KR20160024586A - Method for high-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and cell recycling system - Google Patents

Abstract

The present invention provides a method for producing a target protein via suspension culturing of transgenic rice cells, which comprises the following steps of: (a) suspense-culturing transgenic rice cells in a sugar-rich medium by using an expression vector including a gene encoding a promoter and a target protein operating when sugar is deplete to grow the rice cells; and (b) transferring the rice cells to an alternative carbon source-containing medium having no sugar and culturing the rice cells to produce a target protein from the rice cells. In addition, the present invention provides a method for producing a target protein by recycling transgenic rice cells, which comprises the following steps of: (a) suspense-culturing transgenic rice cells in a sugar-rich medium by using an expression vector including a gene encoding a promoter and a target protein operating when sugar is deplete to grow the rice cells; (b) transferring the rice cells to a medium including a mixture of fumaric acid and sucrose and culturing the rice cells to produce a target protein from the rice cells; and (c) transferring the cultured rice cells to a new medium including a mixture of fumaric acid and sucrose and culturing the rice cells to produce a target protein from the rice cells. According to the present invention, productivity can be improved at low costs and high efficiency in mass-production of recombinant proteins from transgenic rice cells.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing recombinant trypsin using recombinant trypsin in a transgenic rice cell culture,

More particularly, the present invention relates to a method for mass production of recombinant trypsin using a substitute carbon source in a transgenic rice cell culture, and more particularly, to a method for mass production of recombinant trypsin through a cell recycling system, Culturing the rice cells transformed with the expression vector containing the rice cell suspension in a sugar-rich medium to grow the rice cells; And (b) culturing the rice cells in a medium containing no sugar and an alternative carbon source, followed by culturing the rice cells to produce a target protein from the rice cells, and a) suspending the rice cells transformed with the expression vector containing the promoter and the gene encoding the target protein in a sugar-rich medium to induce the rice cell growth; (b) transferring the rice cells to a medium containing a mixture of fumaric acid and sucrose, and then culturing to produce a target protein from the rice cells; And (c) culturing the cultured rice cells in a culture medium containing a mixture of fresh fumaric acid and sucrose to produce a target protein from the rice cells. Provide a production method.

Production of recombinant proteins in plant cells is carried out by transforming plant cells with an expression vector containing a promoter and a target gene capable of expressing in a plant cell. As the basic medium that can be used for plant cell culture, AA, MS, N6 , WHITE, and SH medium. Plant cell culture does not contain animal-derived viruses and toxins compared with animal cell cultures, and many studies have been conducted in that the purification process is simple and economical compared to animal cell culture ought

On the other hand, in the case of rice cells, 20-40% of the total protein secreted into the culture medium in the suspension culture is RAmy3D and RAmy3E, which are α-amylase gene families, and these two genes are expressed in the medium Lt; / RTI > In particular, RAmy3D is known to increase the expression level 165 times when the sugar in the medium is depleted. On the contrary, when the sugar such as sucrose, glucose, fructose and galactose is present in the medium, expression of RAmy3D gene is suppressed. When such an inducible promoter is used for culturing, it is possible to carry out a two-step culture in which the sugar is added to increase the concentration of cells and improve the productivity by depletion of sugar. Thus, recently, As a representative example of a cell, a recombinant protein is produced using RAmy3D promoter in rice cells.

However, the RAmy3D promoter system remains a limitation in many biological and technical terms, because the method of medium exchange leading to glucose depletion in the culture medium may lead to apoptosis due to loss of carbon source for energy metabolism . This may also lead to cell rupture due to changes in osmotic pressure, various factors may cause apoptosis, and since the proteolytic enzyme may accumulate in the cells during production and be secreted into the culture medium, the production of the desired recombinant protein . Thus, the addition of some alternative carbon sources has been made to improve recombinant protein production, and in recombinant antitrypsin (rAAT) production, glucose and pyruvate have been shown to enhance rAAT production in transgenic rice cell suspension cultures Terashima et al., Biotechnol Progt 2001; 17: 403-406). In order to improve the synthesis of alkaloids in Catharanthus roseus suspension cell cultures, both succinic acid, malic acid and citric acid participating in the citric acid cycle were added to the medium, but each showed very different effects in alkaloid production. Succinic acid and malic acid significantly increased alkaloid production, but citric acid had no significant effect (Zhao et al. Biotechnol Lett 2000; 22: 1221-1226).

