KR20140078285A - Method for effective purification of recombinant trypsin produced in transgenic rice cell suspension culture - Google Patents

Abstract

The present invention relates to a method for an effective purification of a recombinant trypsin produced by transgenic rice cell suspension culture. According to the present invention, a recombinant trypsin is simply and effectively purified with the low cost by effectively removing an amylase in a culture liquid by adjusting pH and a temperature of the culture liquid comprising the recombinant trypsin produced while adjusting of a rice amylase 3D promoter expression system, and by carrying out the trypsin affinity chromatography.

Description

[0001] The present invention relates to a method for efficiently purifying recombinant trypsin produced by transgenic rice cell suspension culture,

The present invention relates to a method for efficiently purifying recombinant trypsin produced by transgenic rice cell suspension culture, more particularly, to a method for producing recombinant trypsin by suspension culture of transformed plant cells producing recombinant protein, And removing the amylase in the culture medium, followed by concentrating and dialyzing the culture to carry out affinity chromatography, and a recombinant protein purified by the above method.

Commercial production of recombinant medical proteins has been largely dependent on microbial fermentation or animal cell culture systems. To date, more than 100 proteins have entered the market in the form of human medicines, and over 370 recombinant proteins have been under development (J. Xu et al . , Biotechnol. Adv . 29 (2011) 278-299). The use of transgenic plant or plant cell cultures for the production of pharmaceuticals, functional proteins, industrial enzymes and functional secondary metabolites has recently been referred to as molecular farming. Transgenic plant or plant cell culture methods are alternative systems with several advantages including cost of production, scalability and safety. Because plants lack human pathogens, oncogenic DNA sequences, prions, and endotoxins, plant molecular agriculture is currently being used to produce pharmaceuticals using bacteria, yeast, and cultured animal cells. It is anticipated that it will challenge the established technology. Although in its early stages, the plant molecular agriculture industry has shown considerable growth in recent research and development activities worldwide. More than 120 small-scale enterprises, universities and research institutes active in plant molecular agriculture have been identified (P. Basaran et al . , Critic . Rev. Biotechnol . 28 (2008) 153-172). Advanced plant systems have evolved from the conceptional stage for commercial production of industrial enzymes such as avidin, aprotinin and trypsin. Currently, at least six medical proteins are in preclinical testing or clinical trials (Y. Shin et al . , Plant Biotechnol. J. 9 (2011) 1109-1119).

The advantages of plant cell culture in the production of human-like proteins have long been recognized. Plant cell culture systems have advantages and disadvantages over whole transgenic plant systems. While both systems use plant cells as bioreactors, plant cell culture systems have advantages in terms of production time, contamination risk, and purification costs. The entire transgenic plant system has advantages in terms of overall cost and scalable capacity (J. Xu et al . , Biotechnol . Adv . 29 (2011) 278-299). Since plant cells are eukaryotic cells, almost all post-translational modifications can be performed. As a result, plant cell systems have been recognized as an alternative despite having some disadvantages including low yield and human and slightly different glycan modifications. The best advantage of plant cell systems over animal cell systems is the risk of contamination, because plant cells do not have any human pathogens. Plant cells can replace CHO (Chinese hamster ovary) cells infected with calicivirus producing CerezymeR used in the treatment of Gaucher disease and can be a safe means for the production of therapeutic recombinant proteins have. The world's largest pharmaceutical company, Pfizer, recently announced a $ 60 million upfront payment for a production system using the glucocerebrosidase carrot cell suspension culture system, a rare drug for Goscher's disease, Paid a million dollar milestone payment and acquired a licensing right from Protalix Biotherapeutics, Israel (D. Aviezer et al . , Mol . Genet . Metab . 96 (2009) S13-14). This case focused on the interests of large international pharmaceutical companies in the commercialization of plant molecular agriculture for the production of human medical proteins. This plant-derived glucocerebrosidase was approved by the French regulatory authority in France in July 2010 for the treatment of Goschler's disease and is the first plant-derived human disease drug to enter the commercial market. Israel has been designated as Phase III Fast Track Designation by the US Food and Drug Administration (FDA) in August 2009. In February 2012, They reported the overall results of their switchover trials and the FDA approved them (www.protalix.com).

