JP6578681B2 - Plant cultivation method and production of useful protein using the same - Google Patents

Description

  The present invention relates to a method for cultivating a plant, a method for introducing a polynucleotide encoding the protein into a plant, and a method for producing a protein using a plant cultivated by these methods, for efficiently producing useful proteins. About.

  In recent years, protein production methods using plants have attracted attention for reasons such as the ability to express complex proteins, mass production at low cost, easy separation and purification, and safety. Various methods for producing proteins using plants have been studied, and a cultivation method suitable for protein production and the like have been reported while increasing the expression efficiency of foreign genes.

Patent Document 1 describes a method for producing influenza virus-like particles (VLP) such as H1 protein by cultivating Nicotiana benthamiana infected with transformed Agrobacterium in a greenhouse. .
In Non-Patent Document 1, in a method for producing a protein in a Nicotiana plant by transient expression via Agrobacterium, a virus is infiltrated into a tobacco genus species, a virus strain type, and a tobacco leaf ( The result of studying the degree of pressure reduction during infiltration is disclosed. According to this, if the degree of decompression when infiltrating the Agrobacterium solution is too high, the plant will be mechanically damaged, and then it will wither in a short period of time, resulting in plant death. Infiltrate only about 50% of the protein, and the protein expression level decreases. As a result, it has been proposed that proteins can be expressed without adversely affecting plant growth by infiltrating Agrobacterium under relatively mild conditions (50 to 200 mbar, 30 to 60 seconds).

  Non-Patent Document 2 discloses a method of mechanically infecting Agrobacterium from the back of a leaf and expressing GFP as a foreign protein with high efficiency in tobacco by hydroponics. To achieve high-efficiency expression of foreign proteins, conditions such as light intensity, light irradiation time, day and night temperature, relative humidity, infestation time, and harvest time of tobacco hydroponics are optimized. If so, it is disclosed that the growth of tobacco is strong and leaves are flat, the color of the leaves is pale green to green, and the expression of foreign protein (GFP) by infection is highly efficient.

Special table 2010-533001 gazette

Journal of Visualized Experiments, 2014, 86, e51204, pp.1-13 Journal of Southern Medicine University, 2012, 32 (6), pp. 772-7

As described above, a technique for producing a protein using a plant is known, but examination of conditions for improving the production efficiency is not yet sufficient.
That is, in Patent Document 1, detailed studies have been conducted on the expression regulation mechanism of a gene encoding hemagglutinin (HA) introduced into a plant. This transduced plant is cultivated under normal conditions. Only.

Non-Patent Document 1 discloses the effect of reduced pressure conditions on the leaves of Bensamiana tobacco when infecting Agrobacterium by the vacuum infiltration method. However, the only approach to optimizing protein production efficiency is the selection of the types of tobacco plants and Agrobacterium strains and examination of external factors such as physical conditions at the time of infection. It is enough. This document does not mention a plant cultivation method that ensures a sufficient protein expression level. In addition, there is no description of a method for ensuring a sufficient protein expression level even when the degree of decompression is increased to increase the infection efficiency of Agrobacterium.
Further, in Non-Patent Document 2, various studies have been made on the plant cultivation conditions, but only the plant cultivation conditions in an artificial meteorological device, which are Agrobacterium infection by decompression treatment and protein expression thereby. No effect on the amount has been studied.

In this way, in transient expression of proteins by plants, conditions for decompression at the time of Agrobacterium infection, cultivation conditions, etc. have been studied, but these are integrated and Agrobacterium by decompression treatment is integrated. There has been no report on a plant cultivation method that can secure a sufficient protein expression level after um infection.
Accordingly, an object of the present invention is to improve the conditions for growing plants when producing useful proteins using plants, and to optimize the production efficiency of useful proteins.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have paid attention to the plant before Agrobacterium infection, and surprisingly the infection method by optimizing the cultivation method of the plant before the infection treatment. It has been found that the production efficiency of useful proteins can be improved after the treatment. By controlling the light irradiation cycle and temperature fluctuations in particular, it is possible to grow robust plants that are resistant to mechanical and biological stress during Agrobacterium infection, thereby increasing the protein production using the plant. The present inventors have found that expression efficiency can be improved and completed the present invention.

That is, the gist of the present invention is as follows.
[1] A plant cultivation method for infecting a plant with Agrobacterium having a polynucleotide encoding a useful protein by pressure cycle treatment to express the useful protein, the method comprising:
Hydroponically cultivating the plant in a closed plant factory;
In at least a part of the cultivation process, including the step of controlling the irradiation cycle and temperature,
The irradiation cycle is one cycle or more during the cultivation process by lighting and extinguishing of artificial light, and the temperature of lighting time is controlled to be at least 1 ° C. higher than the temperature of lighting time.
[2] A method for introducing a polynucleotide encoding a useful protein into a plant, comprising:
Hydroponically cultivating the plant in a closed plant factory;
In at least a part of the cultivation process, the irradiation cycle and the temperature are controlled, and the irradiation cycle is set to one cycle or more during the cultivation process by turning on and off the artificial light, and the lighting temperature is turned off. Controlling at least 1 ° C. higher than the temperature of
Infecting the plant with Agrobacterium having the polynucleotide by pressure cycle treatment;
A method for introducing the polynucleotide into a plant, comprising:
[3] A method for producing useful proteins using plants,
Hydroponically cultivating the plant in a closed plant factory;
Infecting the plant after the cultivation process with Agrobacterium having a polynucleotide encoding a useful protein by pressure cycle treatment;
Expressing a useful protein in the plant after the infection step,
In at least a part of the cultivation process, having an irradiation cycle of 1 cycle or more during the cultivation process by turning on and off artificial light, and controlling the temperature of the lighting time at least 1 ° C. higher than the temperature of the lighting time A method for producing a useful protein, which is characterized.
[4] The method according to any one of [1] to [3], wherein the temperature during the lighting time is controlled within a range of 2 to 30 ° C. higher than the temperature during the lighting time.
[5] The method according to any one of [1] to [4], wherein the temperature of the nutrient solution used for hydroponics is controlled at a temperature equal to or higher than the air temperature during the light-off time.
[6] The method according to [5], wherein the temperature difference between the lighting time and the lighting time of the nutrient solution is controlled within a range of ± 1 ° C.
[7] The method for producing a useful protein according to any one of [3] to [6], further comprising a step of purifying and recovering the useful protein after the expression step.
[8] The method for producing a useful protein according to any one of [3] to [7], wherein the useful protein is a protein for medical use.
[9] The method for producing a useful protein according to any one of [3] to [8], wherein the plant is Bensamiana tobacco.