Thus, the present inventors have added succinic acid, fumaric acid and malic acid to fructose and pyruvic acid in order to analyze intermediates in the citric acid circuit, confirming that they have a positive effect. Of these, fumaric acid was selected for the next experiment because it showed the best effect on the production of recombinant trypsin. If the present inventor can bypass the reaction step of producing such mediator by adding a suitable carbon source, the energy source for recombinant protein synthesis may be supplied at a high concentration. In the present invention, a method for improving the yield of recombinant trypsin using several alternative carbon sources and a recycling batch culture system at the flask size have been developed and the possibility of using a combination of sucrose and fumaric acid for economical recombinant trypsin production in a large- .

Korean Patent No. 1171269 discloses a plant cell culture medium containing a plant-derived protein hydrolyzate and a method for mass production of recombinant protein, and in Korean Patent No. 1244025, a recombinant vector for transformation of rice and a A method for mass production of trypsin has been disclosed. However, as described in the present invention, there is no known method for mass production of recombinant trypsin through the use of an alternative carbon source and a cell recycling system in a transgenic rice cell culture.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide a recombinant trypsin capable of producing a recombinant trypsin which is effective for the production after suspension of transgenic rice cells, In order to select the alternative carbon source and induction time, trypsin production was investigated by adding fructose, pyruvic acid, citric acid, succinic acid, fumaric acid and malic acid after various starvation conditions. As a result, 40 mM fumaric acid was added for 5 days, It was confirmed that the highest yield was obtained.

In addition, in order to overcome the problem that the suspension cultured cells can not be reused after sugar depletion, which is one of the biggest problems in the development of mass production system of recombinant trypsin in transgenic rice cell suspension culture, in a medium containing 3% sucrose After 4 days, the culture medium of the suspension culture cells was removed by inhalation, followed by addition of a medium containing 40 mM fumaric acid and 1% sucrose. After culturing for 4 days, the rice cultured cells were collected again and fresh 40 mM fumaric acid And 1% sucrose was added to the culture medium. As a result, it was confirmed that the suspension cultured cells could be used five times for the production of recombinant trypsin. Thus, the present invention was completed.

In order to achieve the object of the present invention,

(a) suspending the rice cells transformed with an expression vector containing a promoter operable at the time of starvation and a gene encoding a target protein in a sugar-rich medium to grow the rice cells; And

(b) culturing the rice cells in a medium containing no saccharide and an alternative carbon source, followed by culturing the rice cells to produce a target protein from the rice cells, and .

In addition,

(a) suspending the rice cells transformed with an expression vector containing a promoter operable at the time of starvation and a gene encoding a target protein in a sugar-rich medium to grow the rice cells;

(b) transferring the rice cells to a medium containing a mixture of fumaric acid and sucrose, and then culturing to produce a target protein from the rice cells; And

(c) culturing the cultured rice cells in a culture medium containing a mixture of fresh fumaric acid and sucrose, and then producing the desired protein from the rice cells. ≪ / RTI >

According to the present invention, 40 mM fumaric acid is most suitable as an alternative carbon source in a transformed rice cell culture in which the expression is regulated by the RAmy3D promoter to produce recombinant trypsin, and using a medium containing 40 mM fumaric acid and 1% sucrose It is possible to provide a simplified recombinant protein production process that does not require a medium exchange operation in the stage of cell growth and the stage of production of recombinant protein and by allowing the suspension culture rice cell to be used five times, It can be used to increase the productivity at low cost and high efficiency in the mass production of recombinant protein in transgenic rice plant cell culture through the development of a cheaper production cost and a simplified mass production process.