Trypsin is a serine protease that plays an important role in the activation of pancreatic enzymes involved in digestion. It is secreted from zymogen granules stored in pancreatic acinar cells and is activated by enterokinase as it is secreted. The enterokinase recognizes the (ASP) 4-Lys sequence of the peptide and cleaves the following lysine residues to secrete active trypsin. Trypsin is one of several versatile enzymes used as detergents for animal cell culture and leather processing, as dietary supplements in the food industry, and as substitutive therapy for patients with pancreatic disease. Currently, small trypsin is extracted from small pancreas. However, the contamination of enzymes with human pathogens including bovine spongiform encephalopathy has received considerable attention, underscoring the urgency of continued efforts to improve plant cell systems as an alternative expression system for pharmaceutical recombinant proteins. As a result, recombinant small trypsin was produced in transgenic maize (Zea mays) seeds and was used as a commercial protease. However, the recovery and purification of recombinant proteins in plants is expensive and technically challenging, accounting for 80-90% of the final production cost (S. Woodard et al . , Biotechnol . Appl . Biochem . 38 (2003) 123-130). The present inventors have studied the use of transgenic rice cell suspension cultures in place of transgenic whole plants to produce recombinant small trypsin and have reported the production of functional recombinant trypsin in transgenic rice cell suspension cultures (N. Kim et < RTI ID = 0.0 > al . , Protein Exp . Purif . 76 (2011) 121-126).

The present inventor intends to provide a simple purification method for purifying recombinant trypsin by controlling the pH and temperature of a rice cell culture medium in which recombinant trypsin is produced and carrying out affinity chromatography.

Korean Patent Publication No. 2007-0091081 discloses a method for purifying and purifying human granulocyte-macrophage colony stimulating factor from a rice cell liquid suspension culture. Korean Patent Registration No. 0488287 discloses a method for purifying recombinant human epithelial cells A method for purifying a growth factor '. However, as in the present invention, a method for efficiently purifying recombinant trypsin produced by transgenic rice cell suspension cultures has not been disclosed.

The present invention provides a recombinant trypsin-producing rice cell cultured in the absence of a sugar under the control of a rice amylase 3D promoter. In order to remove amylase in the culture broth and to purify recombinant trypsin, Was adjusted to pH 3.0, incubated at 57 ° C for 3 hours, and then subjected to benzamidine affinity chromatography to obtain 28.1-fold purified recombinant trypsin at a yield of 39% to complete the present invention .

In order to solve the above problems,

(a) suspending a transformed plant cell producing a recombinant protein; And

(b) culturing the cell culture solution of step (a) at a pH of 2.5 to 4.5 at 50 to 60 ° C to remove amylase.

In addition,

(a) suspending a transformed plant cell producing a recombinant protein;

(b) culturing the cell culture medium of step (a) at a pH of 2.5 to 4.5 and culturing the cells at 50 to 60 ° C for 2 to 4 hours to remove amylase;

(c) concentrating the culture medium in step (b) and then dialyzing the culture medium; And

(d) performing affinity chromatography with the dialysis solution of step (c).

In addition, the present invention provides a purified recombinant protein.

According to the present invention, it is possible to effectively remove amylase in a transgenic rice cell culture medium, which is a problem in purification of recombinant trypsin, by controlling pH and temperature, and purify trypsin by performing affinity chromatography with a culture medium in which amylase has been removed Since the method is simple and efficient, it is possible to produce recombinant trypsin derived from plants which is industrially useful and safe, at a lower cost than the existing production system.