  In general, when a plant is cultivated under continuous light irradiation conditions for 24 hours, the plant itself grows greatly, so that the amount of biomass increases and the total amount of protein to be recovered tends to increase. On the other hand, according to the method of the present invention that controls the temperature together with the light irradiation cycle, the mechanical and biological resistance to Agrobacterium infection is improved by improving the robustness of the plant, and the biomass amount of the plant itself Since protein can be accumulated at a higher concentration in the plant than in the case of increasing the amount of protein, the purification load is reduced in the protein purification step, and the effect is great in terms of reduction in production cost. Furthermore, since the plant having improved robustness can sufficiently increase the amount of biomass after Agrobacterium infection, the total amount of protein finally recovered can also be increased.

The structure of plasmid pGFP / MM444 is shown. The structure of plasmid p19 / MM444 is shown.

Among the methods of the present invention, the first aspect is a plant cultivation method for expressing a useful protein by infecting a plant with Agrobacterium having a polynucleotide encoding a useful protein by pressure cycle treatment. It includes a process for cultivating plants (cultivation process).
Among the methods of the present invention, the second aspect is a method for introducing a polynucleotide encoding a useful protein into a plant, the step of cultivating the plant (cultivation step), and then encoding the useful protein into the plant. A step of infecting Agrobacterium having the polynucleotide to be infected (infection step).
Among the methods of the present invention, the third aspect is a method for producing a useful protein using a plant, the step of cultivating the plant (cultivation step), and then a polynucleotide encoding the useful protein in the plant And a step of cultivating the plant after the infection step and expressing the useful protein (expression step).

  Hereinafter, embodiments of the present invention will be described in detail according to the flow of a method for producing useful proteins, which is the third aspect of the method of the present invention, including the most steps. The same applies to the second embodiment.

  The plant that can be used in the present invention is a plant that is infected with Agrobacterium and is not particularly limited as long as it is a plant that expresses a useful protein. Examples include dicotyledonous plants and monocotyledonous plants. Specifically, dicotyledonous plants include solanaceous plants such as tobacco, potato, tomato, etc., cruciferous plants such as arugula, komatsuna, mizuna, mustard, white lizards, etc. However, as a leguminous plant, alfalfa, mungbean, soybean and the like are exemplified, spinach, sugar beet and the like are used as a red crustaceae plant, perilla, basil and the like are exemplified as a family Lamiaceae, and bees and the like are exemplified as a celery family. Examples of monocotyledonous plants include gramineous plants such as rice, wheat, barley and corn, and examples of mallowaceae include cotton and the like. Among these, solanaceous plants are preferable, and tobacco is more preferable.

  As tobacco, Nicotiana tabacum, N. benthamiana, H. tobacco (N. alata), N. glauca, N. longiflora, N. persica, Nicotiana rustica (N. rustica), Nicotiana sylvestris (N. sylvestris) and the like. Preferred is Bensamiana cigarette.

In the present invention, the useful protein is not particularly limited as long as it is a protein used for medical or industrial purposes. Preferably, it is a protein for medical use.
Medical proteins are classified into therapeutic proteins and diagnostic proteins. Examples of therapeutic proteins include peptides, vaccines, antibodies, enzymes, hormones (preferably peptide hormones), and more specifically, as vaccines. Viral proteins used, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin and other hematopoietic factors, interferon, interleukin (IL) -1, Examples include cytokines such as IL-6, monoclonal antibodies or fragments thereof, tissue plasminogen activator (TPA), urokinase, serum albumin, blood coagulation factor VIII, leptin, insulin, stem cell growth factor (SCF) and the like. . Examples of diagnostic proteins include antibodies, enzymes, hormones and the like.

  The virus protein used as a vaccine is preferably a virus-like particle (VLP) constituent protein. A VLP component protein may be a single protein or may include one or more proteins. Examples of the virus include influenza virus, norovirus, human immunodeficiency virus (HIV), human hepatitis C virus (HCV), human hepatitis B virus (HBV), etc. Influenza hemagglutinin is a constituent protein of VLP of influenza virus. Examples of (HA) protein and norovirus VLP-constituting protein include Norwalk virus capsid protein (NVCP).

  The industrial protein is a protein used for food, feed, cosmetics, fibers, detergents, chemicals, and the like, and examples thereof include peptides, enzymes, and functional proteins. Specific examples include protease, lipase, cellulase, amylase, peptidase, luciferase, lactamase, collagen, gelatin, lactoferrin, jellyfish green fluorescent protein (GFP) and the like.

  Hereinafter, each process of the present invention will be described.

<Cultivation process>
As will be described in detail later, an important point of view in this step is to grow the plant under conditions that control the light irradiation cycle and temperature fluctuation, which will be described in order.
This step includes a step of cultivating the plant by hydroponics. In this specification, hydroponics refers to hydroponics that does not use soil, and is a method of cultivating plants fixed on a solid medium such as rock wool in a nutrient solution, or on a gently inclined plane. There are a thin film hydroponic method (NFT: Nutrient Film Technique) in which the liquid flows down thinly, a drip type hydroponic method (DFT: Deep Flow Technique) in which the liquid is cultivated in the stored nutrient solution. Thin-film hydroponics (NFT) is a method in which a panel with a plurality of holes for holding plants is placed on a cultivation stand, the plants are inserted and held in the holes, and a nutrient solution is supplied for cultivation. It is. Drip-type hydroponics (DFT) supplies enough oxygen to the roots by the flow of nutrient solution (liquid fertilizer) to promote nutrient absorption, and also stabilizes the rhizosphere environment including the temperature and concentration of liquid fertilizer. There is an advantage that the environment is fixed.

Hydroponics is preferable because it facilitates multi-stage cultivation shelves, nutrient solution recycling, fertilizer components and pH management, and among these, the root-bare is exposed in the nutrient solution. The method is preferred. This is because roots can freely grow in the water and increase the contact area with the nutrient solution, so that a sufficient amount of moisture and nutrients can be absorbed.
In hydroponics, a method of cultivating while constantly supplying nutrient solution to the plant by pouring or circulating a nutrient solution, which is water containing fertilizer, and storing the nutrient solution for a certain period of time The method of cultivating is mentioned. Since oxygen is abundantly supplied, it is possible to grow plants at high speed, so a method of cultivating while always supplying nutrient solution is preferable, and a method of cultivating by circulating the nutrient solution is more preferable in terms of cost merit. . Under such high-speed cultivation conditions, the effects of the present invention are remarkably exhibited.