Figure 1 shows the result of selection of an alternative carbon source (80 mM) effective for production of recombinant trypsin. (A) The production of recombinant trypsin was measured by ELISA directly during the batch suspension culture with the addition of the alternative carbon source (80 mM). Ctrl (control) represents a culture medium containing no alternative carbon source in which transgenic rice cells (T111-04) expressing high levels of recombinant trypsin were cultured. The error bars represent the standard error of the results of three replicate cultures. (B) Northern blot analysis was used to measure the expression of recombinant trypsin gene in transgenic rice cell suspension cultures. Lane NC is a total RNA extract of untransformed rice cells as a control. Lane 1 is a total RNA extract of transgenic rice cells (T111-04) cultured in medium containing no alternative carbon source. Lanes 2 to 6 are each a medium containing an alternative carbon source (fructose, pyruvic acid, citric acid, succinic acid, fumaric acid and malic acid) The total RNA extracts of the transgenic rice cells cultured in the medium were used. (C) Standard loading was expressed as EtBr stained rRNA.
Figure 2 shows the effect of alternative carbon sources on the production and activity of recombinant trypsin. (A) Various concentrations of succinic acid, fumaric acid and malic acid were added to the culture medium to determine the optimal concentration of each alternative carbon source. ELISA, SDS-PAGE, western blot analysis and zymography over time (B, C, D and E) were performed to ascertain maximum trypsin yield and activity. Ctrl (control) represents a culture medium containing no alternative carbon source cultured transgenic rice cells (T111-04) expressing high levels of recombinant trypsin. The error bars represent the standard error of the results of three replicate cultures. Lane M is a pre-stained molecular weight standard (PageRuler ™ Protein Ladder). Lane Bt is a positive control and 5 ng of purified trypsin from Bos taurus . Lane Zm is 5 ng of purified trypsin from maize ( Zea mays ) as a positive control. Lane NC is a negative control and is a culture of 5 ug of untransformed rice cells. Lanes 1, 2, 3, and 4 in the following (C), (D), and (E) show no sucrose (lane 1), no sucrose and no succinic acid, fumaric acid and malic acid The culture medium of the producer is 5 占 퐂. (C), the arrow indicates the position of the recombinant trypsin. A sharp band on a dark background gel shows proteolytic enzyme activity (E).
Figure 3 shows ELISA (A), cell weight (B) and cell viability (C) in combination with various concentrations of sucrose and 40 mM fumaric acid for trypsin production in transgenic rice cell suspension cultures. The reduction degree of TTC was regarded as 100% of the cells cultured in the medium containing 3% sucrose, and the degree of reduction of the cells cultured in the culture medium containing various concentrations of sucrose was calculated as a percentage Respectively. The error bars represent the standard error of the results of three replicate cultures.
Figure 4 shows the time course of the effect of 1% sucrose and 40 mM fumaric acid addition on the production of recombinant trypsin during batch suspension culture. Western blot analysis (A) and ELISA (B) were performed on the culture medium. Lane M is a pre-stained molecular weight standard (PageRuler ™ Protein Ladder). Lane PC is 100 ng of purified trypsin from maize ( Zea mays ) as a positive control. Lane NC is a negative control and 5 ug of untransformed rice cell culture. Lanes 1, 3, 5, 7, 9, and 11 are each 5 μg of the trypsin production medium. The error bars represent the standard error of the results of three replicate cultures.
FIG. 5 shows the result of confirming the repetitive production of recombinant trypsin in transgenic rice cells in medium containing 1% sucrose and 40 mM fumaric acid. T111-04 suspension cultured cells were maintained in subculture every 7 days with 3% sucrose medium. For cell re-use experiments, transgenic rice cells were cultured in 3% sucrose medium for 4 days, then the suspension culture was inhaled and removed, and then 5 g of fresh cells were treated with 0% sucrose (-S), 0% 40 mM fumaric acid, or 1% sucrose and 40 mM fumaric acid, respectively. Each culture was harvested 4 days after the culture, and the three kinds of fresh media were added again to each flask. Cell re-use was repeated 5 times over a period of 20 days. The culture medium and cells were collected for each incubation period for determination of concentration of recombinant trypsin by ELISA (A) and measurement of cell viability (B). The reduction degree of TTC was regarded as 100% of the cells cultured in the medium containing 3% sucrose, and the degree of reduction of the cells cultured in the culture medium containing various concentrations of sucrose was calculated as a percentage Respectively. The error bars represent the standard error of the results of three replicate cultures.