Figure 1 shows the SDS-PAGE of the reaction solution cultured at 2 ° C for 12 hours at various pH levels. The reaction solution was concentrated 10 times and loaded in each well 30 times. Lane 1 was treated with pH 2, lane 2 with pH 3, lane 3 with pH 4, lane 4 with pH 5, lane 5 with pH 7, lane 6 with pH 8, lane 7 with pH 10 and lane 8 with pH 11 It is a reaction liquid. Molecular weight standard markers were indicated on both sides of the gel. Arrows indicate amylase produced in the culture medium.
Figure 2 shows an SDS-PAGE analysis of the reaction solution cultured for 12 hours at various temperature and pH levels. The reaction solution was concentrated 20 times and loaded 30 times into each well. Lanes 1-4 are reaction solutions treated at pH 3 and 2 ° C, 20 ° C (RT), 57 ° C or 67 ° C, respectively. Lane 5 is a reaction solution which has been treated at pH 7 and 2 ° C. Lanes 6-8 are reaction solutions treated at pH 10.5 and 2 ° C, 57 ° C or 67 ° C, respectively. Molecular weight standard markers were indicated on both sides of the gel. Arrows indicate amylase produced in the culture medium.
Figure 3 shows the elution profile of recombinant rice-derived trypsin for a benzamidine affinity column.
Figure 4 shows SDS-PAGE analysis of each purification step. The reaction solution was concentrated 20 times and loaded 30 times into each well. Lane 1 is a reaction solution treated at pH 7 and 2 ° C, lane 2 is a reaction solution treated at pH 3 and 2 ° C, and lane 3 is a recombinant trypsin purified by benzamidine affinity chromatography. Molecular weight standard markers were indicated on both sides of the gel. The upper arrow indicates the amylase produced in the culture medium, and the lower arrow indicates the recombinant trypsin (rTrypsin).

In order to achieve the object of the present invention,

(a) suspending a transformed plant cell producing a recombinant protein; And

(b) culturing the cell culture solution of step (a) at a pH of 2.5 to 4.5 at 50 to 60 ° C to remove amylase.

In one embodiment of the present invention, the culturing of step (b) may be performed by adjusting the cell culture liquid to pH 2.5 to 4.5 and culturing the cells at 50 to 60 ° C for 2 to 4 hours. Preferably, pH 3.0 and incubating at 57 ° C for 3 hours, but it is not limited to the pH, temperature and time at which the amylase can be effectively removed.

In one embodiment of the present invention, the plant cell may be a monocot plant cell, more preferably a rice cell, but is not limited thereto.

In one embodiment of the present invention, the production of the recombinant protein may be performed by culturing under the condition of sugar free amylase 3D promoter, but is not limited thereto.

The rice amylase 3D promoter induces the expression of a recombinant protein coding gene operably linked to a promoter in the absence of a sugar as an inducible promoter. Plant cells producing the recombinant protein under the control of rice amylase 3D promoter can be grown and proliferated by culturing in a sugar-containing medium. In addition, production of the recombinant protein can be induced by exchanging the cell culture medium with a glucose-free medium or sugar-free medium in the medium.

In one embodiment of the present invention, the recombinant protein may be an enzyme, preferably a serine protease, more preferably a mammalian recombinant trypsin, and most preferably, May be recombinant bovine trypsin but is not limited to a recombinant protein that can be produced by the regulation of the rice amylase 3D promoter in plant cells and can be stably maintained at an acidic pH and a temperature of 50 to 60 ° C .

 In addition,

(a) suspending a transformed plant cell producing a recombinant protein;

(b) culturing the cell culture medium of step (a) at a pH of 2.5 to 4.5 and culturing the cells at 50 to 60 ° C for 2 to 4 hours to remove amylase;

(c) concentrating the culture medium in step (b) and then dialyzing the culture medium; And

(d) performing affinity chromatography with the dialysis solution of step (c).

In the method for purifying a recombinant protein according to an embodiment of the present invention, the step (a) specifically comprises transforming the transgenic monocotyledonous plant cells producing the recombinant protein under the condition of no sugar under the control of the rice amylase 3D promoter, And preferably transgenic rice cells that produce recombinant trypsin under the condition of no sugar under the control of rice amylase 3D promoter can be suspended and cultured in a sugar-free medium, more preferably rice amylase 3D But not limited to, culturing transgenic rice cells that produce recombinant trypsin under conditions without sugar under the control of a promoter, for 10 days in a sugar-free medium.