  Next, the cultivation days in the cultivation process are usually 10 days or more, preferably 15 days or more, more preferably 17 days or more, and usually 45 days or less, preferably 40 days or less, more preferably 35 days or less. is there. In addition, when performing the seedling process mentioned later by hydroponics here, it shall include in the cultivation days of a cultivation process.

  In the cultivation process, transplantation may be performed as many times as necessary. The time for transplantation is preferably 3 to 18 days after sowing or transplantation.

  In a cultivation process, as long as a plant can be grown on the said conditions, any plant production system may be used and it is not specifically limited. A semi-closed or closed plant factory is preferable because of easy adjustment of light energy during cultivation, and as described later, strict control of light energy by the light irradiation cycle is effective. At least part of it takes place in a closed plant factory. Examples of the semi-closed type include horticultural facilities and solar type plant factories.

  Here, the closed type plant factory means a plant factory not exposed to sunlight, and is a system for cultivating plants in a space in which temperature, humidity, carbon dioxide concentration, wavelength of artificial light, irradiation time, and the like are controlled. By using a closed plant factory, it is possible to control light, so that there is an effect that the quality of the plant and the substance produced by the plant is stabilized, and an effect that infection of pathogenic bacteria contained in the outside air can be prevented. Furthermore, it is preferable because precise control of cultivation environment conditions such as temperature, humidity, and airflow is possible, and the effects of improving the growth rate of plants in the cultivation process and the expression of useful proteins in the expression process are obtained.

  The closed plant factory includes an environment-controlled room, a plant cultivation container shelf installed in the environment-controlled room for placing a plant cultivation container, and a plant body disposed in the vicinity of the plant cultivation container shelf. Illustrated is a system including illumination that closely irradiates light. Plant cultivation container shelves can be arranged in multiple stages.

The plant cultivated in the cultivation process preferably has a plant height (cm) of 2 cm or more, more preferably 3 cm or more, and preferably 25 cm or less, more preferably 15 cm or less. . When the plant height is within the above-mentioned range, it is advantageous for improving STY (space time yield) of the closed plant factory by increasing the number of cultivation shelves.
In addition, when a plant capable of accumulating a high concentration of protein in the plant is used, if the plant height is less than or equal to the upper limit, the ratio of leaves in the entire above-ground part of the plant increases, and leaves per plant strain. The amount of harvested can be increased, and a sufficient amount of protein can be secured. In addition, an increase in the weight ratio of leaves that are easy to handle in the purification step can reduce the purification load, and as a result, it is preferable because proteins can be easily secured.
In addition, the "plant height" here means the length from the lower end of the above-ground part to the growth point, and is obtained by measuring the length of the plant height after excising the underground part of the plant immediately after harvesting. it can.

The plant cultivated in the cultivation process preferably has an above-ground fresh weight (g) of 3 g or more, more preferably 10 g or more, and preferably 100 g or less, and 70 g or less. Is more preferable.
A plant having an above-ground fresh weight within the above-mentioned range has a high growth rate, and it is preferable to use a plant in such a high growth rate because the production efficiency of useful proteins is improved. In particular, when the cultivation process is carried out under conditions that enable high-speed cultivation of plants, the effects of the present invention can be remarkably obtained by controlling the light irradiation cycle and temperature fluctuation described later. Conditions that enable high-speed cultivation include cultivation while constantly supplying the aforementioned nutrient solution, increasing the light irradiation time, increasing the amount of fertilizer components in the nutrient solution, adopting the bare-root method, etc. By appropriately combining these conditions, for example, tobacco, it is possible to achieve a fresh weight of 60 g or more in the above-ground part in a cultivation period of 28 days including raising seedlings.

  The plant cultivated in the cultivation process preferably has a leaf weight (g) of 2.5 g or more, more preferably 7.5 g or more, and preferably 80 g or less, and 60 g or less. More preferably. A plant having a leaf weight within the above-mentioned range has a high growth rate, and it is preferable to use a plant in such a high growth rate because the production efficiency of useful proteins is improved.

  Although it does not restrict | limit especially as a light source used in a cultivation process, Sunlight, a fluorescent lamp, LED, a cold cathode fluorescent lamp (CCFL, HEFL), inorganic, organic EL, etc. can be illustrated. Preferably, a fluorescent lamp, LED, and cold cathode fluorescent lamp are mentioned. The LED is preferable because it has high light conversion efficiency and power saving as compared with an incandescent bulb and an HID lamp. It is also preferable in that the amount of heat rays that cause leaf burning damage to plants is small.

  In the present invention, among the above light sources, a lighting time (hereinafter sometimes referred to as daytime) by turning on artificial light such as a fluorescent lamp, an LED, a cold cathode fluorescent lamp, and a turn-off time by turning them off. (Hereinafter, sometimes referred to as night time) Irradiation cycle (hereinafter sometimes referred to as day / night cycle) is provided at least one cycle during the cultivation process, and at least the daytime temperature is higher than the nighttime temperature. It is characterized by being controlled to 1 ° C higher. Here, one cycle of irradiation (day and night) cycle is a period including one lighting time and one light-off time, and includes one time each of lighting and light-off.

First, control of the light irradiation cycle will be described.
The lighting (daytime) time in one cycle of the irradiation cycle is preferably about 10 hours or more, more preferably 12 hours or more, further preferably 14 hours or more, particularly preferably in the case of one cycle per day. It is preferably 15 hours or longer and 23 hours or shorter, more preferably 22 hours or shorter, still more preferably 21 hours or shorter, particularly preferably 20 hours or shorter. On the other hand, the turn-off (night) time in one light irradiation cycle is preferably about 1 hour or more, more preferably 2 hours or more, still more preferably 3 hours or more, and particularly preferably 4 hours or more. 14 hours or less, more preferably 12 hours or less, still more preferably 10 hours or less.