In order to achieve the object of the present invention,

(a) suspending the rice cells transformed with an expression vector containing a promoter operable at the time of starvation and a gene encoding a target protein in a sugar-rich medium to grow the rice cells; And

(b) culturing the rice cells in a medium containing no saccharide and an alternative carbon source, followed by culturing the rice cells to produce a target protein from the rice cells, and .

In a method according to an embodiment of the present invention, the alternative carbon source may be fumaric acid, succinic acid or malic acid, preferably fumaric acid, but is not limited thereto.

In the method according to an embodiment of the present invention, the concentration of fumaric acid may be 30 to 70 mM, preferably 30 to 50 mM, and most preferably 40 mM, but is not limited thereto. When the concentration of fumaric acid was 20 mM or less or 80 mM or more, the production amount of the target protein was remarkably decreased.

In the method according to an embodiment of the present invention, the step (b) may be a step of culturing the rice cells for 3 to 7 days in a medium containing no sugar and containing 30 to 50 mM of fumaric acid, But may be, for example, culturing rice cells for 6 days, and most preferably for culturing rice cells for 5 days. It was confirmed that when the rice cells were cultured for 2 days or less or 9 days or more in a culture medium containing 30 to 50 mM of fumaric acid, the production amount of the target protein was remarkably decreased.

In the method according to one embodiment of the present invention, the promoter that operates upon sugar deprivation may be, but is not limited to, the Rmy3D (Rice alpha-amylase 3D) promoter.

In a method according to an embodiment of the present invention, the target protein may be buttrysin, but is not limited thereto.

In addition,

(a) suspending the rice cells transformed with an expression vector containing a promoter operable at the time of starvation and a gene encoding a target protein in a sugar-rich medium to grow the rice cells;

(b) transferring the rice cells to a medium containing a mixture of fumaric acid and sucrose, and then culturing to produce a target protein from the rice cells; And

(c) culturing the cultured rice cells in a culture medium containing a mixture of fresh fumaric acid and sucrose, and then producing the desired protein from the rice cells. ≪ / RTI >

In the method according to one embodiment of the present invention, the suspension culture period in the sugar-rich medium of step (a) is preferably 4 days.

The method according to an embodiment of the present invention may be repeated three to five times, preferably four times, but is not limited thereto.

In the method according to one embodiment of the present invention, the cultured rice cells may be transferred to a medium containing fresh fumaric acid and sucrose mixture, followed by culturing for 3 to 5 days, preferably by culturing for 4 days But is not limited thereto.

When the step (c) is repeated 4 times, the total incubation time from the step (a) to the step (c) for producing the target protein of the present invention may be 20 days.

In the method according to an embodiment of the present invention, the fumaric acid and sucrose mixture in steps (b) and (c) are mixed in a concentration of 30 to 50 mM of fumaric acid and 0.5 to 1.5% (w / v) And preferably a mixture of 40 mM fumaric acid and 1% (w / v) sucrose, but is not limited thereto.

In the method according to one embodiment of the present invention, the promoter that operates upon the sugar depletion may be, but is not limited to, the RAmy3D promoter.