In step (b), the cell culture medium of step (a) may be adjusted to pH 2.5 to 4.5, and the cells may be cultured at 50 to 60 ° C for 2 to 4 hours The amylase may be removed by adjusting the pH of the rice cell culture medium of step (a) at 57 ° C for 3 hours, and more preferably, The amylase may be removed by adjusting the pH of the rice cell culture solution of step a) to 57 ° C for 3 hours, but not limited thereto.

In the method for purifying a recombinant protein according to an embodiment of the present invention, the step (c) may be performed by concentrating the culture medium of step (b) and then dialysis, the culture medium of step (b) may be concentrated 10 to 25 times and dialyzed with Tris-HCl buffer. More preferably, the culture medium of step (b) is concentrated 20 times by ultrafiltration, 50 mM Tris-HCl (pH 7.4) buffer, but is not limited thereto, as long as it is a concentration and dialysis condition of a culture solution which can be used for purification of recombinant trypsin.

In the method for purifying a recombinant protein according to an embodiment of the present invention, the step (d) may be performed by performing chromatography using an affinity column with the dialysis solution of step (c) (FPLC) using a benzamidine column having an affinity for trypsin as the dialysis solution of step (c). However, affinity chromatography to purify recombinant trypsin Use is not limited.

In addition, the present invention provides a purified recombinant protein. The recombinant protein may be an enzyme, preferably a serine protease, more preferably a mammalian recombinant trypsin, and most preferably a recombinant bovine trypsin. However, it is not limited to a recombinant protein that can be produced by controlling rice amylase 3D promoter in plant cells and can be stably maintained at an acidic pH and a temperature of 50 to 60 ° C.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Preparation of recombinant trypsin

Methods for the cloning and expression of recombinant trypsin in transgenic rice cell suspension cultures have been described previously (N. Kim et al . , Protein Exp . Purif . 76 (2011) 121-126). The supernatant of the medium cultured for 10 days under induction conditions was used for purification.

2. Enzyme activity assay and protein amount and Amylase  dose

Trypsin activity was measured according to the following steps. The substrate BAEE (sodium-benzoyl-L-arginine ethyl ester) was reacted with the sample and the absorbance of the reaction solution was measured at 253 nm. Standard trypsin is trypsin from small pancreas (Sigma, product number T1426, Sigma-Aldrich Korea). The amount of protein was determined using the Bradford method (M. Bradford, Anal . Biochem . 72 (1976) 248-54). The amount of amylase was determined by absorbance at 570 nm using the DNS (3,5-dinitrosalicylic acid) method (P. Bernfeld, Meth . Enzymology 1 (1955) 149-158).

3. SDS - PAGE

Electrophoresis was performed according to the manufacturer's instructions for mini-PROTEAN TGX precast gel (Bio-Rad Laboratories, Inc., USA). 30 [mu] l of the sample was loaded onto 10% gel, and then electrophoresed at 200 V for 30 minutes.

4. Purification step

Since the present inventor used the rice amylase 3D promoter, amylase appears to be a major product during trypsin production through transgenic rice cell suspension cultivation. As a result, the present inventor sought to identify the best purification conditions for recovering the maximum amount of trypsin and removing the amount of amylase as much as possible.

5. pH Control of

The culture medium was titrated to various levels of pH and incubated at 2 ° C for 12 hours. After incubation, the culture was centrifuged at 12,000 xg for 20 minutes, and the supernatant was used for reactions to determine trypsin activity, total protein and total amylase.

6. Optimum temperature

The pH of the culture medium was titrated to 3.0, 7.0 and 10.5 and cultured at 2 ° C, 20 ° C (RT), 37 ° C, 47 ° C, 57 ° C and 67 ° C for 12 hours. After incubation, the culture was centrifuged at 12,000 xg for 20 minutes, and the supernatant was used to determine the amount of trypsin activity, total protein and total amylase.