In the case of an irradiation cycle exceeding one cycle per day, the lighting (daytime) time in one cycle of the irradiation cycle is preferably 40% or more, more preferably 50% or more, and more preferably 98%. The following is preferable, and 95% or less is more preferable.
When one cycle is an irradiation cycle that is performed for more than one day, the lighting (daytime) time in one cycle of the irradiation cycle is preferably 45% or more of the total time of day and night of one cycle, and more preferably 55% or more. 98% or less is preferable, and 95% or less is more preferable.

  The total time of day and night is not particularly limited, but is preferably at least 6 hours or more, more preferably 12 hours or more, preferably 72 hours or less, more preferably 48 hours or less. That is, it is preferably 4 cycles or less per day and 1 cycle or more in 3 days.

  Note that the light irradiated here may be pulsed light. Pulsed light is obtained by blinking an LED or the like at short intervals of 1 microsecond to 1 second. By using such pulsed light, light is emitted at a time when a plant does not need light physiologically. Since the light can be applied only for the time required for light, the photosynthesis speed can be increased and the power cost can be reduced. In this case, the irradiation time includes the time when the LED is not turned on, and is the total of the irradiation time of the pulsed light per day.

  Moreover, it is not necessary to set the conditions of the irradiation (day and night) cycle over the entire period of the cultivation process as described above. For example, the cultivation process is divided into the first half and the second half, and the conditions of the irradiation cycle are set as the above conditions only in the second half. The plant may be cultivated under the above conditions only for a certain period in the cultivation process. In this case, as a period set as said conditions, the period of 3% or more of the whole cultivation period is preferable, and the period of 10% or more is more preferable. Moreover, it is preferable that the period used as conditions of said irradiation cycle shall be the second half of a cultivation period, and it is more preferable to set it as 3% or more until the completion | finish of a cultivation period at least. This is because plants often grow rapidly at the final stage of the cultivation period, so in order to improve the robustness of the plant, it is necessary to control the light irradiation cycle and temperature fluctuation during this rapid growth period. It is particularly effective.

  Of the entire period of the cultivation process, there is no particular limitation on the conditions of the irradiation cycle when the irradiation cycle condition is not the above-mentioned condition, and this period may be 24 hours continuous daytime in a closed plant factory, or open type The plant may be cultivated in a 24-hour cycle a day under sunlight.

  Next, control of temperature fluctuation will be described. In the present specification, “air temperature” in the cultivation process means the temperature of air near the plant to be cultivated. Therefore, for example, the temperature measured at a height of 6 cm to 12 cm from the shelf surface of the plant cultivation container shelf by a thermometer installed within a radius of 30 cm of the plant is the temperature. When the temperature of the entire environment-controlled room is constant, the temperature of the entire room is referred to as air temperature, and when the temperature is set for each plant cultivation container shelf installed in the room, the temperature of each shelf is referred to as air temperature. .

  The temperature control in the cultivation process can be performed by any method in a closed plant factory, for example, an air conditioner, a cooler, a heater, or a combination thereof. As a temperature control means in a cold region, a heating device that warms the plant factory to a predetermined temperature is usually used. Specifically, for example, a heating device or a heating device using a boiler, a warm air heater, an electric heater, a heating lamp, a heat pump, or the like is used, and air in a plant factory or a nutrient solution used for hydroponics Can be heated to a predetermined temperature and temperature controlled.

  The temperature in the cultivation process is not particularly limited as long as the daytime temperature is controlled at least 1 ° C higher than the nighttime temperature, but the daytime temperature may be 2 ° C higher than the nighttime temperature. More preferably, it is 3 ° C. or more, more preferably 4 ° C. or more, further preferably 5 ° C. or more, and particularly preferably 8 ° C. or more, since the expression efficiency of the target protein is improved. Moreover, as an upper limit of the temperature difference of daytime and night time, Preferably it is 30 degrees C or less, More preferably, it is 25 degrees C or less, More preferably, it is 20 degrees C or less, Preferably it is 15 degrees C or less. Therefore, when the daytime air temperature is set to 25 ° C, the nighttime air temperature is preferably 5 to 24 ° C, more preferably 10 to 23 ° C. The temperature of each daytime or nighttime does not need to be always constant, and may be within the above range as an average value, but preferably within ± 5 ° C. within each period of daytime and nighttime, preferably Is preferably controlled within ± 2 ° C., more preferably within ± 1 ° C.

  The temperature in the cultivation process is not particularly limited, but the temperature during lighting (daytime) in the irradiation (day and night) cycle is preferably 18 ° C or higher, more preferably 20 ° C or higher, and preferably 35 ° C or lower. More preferably, it is 33 degrees C or less. The temperature of the light-off (night) time in the irradiation cycle is preferably 5 ° C or higher, more preferably 10 ° C or higher, preferably 28 ° C or lower, more preferably 26 ° C or lower.

  The cultivation environment for growing the plant so as to satisfy these conditions is not particularly limited as long as it is a condition suitable for the growth of the plant and the production of useful proteins. For example, it can be cultivated under the following conditions.

Based on the fluctuation of the air temperature in the day-and-night cycle, the temperature of the nutrient solution used for hydroponics (hereinafter also referred to as “liquid temperature”) usually varies accordingly. However, the liquid temperature is not necessarily changed, and the liquid temperature may be constant throughout the day or night, or may be controlled so that the liquid temperature fluctuates in a range different from the temperature fluctuation of the day / night cycle. “The liquid temperature is constant” means that the temperature difference in a specific period is within ± 2 ° C., preferably within ± 1 ° C.
At this time, it is preferable that the liquid temperature at night time is controlled to be higher than the temperature at night time, and more preferably higher than the air temperature. For example, the liquid temperature during the night time is preferably controlled within the range of 10 ° C. to 10 ° C., more preferably 1-5 ° C. higher. Specifically, when the nighttime air temperature is 20 ° C, the liquid temperature is 20-30 ° C, and preferably 21-25 ° C.
Moreover, it is preferable that the difference between the liquid temperature and the air temperature during night time is smaller than the difference between the liquid temperature and the air temperature during daytime.

The liquid temperature does not always need to be constant, and may vary within a range of 10 to 30 ° C., preferably 15 to 27 ° C. Therefore, the temperature difference between the nighttime temperature and the liquid temperature also varies, It may be controlled by an average value throughout the night time.
In addition, when controlling the liquid temperature to fluctuate day and night, it is only necessary that the liquid temperature at night time is controlled to be higher than the air temperature. Like the air temperature, the liquid temperature at daytime is higher than the liquid temperature at night time. Alternatively, on the contrary, the liquid temperature at nighttime may be higher than the liquid temperature at daytime.