In a method according to an embodiment of the present invention, the target protein may be buttrysin, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples

Materials and methods

Expression systems and plant cell lines

Recombinant trypticin is a powerful promoter developed to enhance the expression of recombinant proteins and has been expressed under the control of the rice α-amylase 3D (RAmy3D) promoter induced by sucrose depletion (Shin et al. Biotechnol Bioeng 2003; 82: 778 -783). The transformed rice cell line pMYT111-04 was established and maintained in our laboratory by the methods previously described (Kusnadi et al. Biotechnol Bioeng 1997; 56: 473-484).

Plant cell culture

Transgenic rice cells were proliferated and cultured using a rotary stirrer at a rotating speed of 110 rpm under a dark condition at 28 ° C. To maintain the cell line, the suspension culture cells were suspended in 50 mL of N6 medium (2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0,02 mg / L kinetin and 3% Cross). ≪ / RTI > 10 mL of the inoculum was transferred every 7 days for subculture. In order to induce expression of the trypsin gene under the control of the RAmy3D promoter, N6 medium was removed from the cell suspension culture by inhalation and 10% of the cells (volume of cell biomass / volume of medium) contained fresh N6 (S- ) Medium. The culture supernatant was collected by passing the cell suspension culture from the culture medium of the induced rice cells through 2-3 layers of Myracloth (Calbiochem, La Jolla, CA, USA). Proteins in the culture were collected by centrifugation at 18,000 g for 10 min at 4 ° C to remove cellular precipitate.

Selection of alternative carbon sources and measurement of cell biomass and dry weight

In order to select the optimum alternative carbon source and induction time in the flask culture, the culture medium was inhaled by removing the culture medium from the cell suspension culture 7 days after the culture (growth period), and 5 g of the cells were mixed with various carbon sources In 50 mL of fresh N6 medium and then cultured for 5 days (producer). Fructose, pyruvic acid, citric acid, succinic acid, fumaric acid and malic acid were used to test the effect of alternative carbon sources. Each alternative carbon source was completely dissolved in sucrose-free N6 medium and each medium was adjusted to pH 5.8 using 1N NaOH. Reuse of cultured cells was performed using fresh N6 medium containing 1% sucrose and 40 mM fumaric acid. To determine the change in cell weight after addition of the alternative carbon source, the cell weight and dry weight were determined as follows. The initial transformed rice cells inoculated to induce protein expression in 50 mL medium had a body weight of 5 g. After 7 days of induction, the culture was removed from the cell suspension culture by inhalation and 50 mL of fresh N6 medium (without sucrose) containing various carbon sources was added and incubated for the indicated time. The cells were harvested after removal of the medium using Wattmann No. 2 filter paper under a weak vacuum pressure, and after washing three times with distilled water, the viable cell weight was measured. The dry cell weight was measured after drying the cells in a dryer at 65 ° C until a constant weight was maintained.

Northern blot analysis

For Northern blot analysis, total RNA was extracted from the N6 (-S) liquid medium and an alternative carbon source (fructose, pyruvic acid, citric acid, succinic acid, fumaric acid, and the like) using the RNeasy plant total RNA extraction kit (Qiagen, Valencia, CA, USA) And malic acid) in N6 (-S) liquid medium for 5 days. The RNA was separated by electrophoresis on agarose gel containing formaldehyde. The isolated RNA was transferred to a Hybond N ⁺ membrane (Amersham Pharmacia Biotech RPN82B, Piscataway, NJ, USA). The membrane was then hybridized with a ³² P-labeled trypsin probe in a hybridization reactor (FINEPCR Combi-H, Seoul, Korea) at 65 ° C using the Prime-a gene labeling system (Promega U1100, Madison, WI, USA). The membrane was washed twice with 2 x SSC and 0.1% SDS for 15 minutes each at 65 캜 and then twice with 2 x SSC and 1% SDS. Hybridized bands were detected by self-spinning on X-ray film (Fuji Photo Film Co., HR-G30, Tokyo, Japan).