7. Culture time

Optimal pH (pH 3.0) and temperature (57 ℃) were measured and optimum culture time was measured by incubating the culture medium for 1, 3 and 6 hours. After incubation for various times, the reaction mixture was centrifuged at 12,000 xg for 20 minutes, and the supernatant was used to determine the total amount of trypsin activity, protein and amylase.

8. Benzamidine  Affinity chromatography

The culture medium was titrated to pH 3, incubated at 57 ° C for 3 hours, and then centrifuged at 12,000 xg for 20 minutes to remove cell debris. The supernatant was concentrated 20 times using ultrafiltration (YM 10, raw material: regenerated cellulose, membrane: 10,000 MWCO PES; Millipore, USA) and dialyzed overnight with 50 mM Tris-HCL (pH 7.4) buffer. Samples were applied to a column volume of 1 mL of HiTrap Benzamidine FF (GE Healthcare, Sweden) using fast protein liquid chromatography (AKTAprime, GE Healthcare, Sweden).

Example  1. Optimal pH

To determine optimal pH, the culture medium was titrated to various pH levels and incubated at 2 ° C for 12 hours. After treatment, the enzyme activity, the amount of total protein and the amount of total amylase were measured using a spectrophotometer (Table 1). Protein profiles were analyzed by SDS-PAGE (Figure 1). At pH 3, the total amount of total protein recovered was the lowest and no reducing sugar was detected. This is generally consistent with the data of FIG. As a result, there was no significant difference between pH 3 and 4 in the amount of recovered trypsin, but pH 3 was determined to be the optimum pH for recovering recombinant trypsin. Based on these results, it is assumed that the recombinant trypsin is relatively stable at acidic pH. However, most other proteins, including amylase, were fairly unstable at pH 3. In addition, the reducing sugar was completely degraded at pH 3.

At various pH levels, enzyme activity (A ₂₅₃ ), total protein (A ₅₉₅ ) and total reducing sugar (A ₅₇₀ ) were measured using a spectrophotometer wavelength pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 A ₂₅₃ 0.005 0.007 0.006 0.005 0.005 0.004 0.003 0.003 0.002 A ₅₉₅ 0.24 0.18 0.24 0.27 0.29 0.28 0.27 0.24 0.28 A ₅₇₀ 0.01 - 0.05 0.07 0.09 0.15 0.1 0.1 0.07

Example  2. Optimum temperature

To determine the optimum temperature, the culture medium was titrated to pH 3.0, 7.0 and 10.5 and then cultured at various temperatures for 12 hours. Optimal conditions for maximum tryptic recovery are pH 3 and 57 ° C (Table 2). The amount of recovered trypsin was slightly higher at 57 째 C than at 20 째 C, 47 째 C or 67 째 C (Table 2). Recombinant trypsin was relatively stable at pH 3 regardless of temperature. However, recombinant trypsin was quite unstable at pH 10.5. This phenomenon is consistent with data collected for optimal pH. No reducing sugar was detected and the total protein amount was relatively low at 57 캜. As a result, the present inventors have determined that the optimum temperature is 57 deg.

In the present invention, since rice amylase 3D promoter is used, amylase is the main product in culture. However, amylase is a major problem in the purification step. Fortunately, the target protein, recombinant trypsin, is fairly stable at pH 3, whereas amylase is fairly unstable at pH 3. A trace amount of reducing sugar was detected at 2 캜 and pH 3, but regardless of temperature, the reducing sugar was considerably unstable at pH 3. This property was also confirmed by SDS-PAGE analysis (Fig. 2), indicating that the amylase is very stable at pH 7 and more stable at alkaline pH than acidic pH. Total protein content was highest at pH 7 and more stable at pH 10.5 than pH 3.0.