  On the other hand, the liquid temperature during the daytime is not particularly limited, but is preferably controlled to be equal to or lower than the daytime air temperature. Usually, in order to promote the growth of plants, the daytime temperature tends to be controlled relatively high, so that the liquid temperature is preferably lower than this from the viewpoint of dissolved oxygen in the nutrient solution. For example, the liquid temperature during the daytime is preferably 1 ° C. or more lower than the air temperature during the daytime, more preferably 2 ° C. or more, and further preferably 3 ° C. or more. Specifically, the liquid temperature is 28 ° C. or lower during daytime, preferably 25 ° C. or lower, and 10 ° C. or higher, preferably 15 ° C. or higher during night time.

  The temperature (liquid temperature) of the nutrient solution in the cultivation process can be controlled by any method in a closed plant factory. For example, when the nutrient solution is circulated, a heater or a cooler is installed in the tank. The method of controlling the temperature of the nutrient solution put in and sent out from the tank is mentioned. In addition, you may control liquid temperature to different temperature in each cultivation shelf.

Presumably, plants obtained under the above conditions have improved robustness compared to normal plants and are resistant to mechanical damage caused by reduced pressure treatment when infiltrating Agrobacterium. Is done. In addition, plants with improved robustness are also considered to have biological resistance to Agrobacterium, growth after Agrobacterium infection is promoted, and a sufficient amount of biomass can be expected. It seems that expression is also improving.
Although the reason why the physical strength of the plant is improved by the cultivation method of the present invention is not necessarily clear, for example, the energy transduction synthesized by leaf photosynthesis (the substance synthesized in a certain part of the plant is sieved). It is transported to other parts through the tube), and it is able to withstand mechanical damage when infiltrating Agrobacterium by being promoted during the night time and making the plant itself robust. It is presumed that the plant in the state can be obtained. It is also assumed that biological resistance to Agrobacterium was acquired. As a result, it is considered that the expression efficiency of the target protein in transient expression was improved.

  In general, in the cultivation process, continuous light irradiation for 24 hours is considered preferable when the plant itself grows larger, that is, when the amount of biomass increases, so that the amount of recovered protein is increased. However, in the method of the present invention, even if the amount of biomass is reduced in order to improve the productivity of plant factories (increase in space efficiency due to multi-stage cultivation shelves, etc.), the amount of protein expressed (the amount of target protein per total plant leaf weight) ) Can be increased to improve the final protein recovery. In addition, unexpectedly, it was revealed that the amount of biomass after Agrobacterium infection can be increased and the total amount of the target protein can be increased.

  The promotion of commutation is considered to be influenced not only by turning off the artificial light but also by the temperature. Furthermore, in the case of hydroponics, on the basis of the relationship between the temperature at which the above-ground plant is in contact with the liquid temperature at which the root part is in contact, for example, when the above-ground temperature is lower than the liquid temperature, the commutation is particularly It is considered to be promoted.

  In addition, because it is possible to cultivate highly robust plants by cultivating under the above conditions, it is produced in the expression process by preventing mechanical damage of plants when infiltrating Agrobacterium, especially leaf damage It is considered that impurities can be prevented from being mixed into the target protein and the expression efficiency of the target protein is improved. For example, preventing the generation of denatured proteins due to leaf damage. By suppressing the production of the denatured protein, the purity of the target protein is improved, and as a result, the expression efficiency is improved.

  From the above, the plant obtained in the cultivation process of the present invention has resistance to Agrobacterium infection, and the polynucleotide introduced using Agrobacterium in the infection process expresses the protein with high efficiency. Is possible. As a result, it is considered that the expression efficiency and expression level of the target useful protein can be improved. This effect can be expected regardless of the type of useful protein.

Moreover, such an effect is further effective by controlling daytime and nighttime humidity, carbon dioxide concentration, and fertilizer concentration in circulating water in the cultivation process.
The carbon dioxide concentration in the cultivation process is usually 300 ppm or more, preferably 500 ppm or more, and usually 5000 ppm or less, preferably 3000 ppm or less. For example, when the daytime carbon dioxide concentration is set at least 100 ppm higher than the nighttime carbon dioxide concentration, preferably 500 ppm or more and 1000 ppm or more, the effect of the present invention may be remarkable.

  The relative humidity in the plant factory in the cultivation process is preferably 20% or more, more preferably 30% or more, and is usually 100% or less, preferably 95% or less. For example, by providing a difference within 50% between the relative humidity of daytime and nighttime, the effect of the present invention may become remarkable.

  Moreover, as one preferable embodiment of this process, the fertilizer density | concentration melt | dissolved in the nutrient solution is optimized according to a cycle of day and night, and includes circulating the day and night with a constant liquid temperature. The constant liquid temperature need not always be constant, and may be a predetermined temperature as an average value, but is preferably controlled within ± 2 ° C., more preferably within ± 1 ° C. of the predetermined temperature. Is desirable. The optimum fertilizer concentration differs depending on, for example, the components of the fertilizer, but it is possible to increase the concentration during daytime when photosynthesis is active and relatively reduce the concentration during nighttime. Or about the nutrient for strengthening the structure of a plant body, you may make the nighttime density | concentration relatively high.

  In addition, in the manufacturing method of this invention, it is preferable to perform a seedling raising process in the first half of a cultivation process. The seedling raising process refers to a process until a seedling of a plant is germinated and grown in an artificial environment for a certain period and transplanted to a subsequent cultivation process. Conditions such as temperature and humidity in the seedling raising process can be the same as the conditions in the cultivation process. In addition, light irradiation conditions may be normal conditions using sunlight, fluorescent lamps, LEDs, cold cathode fluorescent lamps (CCFL, HEFL), inorganic / organic EL, etc. It is preferable to grow plants under a cycle in which the irradiation time is 12 hours or more and 24 hours or less per day. The “light irradiation time of 12 hours or more and 24 hours or less per day” is not necessarily continuous irradiation. For example, when the irradiation time is 20 hours per day, continuous irradiation of 10 hours or more is performed. You may do it twice a day. That is, it can be grown in a day / night cycle of one or more times a day.