SDS-PAGE and western blot analysis

For Western blot analysis, 5 μg of cell culture derived proteins were separated by 12% (w / v) SDS-PAGE and electroblotted onto nitrocellulose membranes. The membrane was reacted with rabbit polyclonal anti-trypsin antibody and then reacted with AP (alkaline phosphatase) conjugated anti-rabbit IgG (Calbiochem, San Diego, CA, USA) as secondary antibody. Commercial trypticin (Sigma, St. Louis, Mo., USA) and TrpyZean ™ (Sigma) from small pancreas were used as positive control. SDS-PAGE gels were stained with Coomassie Blue staining solution (0.25% Coomassie Brilliant Blue R-250, 45% methanol and 10% glacial acetic acid). Protein concentration was measured by protein analysis reagent (Sigma) based on the Bradford method, and BSA (bovine serum albumin) was used as a standard.

Quantification of recombinant trypsin

To determine the amount of recombinant trypsin, ELISA was performed in the following manner. Standard material (0.1 μg / mL) and samples were diluted with coating buffer and dispensed into ELISA plates (Thermo Scientific, Roskilde, Denmark) and reacted overnight at 4 ° C. The plate was washed with PBST buffer (2.4 g / L KH ₂ PO ₄ , 11.6 g / L Na ₂ HPO ₄ , 80.0 g / L NaCl, PBS buffer pH 7.4 with 2.0 g / L KCl and 0.05% Tween 20) Washed three times, then 200 μl of 0.1% skim milk as a blocking buffer was added to prevent nonspecific antibody reaction, and reacted at room temperature for 2 hours. After removing the blocking buffer, 100 [mu] l of 2.5 [mu] g / ml biotinylated rabbit anti-trypsin polyclonal antibody (Abcam, ab34676) was added to each well of the plate. The plate was reacted at room temperature for 2 hours and washed with PBST. 100 μl of avidin-HRP (BD Pharmingen, 554058) diluted 1: 1000 was added to each well, Lt; / RTI > Samples were washed 4 times with PBST, and 100 의 of TMB substrate solution was added to each well. The reaction was stopped by adding 20 μl of 6.3% H ₃ PO ₄ at room temperature for 20 minutes, and the absorbance of the sample was recorded at 430 nm using a microplate reader.

Measurement of cell viability

Cell viability was measured by reduction of TTC (triphenyl tetrazolium chlorice) as previously described. For the formazan-forming reaction, 0.1 g fresh cells were added to microtube containing 1.6 mL of TTC solution. The reaction was carried out at 20 ° C for 24 hours, and the supernatant was removed by centrifugation at 15,000 g for 10 minutes at 4 ° C. The live cell-derived formazan was then extracted with 1 mL of 95% ethanol at 60 ° C for 30 minutes. The supernatant was separated by centrifugation at 15,000 g for 30 minutes at 4 ° C, and the absorbance of the extracted formazan was measured at 485 nm.

Example  1. Substitution for trypsin production Carbonic  effect

Fructose, pyruvic acid, citric acid, succinic acid, fumaric acid and malic acid were investigated to identify alternative carbon sources effective in increasing trypsin production. Rice cells were maintained in a 300 mL flask containing 50 mL of N6 medium for 7 days. To induce protein expression, the culture medium was removed by inhalation after 5 days of cell culture. Five grams of cells were inoculated into 50 mL of fresh N6 medium, each containing 80 mM of various alternative carbon sources, without sucrose, and cultured for 5 days. As a control, sugar deficiency conditions without alternative carbon sources were investigated. As shown in Fig. 1, the amounts of recombinant trypsin protein (Fig. 1A) and mRNA transcript (Fig. 1B) were significantly increased in the medium containing succinic acid, fumaric acid and malic acid compared to the control (Ctrl). The three alternative carbon sources were therefore selected for further experiments.

