotor is at least 0.2 kJ per kg of the liquid medium and per minute, and / or the specific heat power entering the medium is at least 0.02 kJ per kg of the liquid medium, per minute and per g / L of the dry mass of the plant cells.

5. The method according to any one of claims 1 to 2, characterized in that The expression material contains protein, and / or the expression material is secreted material.

6. The method according to any one of claims 1 to 2, characterized in that The rotor-stator is introduced into a container containing a medium.

7. The method according to any one of claims 1 to 2, characterized in that The rotor-stator has an interior space with at least one inlet and one outlet, through which the liquid medium is continuously conveyed.

8. The method according to any one of claims 1 to 2, characterized in that The stator is limited to 10cm 3 Up to 1m 3 The volume of the container and / or the amount of liquid medium processed is at most 50 kg.

9. The method according to any one of claims 1 to 2, characterized in that The stator is limited to 10cm 3 Up to 1m 3 The volume of the reaction mixture and / or the amount of the liquid medium to be processed is from 0.5 g to 50 kg.

10. The method according to any one of claims 1 to 2, characterized in that The plant cells are present in the liquid medium at a concentration of 0.2 g / L to 60 g / L, wherein the mass of the plant cells is expressed as dry mass.

11. The method according to any one of claims 1 to 2, characterized in that The plant cell is a Physcomitrella patens cell.

12. The method according to any one of claims 1 to 2, characterized in that The rotor is operated at a maximum speed of 15,000 revolutions per minute.

13. The method according to any one of claims 1 to 2, characterized in that The rotor is operated at 1,000 to 15,000 revolutions per minute.

14. The method according to any one of claims 1 to 2, characterized in that The heat introduced into the rotor-stator by the rotation of the rotor is a maximum of 30 kJ per kg of the liquid medium, and the thermal power introduced into the medium is a maximum of 1.5 kJ per kg of the liquid medium and per minute.

15. The method according to any one of claims 1 to 2, characterized in that The medium has a pH of 5 to 8 and / or an osmotic pressure of at least 0.1 osmol / L.

16. The method according to any one of claims 1 to 2, characterized in that The plant cells are treated with the rotor-stator for 2 minutes to 150 minutes.

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