<Infectious process>
In the infection step, the plant obtained in the cultivation step is infected with Agrobacterium having a polynucleotide encoding a useful protein by pressure cycle treatment. The pressure cycle process includes pressurization or decompression process. In the case of performing a pressure treatment that is performed at a pressure higher than atmospheric pressure (usually 1 atm = 101.325 kPa = about 0.1 MPa), at least 1.1 atm (112 kPa), 1.5 atm (152 kPa), 2 It is processed at atmospheric pressure (203 kPa), 2.5 atmospheric pressure (253 kPa), 3 atmospheric pressure (304 kPa), 4 atmospheric pressure (405 kPa) or 5 atmospheric pressure (507 kPa) or any intermediate value or any value exceeding this. It is preferably in the range of 1.7 atmospheres (172 kPa) to 10 atmospheres (1013 kPa), more preferably in the range of 4 atmospheres (405 kPa) to 8 atmospheres. In the case of performing a pressure reduction treatment at a pressure lower than the atmospheric pressure, a range of 0.005 atm (0.5 kPa) to 0.3 atm (30 kPa) is preferable, and 0.01 atm (1.0 kPa) to 0.1 The range of atmospheric pressure (10.1 kPa) is more preferable, and the range of 0.02 atmospheric pressure (2.0 kPa) to 0.06 atmospheric pressure (6.1 kPa) is more preferable. When the pressure of decompression is too high, infiltration of the infectious solution becomes insufficient, which is not preferable. On the other hand, when the pressure is too low, the liquid reaches the boiling point and remarkably evaporates to decrease the liquid, resulting in insufficient infiltration and a large manufacturing process and equipment, which is also not preferable. Therefore, the time for these pressure treatments or pressure reduction treatments can be appropriately set according to the type of plant and the tissue to be treated, but is 10 seconds to 10 minutes, preferably 20 seconds to 5 minutes, More preferably, it is about 30 seconds to 3 minutes.

  The polynucleotide encoding a useful protein is a polynucleotide encoding a target useful protein. The target useful protein is a protein exemplified as a useful protein. As the polynucleotide, a natural sequence that has been appropriately mutated or modified within the range in which the intended useful protein can be obtained may be used.

  In order to overexpress a polynucleotide encoding a useful protein in a plant, the polynucleotide is operably linked downstream of an appropriate promoter, and the resulting polynucleotide construct is introduced into a plant cell by the Agrobacterium method. That's fine. Examples of the promoter include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the rice actin promoter, the elongation factor 1β promoter, and the corn ubiquitin promoter.

  When performing the Agrobacterium method, a binary vector or an intermediate vector containing T-DNA (transfer DNA) derived from Agrobacterium Ti plasmid or Ri plasmid can be used (Nucl. Acids Res. 12 (22): 8711-8721 (1984), Plasmid, 7, 15-29 (1982)). Specific examples of binary vectors include pBI vectors (for example, pRiceFOX), pPZP vectors (Plant Molecular Biology 25 (6): 989-94. (1994)), pCAMBIA vectors (vector backbone: pPZP vector), pSMA systems A vector (Plant Cell Reports 19: 448-453. (2000)) can be mentioned, but is not limited thereto.

  In order to express the polynucleotide construct, a transient expression method using these vectors can be used (Journal of Virological Methods, 105: 343-348 (2002)). In addition, infection with Agrobacterium is preferably performed under reduced pressure conditions, and it is more preferable to use a transient expression method based on vacuum described by Plant Science, 122, 1: 101-108 (1997)). . By agroinoculation or agroinfiltration, the Agrobacterium containing the polynucleotide construct becomes tissue, eg leaves, parts of the plant's ground structure (including stems, leaves and flowers), other parts of the plant (stems , Roots, flowers), or into the space between whole plant cells. After passage through the epidermis, Agrobacterium infects and transfers the polynucleotide into the cell. The polynucleotide is transcribed as an episome and the mRNA is translated, resulting in production of the protein of interest in the infected cell, but passage of the polynucleotide within the nucleus is transient.

<Expression process>
In the expression step, the plant after the infection step is cultivated to express the useful protein. The cultivation conditions in the expression step are not particularly limited as long as the useful protein can be efficiently expressed. Conditions such as temperature and humidity in the expression step can be the same as the conditions in the cultivation step. Moreover, the normal conditions using sunlight, a fluorescent lamp, LED, a cold cathode fluorescent lamp (CCFL, HEFL), inorganic / organic EL, etc. are employable as light irradiation conditions. Alternatively, it is possible to set conditions such as day / night cycle and temperature / humidity suitable for expression depending on the type of protein to be expressed or not. The cultivation days in the expression step are preferably 3 days or more, more preferably 4 days or more, and preferably 14 days or less, more preferably 10 days or less.

Specific examples of conditions such as temperature and humidity among the cultivation conditions in the expression step are given below, but the cultivation conditions are not limited thereto as long as the useful protein can be efficiently expressed.
The cultivation temperature is usually 10 ° C or higher, preferably 15 ° C or higher, and is usually 40 ° C or lower, preferably 37 ° C or lower. The relative humidity in the plant factory is usually 20% or more, preferably 30% or more, and usually 100% or less, preferably 95% or less. The carbon dioxide concentration in the plant factory is usually 300 ppm or more, preferably 500 ppm or more, and usually 5000 ppm or less, preferably 3000 ppm or less.

<Useful protein recovery process>
In the expression step, the useful protein accumulated in the plant is preferably recovered from the plant. It is preferable to obtain a fraction containing a useful protein from a plant and purify the useful protein by an appropriate method. The polynucleotide encoding the useful protein may contain a tag sequence for purification.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited at all by these Examples.

[Example 1]
1. Preparation of transformed Agrobacterium 1.1 Expression plasmids The following two types of expression plasmids were used for the examination of GFP (jellyfish green fluorescent protein) expression.
A kanamycin resistance expression cassette (consisting of a nopaline synthase gene promoter, a kanamycin resistance gene, and a nopaline synthase gene terminator) of a plant binary vector pMM444 (Japanese Patent Laid-Open No. 9-313059) The hygromycin resistance expression cassette (cauliflower mosaic virus 35S promoter, castor catalase gene first intron, hygromycin resistance gene, and nopaline synthase gene terminator) was substituted. The plasmid thus obtained was further added to a GUS expression cassette derived from pIG221 (Plant Cell Physiol., 31, 805 (1990)) (cauliflower mosaic virus 35S promoter, castor catalase gene first intron, β- An EGFP expression cassette was prepared by introducing an EGFP expression cassette obtained by replacing the β-glucuronidase gene of the glucuronidase gene and the nopaline synthase gene terminator with an EGFP gene (pEGFP-N3: manufactured by Clontech) (hereinafter referred to as “EGFP gene expression plasmid”). This plasmid is called “pGFP / MM444. The structure is shown in FIG. 1).