To determine the optimum concentration and incubation time of each carbon source for trypsin production, three carbon sources at 20, 40, 60, 80 and 100 mM concentrations were investigated, respectively. Apparently, the highest productivity was observed when the cells were cultured for 5 days in a culture medium containing 40 mM fumaric acid (Figs. 2A and B). The highest trypsin production reached 68 mg / L on the 5th day of culture, about 4.3 times higher than the control trypsin production (18 mg / L).

Accumulated trypsin and enzyme activity in culture media using alternative carbon sources was confirmed using SDS-PAGE, Western blot analysis and zymography (FIGS. 2C, D and E). Total secreted proteins collected from the culture medium were analyzed by SDS-PAGE, and the gel was stained with 0.25% Coomassie Brilliant Blue R-250. The 44-46 kDa rice α-amylase protein induced by the sugar-deficient condition was indicated on the gel. Two types of trypsin from rice cell culture fluids corresponding to a protein of 24-26 kDa in molecular weight, as described in the previous paper (Kim et al. Protein Exp Purif 2011; 76: 121-126) It was observed in emography. In the positive control, trypsin from bovine taurus (Bt) only appeared at 24 kDa whereas recombinant trypsin from maize ( Zea mays , Zm) was located at a position of 24-26 kDa molecular weight similar to trypsin from rice cell culture appear. This was due to the nonconsensus N-linked glycosylation site of plant-derived trypsin (Zhang et al. Anal Chem 2010; 82: 10095-10101). In ELISA, similar results were also obtained in western blot analysis and in the case of a culture in which 40 mM fumaric acid was contained in the culture, showing a stronger band than the control culture. This result shows that the highest trypsin production and activity among the cultures containing the alternative carbon source is observed in the culture medium containing 40 mM fumaric acid. The conditions were therefore selected for further analysis.

Example  2. Fumaric acid and various concentrations of Sucrose's  effect

In general, the depletion conditions cause inhibition of cell growth in plant cells, reduced respiration rates, degradation of cellular components such as carbohydrates, lipids, and proteins, and decreased activity of sugar chain enzymes. Therefore, suspension cultured cells can not be reused after glucose depletion. This is one of the biggest problems in developing a mass production system of recombinant trypsin in transgenic rice cell suspension culture. To determine the combined effect of sucrose and fumaric acid, a combination of 0, 0.5, 1 and 2% sucrose and 40 mM fumaric acid was investigated and the results were analyzed by ELISA (Figure 3A), dry cell weight (Figure 3B) Survival was also assessed by measurement of (Fig. 3C). As shown in Figures 3A, 3B, and 3C, the combination of 0% sucrose and 40 mM fumaric acid showed the highest trypsin yield (68.2 mg / L) on day 5 of culture, and dry cell weight and cell viability were maintained for 5 days Were 21.2% and 31% of the control (3% sucrose, data not available), respectively. Although sucrose medium at 40 mM fumaric acid and 0-2% concentration resulted in reduced trypsin production from 62.8 mg / L to 12 mg / L at 5 days of culture, dry cell weight and cell viability Respectively. According to the results, the combination of 1% sucrose and 40 mM fumaric acid enhanced trypsin production, dry cell weight and cell viability by synergistic action. This was in contrast to the results of sucrose-free culture experiments, and for this reason the above conditions were selected for cell reuse studies.

Example  3. Culture time for cell re-use and trypsin production

Transgenic rice cell suspension cultures were subcultured every 7 days in basal medium containing 3% sucrose. To determine the optimal time for medium exchange, the culture medium containing 3% sucrose was removed after various incubation periods (0, 2, 4, 6 and 8 days), followed by 1% sucrose and 40 mM fumaric acid Was added. After the designated incubation period, trypsin yield was confirmed by Western blot analysis and ELISA. As shown in Figure 4, when 1% sucrose and 40 mM fumaric acid were added at the indicated times, trypsin production was increased under all conditions. When the cells cultured for 4 days were used, the highest trypsin production (55 mg / L) was observed on the fifth day of culture. Thus, cells cultured for 4 days in 3% sucrose medium were used to test the possibility of repeated use of suspension cultured cells in the rice amylase 3D system.