In FIG. 1 and FIG. 2, the meanings of symbols indicating genes and their control regions are as follows.
35SP: cauliflower mosaic virus 35S promoter int: castor bean catalase gene first intron Nos ^t: nopaline synthase terminator Spec ^R: spectinomycin resistance gene Tc ^R: tetracycline resistance gene Hm ^R: hygromycin resistance gene ^Ori pBR322: pBR322 ori
Ori ^pRK2 : pRK2 ori
BL: T-DNA left border
BR: T-DNA right border

  Further, the hygromycin resistance expression cassette in pGFP / MM444 was deleted, and then the EGFP gene in the EGFP expression cassette was replaced with the P19 gene (derived from tomato bushy stunt virus) to prepare a P19 gene expression plasmid (hereinafter referred to as this plasmid). Is referred to as “p19 / MM444.” The structure is shown in FIG. The P19 gene has a function of enhancing the expression of the EGFP gene, and this p19 / MM444 was subjected to co-expression with pGFP / MM444.

1.2 Transformation of Agrobacterium and preparation of transformed Agrobacterium glycerol stock Each of the above plasmids (pGFP / MM444, p19 / MM444) was transformed into Agrobacterium (Mattanovich et al. 1989) by electroporation (Mattanovich et al. 1989). Agrobacterium tumefaciens AGL1: Rhizobium radiobacter ATCC BAA-101; American Type Culture Collection (ATCC), Manassas, VA20108, USA) strain (obtained transformed Agrobacterium, GFP-Agrobacterium, Called P19-Agrobacterium).

  After culturing the transformed Agrobacterium (GFP-Agrobacterium, P19-Agrobacterium) in LB medium (SIGMA-ALDRICH) containing carbenicillin 25 μg / ml and spectinomycin 50 μg / ml, glycerol was added. In addition, the glycerol concentration was finally adjusted to 30% and stored at −80 ° C. to obtain each transformed Agrobacterium glycerol stock.

1.3 Preparation of transformed Agrobacterium for infection process The glycerol stock of transformed Agrobacterium (GFP-Agrobacterium, P19-Agrobacterium) prepared in 1.2 above was inoculated in LB medium, Culture was performed.
After culturing, the cells were collected by centrifugation, and the obtained cells were suspended in an infiltration buffer (5 mM MES, 10 mM MgCl ₂ , pH 5.6) to obtain a concentrated bacterial solution. The concentrated bacterial cells obtained were adjusted to pH 5.6 by adding to a 4 L infiltration buffer so that the OD600 of a 1: 1 mixed bacterial solution of GFP-Agrobacterium and P19-Agrobacterium was 0.8. Agrobacterium solution for infection process (for GFP / P19 co-expression) was used.

2. Preparation of plant biomass 2.1 Sowing Liquid fertilizer for sowing (Otsuka House S1 (Otsuka Agritechno Co., Ltd.) 0.78 g / L, Otsuka House 2 (Otsuka Agritechno Co., Ltd.) 0.25 g / L, pH 5.0 ) Into a urethane mat for hydroponics (Ematsu Kasei W587.5mm x D282mm x H28mm: 12 x 2 mass, hole diameter φ9mm), placed in a seedling tray (W600mm x D300mm x H300mm), and benthamiana tobacco ( Nictiana benthamiana) seeds were sown.

2.2 Raising seedlings Plants after sowing were grown for 12 days in an artificial meteorological device (NC-410HC) (Nippon Medical Instruments Co., Ltd.) at an air temperature of 28 ° C., 16 hours day / 8 hours night light cycle. Here, a constant temperature was maintained in the space inside the meteorological device.

2.3 Cultivation (previous term)
The urethane mat used for raising seedlings in 2.2 was separated one by one and transplanted to a cultivation (previous term) panel (W600 mm × D300 mm, 30 holes). The planting (first term) panel after transplanting was set in a cultivation device, and cultivated for 9 days by a deep flow technique (DFT system). Environmental conditions and nutrient solution conditions were controlled as follows. In addition, the thermometer for measuring air temperature is a position where a plurality of plants exist within a radius of 5 cm, and is installed so that the temperature sensor comes to a height of 6 cm to 12 cm from the shelf surface of the plant cultivation container shelf. .

"Environmental condition"
-Temperature: 28 ° C
-Relative humidity: 60-80%
-CO ₂ concentration: 500ppm
Illumination: Average photosynthetic photon flux density (PPFD): 160 μmol / m ² · second, 24 h continuous irradiation, three-wavelength fluorescent lamp “Lupica Line” (Mitsubishi Electric Corporation)

《Nutrient conditions》
As liquid fertilizer, fertilizer A liquid (Otsuka House S1 150g / L, Otsuka House 5 (Otsuka Agritechno Co., Ltd.) 2.5g / L) and Fertilizer B Liquid (Otsuka House 2 100g / L) are dechlorinated, respectively. Dissolved in water and mixed in equal amounts. For pH adjustment, a pH adjuster down (Otsuka Agritechno Co., Ltd.) and a 4% KOH aqueous solution were used. The electrical conductivity (EC) and pH of the nutrient solution were adjusted to EC: 2.3 mS / cm and pH 6.0 using “Raku-Raku Fertilizer Manager 3” (Sem Corporation). The temperature of the nutrient solution was controlled at 25 ° C. The nutrient solution temperature was controlled at 25 ° C. by cooling using a carry cool (Orion Machinery Co., Ltd.). Plants were cultivated by circulating a nutrient solution having a constant EC, pH and temperature as described above.

2.4 Cultivation (late stage)
A plant body was taken out from the panel for cultivation (first half) and planted in a panel for cultivation (late stage) (W600 mm × D300 mm, 6 holes). A panel for cultivation (late stage) after transplantation was set in a cultivation apparatus and cultivated for 7 days (28 days after sowing) by the DFT method. Environmental conditions were controlled as follows. The nutrient solution conditions were controlled to the same conditions as in cultivation (first half). In addition, temperature was measured by the method similar to cultivation (pre-term).
Table 2 shows the total weight of plants after completion of cultivation (late stage) and before performing 3.1 infection described later.