Example  4. 1% Sucrose  And 40 mM  Improved trypsin production by repeated use of transgenic rice cells in medium containing fumaric acid

To investigate the possibility of repeated use of cells to increase trypsin production, transgenic rice cells were treated with 0% sucrose (-S), 0% sucrose, 40 mM fumaric acid, 1% sucrose and 40 mM fumaric acid respectively In a 300 mL flask containing 50 mL of the culture medium. Cells were subcultured every 7 days using 3% sucrose medium. To test the process of cell recycling, culture of suspension culture cells cultured for 4 days in medium containing 3% sucrose was removed by inhalation. Then, 50 mL of three fresh culture media was added to each flask and incubated for a specified period of time. Recombinant trypsin accumulation and cell viability were observed after the designated incubation period. Suspension cultured cells were reused five times to confirm the possibility of repeated use of suspension cultured cells for recombinant trypsin production. In samples containing 0% sucrose, trypsin accumulation rapidly decreased to about 33% after the first use of the cells and below 22.2% after the second use. In the case of 0% sucrose, cell viability was about 26.7% at the first use and then rapidly decreased to below 16.7%. Also, when sucrose was re-fed, cell damage was irreversible (data not shown) because there was no recovered cell growth. In the case of 0% sucrose and 40 mM fumaric acid, a phenomenon similar to that of 0% sucrose was observed despite the delayed reduction of trypsin production and cell viability after first use. However, in the case of 1% sucrose and 40 mM fumaric acid, there was a slight decrease in recombinant trypsin production and cell viability after 5 repeated uses of cells. After 5 cell re-use, the yield of recombinant trypsin was 73.6% of the initial yield and the cell viability was 81.3% (Fig. 5A and B).

Claims (10)

(a) suspending the rice cells transformed with an expression vector containing a promoter operable at the time of starvation and a gene encoding a target protein in a sugar-rich medium to grow the rice cells; And
(b) transferring the rice cells to a culture medium containing no saccharide and an alternative carbon source, followed by culturing to produce a target protein from the rice cells. The method of claim 1, wherein said alternative carbon source is fumaric acid, succinic acid or malic acid. 3. The method according to claim 2, wherein the concentration of fumaric acid is 30 to 50 mM. The method according to claim 1, wherein the step (b) comprises culturing the rice cells for 4 to 6 days in a medium containing no sugar and containing 30 to 50 mM of fumaric acid. 2. The method of claim 1, wherein the promoter operative upon depletion of the sugar is an RAmy3D promoter. 2. The method of claim 1, wherein the target protein is small trypsin. (a) suspending the rice cells transformed with an expression vector containing a promoter operable at the time of starvation and a gene encoding a target protein in a sugar-rich medium to grow the rice cells;
(b) transferring the rice cells to a medium containing a mixture of fumaric acid and sucrose, and then culturing to produce a target protein from the rice cells; And
(c) culturing the cultured rice cells in a culture medium containing a mixture of fresh fumaric acid and sucrose, and then producing the desired protein from the rice cells. Way. The method according to claim 7, wherein the step (c) is repeated three to five times. The method according to claim 7, wherein the mixture of fumaric acid and sucrose in steps (b) and (c) is a mixture of 30 to 50 mM of fumaric acid and 0.5 to 1.5% (w / v) . [8] The method according to claim 7, wherein the step (c) comprises culturing the cultured rice cells for 3 to 5 days after transferring the cultured rice cells to a medium containing fresh fumaric acid and sucrose mixture.

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