"Environmental condition"
-Temperature: 28 ° C (daytime) / 26 ° C (nighttime)
-Relative humidity: 45-65%
-CO ₂ concentration: 500ppm
Illumination: Average PPFD: 180 μmol / m ² · second, 20 h continuous light irradiation, 4 h extinction light / dark cycle, three-wavelength fluorescent lamp “Lupica Line (registered trademark)” (Mitsubishi Electric Corporation)

3. 3. Agrobacterium infection in plants and plant collection 3.1 Infection by Vacuum Infiltration (Vacuum Infiltration Method) Agrobacterium in a beaker by turning upside down the benthamiana cigarette 28 days after sowing obtained in 2.4 above All leaves were submerged in the um fungus solution (prepared in 1.3 above) so that it was completely immersed in the solution.
Then, this beaker was put into a vacuum desiccator (FV-3P) (Tokyo Glass Instrument Co., Ltd.), left at 19 to 40 Torr for 1 minute, and decompressed. After that, the valve was opened at once to restore the pressure.
After completion of the decompression, the plant was returned to an upright position and fitted into a cultivation (late stage) panel, and then set in a cultivation apparatus.

3.2 Cultivation of plants after infection (expression process)
Cultivation after infection was carried out using a cultivation device in an artificial meteorological instrument (manufactured by Nippon Medical Instruments Co., Ltd.). A three-wavelength fluorescent lamp “Lupica Line (registered trademark)” (Mitsubishi Electric Corporation) was used as a light source, and was cultivated at an average PPFD of 125 μmol / m ² · sec with a light cycle of 16 hours day / 8 hours. The nutrient solution was cultivated as EC: 2.1-2.4 mS / cm, pH 5.6 by the dripping method (deep flow technique, DFT method). The temperature was a cycle of 25 ° C. day / 20 ° C. night. The relative humidity was 60 to 85%. The temperature of the nutrient solution was controlled at 20 ° C. The liquid fertilizer was cultivated in the same manner as in the cultivation (previous term) with the EC, pH and temperature being constant as described above, and the nutrient solution was circulated.

3.3 Collection of cultivated infected leaves Agrobacterium-infected Bensamiana tobacco that had undergone the above-described expression process over 6 days harvested all leaves without petioles and stored them at -80 ° C. .
Table 2 shows the total plant weight and the harvested leaf weight after cultivation in the expression step of 3.2.

4). 4. Measurement of GFP expression level 4.1 Preparation of crude extract The GFP / P19 co-expressing Agrobacterium-infected leaves cryopreserved in 3.3 were transferred to a mortar and ground in liquid nitrogen. Then, 6 times the sample fresh weight of GFP assay extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 0.1% Triton-X100 (pH 7.25)) was added and the protein was suspended vigorously. Crude extraction was performed. 1 ml of the crude extract was transferred to a 1.5 ml Eppendorf tube, centrifuged at 4 ° C., 20,400 × g for 10 minutes, and the supernatant was collected and used for GFP quantification described later.

4.2 GFP quantification For detection of GFP fluorescence, 507 nm emission generated by excitation light of 485 nm was detected using Wallac ARVO SX 1420 Multilabel counter (Perkin-Elmer Life Sciences). For quantification, serially diluted GFP standard (rAcGFP1 Protein: manufactured by Takara Bio Inc.) was used. The measurement sample was diluted 5-fold with GFP extraction buffer, and 100 μL each was dispensed into a 96-well microplate (Nunc fluoronunk plate: manufactured by Thermo Fisher Scientific Co., Ltd.), measurement was performed, and GFP expression per fresh weight The amount (mg / kg-FW) and the amount of GFP expression per strain (mg) were calculated. The measurement results of the three strains were averaged, and the ratio when the value (the following Comparative Example 1) at 24 h continuous light irradiation and constant temperature in cultivation (late stage) was 100% is shown in Table 1.

[Comparative Example 1]
In the adjustment of plant biomass, sowing, raising seedling, and cultivation (first half) are performed under the same conditions as in Example 1. In 2.4 cultivation (second half), there is no day / night cycle, continuous light irradiation for 24 hours, temperature difference between day and night The experiment was conducted at a constant 27 ° C.
The measurement results of the three strains were averaged for GFP quantification, and the results are shown in Table 1 with this value taken as 100%. Table 2 shows the total plant weight and the harvested leaf weight measured in the same manner as in Example 1.

From the above results, it was confirmed that the expression of the target protein was improved in the subsequent expression step when the irradiation cycle and temperature were controlled in the cultivation step. In addition, in the cultivation process, the amount of biomass was increased by continuous light irradiation, whereas after passing through the infection process and the expression process, Example 1 in which the irradiation cycle and temperature were controlled in the cultivation process was final. As a result, the amount of biomass was increased. This is presumably because the growth of the plant after the infection process progressed well due to the fact that a highly robust plant was obtained in the cultivation process.

Claims (5)

A method for producing a useful protein using a plant,
Hydroponically cultivating the plant in a closed plant factory;
Infecting the plant after the cultivation process with Agrobacterium having a polynucleotide encoding a useful protein by pressure cycle treatment;
Expressing a useful protein in the plant after the infection step,
In at least a part of the cultivation process, there is an irradiation cycle of 1 cycle or more in the cultivation process by turning on and off the artificial light, and the lighting time temperature is within a range of 2 to 30 ° C. higher than the lighting time temperature. Control
The temperature difference between the turn-on time and the turn-off time of the nutrient solution used for hydroponics is controlled within a range of ± 1 ° C.,
The said plant is tobacco, The manufacturing method of useful protein characterized by the above-mentioned. Temperature of nutrient solution used for the hydroponic cultivation, during the off time, the production method of a useful protein of claim 1 which is controlled by the temperature or higher. The method for producing a useful protein according to claim 1 or 2 , further comprising a step of purifying and recovering the useful protein after the expression step. The method for producing a useful protein according to any one of claims 1 to 3 , wherein the useful protein is a protein for medical use. The method for producing a useful protein according to any one of claims 1 to 4 , wherein the tobacco is Bensamiana tobacco.