Enzyme activity (A ₂₅₃ ), total protein (A ₅₉₅ ) and total amylase (A ₅₇₀ ) at various temperatures were measured using a spectrophotometer wavelength pH 3.0 pH 7.0 pH 10.5 A ₂₅₃ 2 ℃ 0.006 0.004 0.002 20 ℃ 0.007 0.005 0.003 37 ℃ 0.006 0.003 0.005 47 C 0.007 0.005 0.001 57 ℃ 0.008 0.006 0.003 67 ° C 0.007 0.005 0.004 A ₅₉₅ 2 ℃ 0.12 0.30 0.25 20 ℃ 0.1 0.28 0.23 37 ℃ 0.09 0.27 0.22 47 C 0.09 0.29 0.20 57 ℃ 0.07 0.28 0.22 67 ° C 0.06 0.30 0.15 A ₅₇₀ 2 ℃ 0.001 0.252 0.05 20 ℃ - 0.257 0.03 37 ℃ - 0.275 0.03 47 C - 0.272 0.05 57 ℃ - 0.314 0.02 67 ° C - 0.352 0.03

Example  3. Optimal incubation time

When the culture medium was cultured at pH 3 and 57 ° C for various times, the optimal incubation time was found to be 3 hours (Table 3). A solution incubated at 2 캜 for 6 hours was used as a control. Enzyme activity was slightly higher after 3 hours compared to 1 or 6 hours culture. The total amount of protein after 3 hours was much lower than that after 1 hour or 6 hours. After 6 hours, the amount of reducing sugar was the lowest, but after 3 hours, the enzyme activity was the highest. Especially, the enzyme activity was highest in proportion to the amount of protein, and the most suitable incubation time was determined (Table 3). These results indicate that culturing for 3 hours is optimal.

Enzyme activity (A ₂₅₃ ), total protein (A ₅₉₅ ) and total amylase (A ₅₇₀ ) for various incubation times were measured using a spectrophotometer wavelength Control Trypsin culture medium (pH 3.0, culture at 57 캜) 2 ° C, culture for 6 hours 1 hours 3 hours 6 hours A ₂₅₃ 0.007 0.008 0.009 0.008 A ₅₉₅ 0.14 0.12 0.08 0.11 A ₅₇₀ 0.12 0.019 0.01 0.003

Example  4. Benzamidine  Affinity chromatography

The column was equilibrated with 5 times column volumes of binding buffer (0.005 M Tris-HCl, 0.5 M NaCl, pH 7.4) at a flow rate of 1 mL / min. Samples were injected using a syringe mounted on a luer connector and the column was washed with 25-fold column volumes of binding buffer or eluent until no material appeared. The sample was then eluted with 15 column volumes of elution buffer (0.05 M glycine, pH 3.0) (Figure 3). During affinity chromatography, there were two large protein peaks. The first peak appeared as the binding buffer and the second peak as the elution buffer. One mL of each fraction was collected and enzyme activity was measured. Fractions representing enzyme activity were collected and the buffer was exchanged using a PD-10 desalting column if necessary. Samples were treated at 2 < 0 > C and pH 7.0.

Samples extracted with affinity chromatography as well as the original culture medium samples were analyzed by SDS-PAGE (Figure 4). Recombinant trypsin appeared in fraction 2 (Figure 4). The apparent molecular weight of the recombinant trypsin according to SDS-PAGE was about 26 kDa, slightly higher than the standard trypsin molecular weight of 24 kDa. This property has been reported in a previous study of the inventors group that identified the production of functional recombinant trypsin in transgenic rice cell suspension cultures. A slight increase in the molecular weight of the recombinant protein produced in plants is also reported in corn and is apparent due to the O-linked glycosylation sequence in plant-derived proteins (N. Kim et < RTI ID = 0.0 > al ., Plant Mol . Biol . 68 (2008) 369-377). The two steps, purified to a final yield of 39% and 28.1 fold, are summarized in Table IV. One unit is defined as a change in absorbance at 0.001 / min at 253 nm.

In the present invention, under the control of the rice amylase 3D promoter expression system, a simple purification step related to the modified recombinant trypsin was constructed based on the rice-optimized codon usage frequency. The method of the present invention for purifying recombinant trypsin by performing affinity chromatography after adjusting the pH and temperature of the culture medium is sufficiently effective for trypsin purification produced by rice cell suspension culture.

Purification of recombinant trypsin from transgenic rice cell suspension cultures step Total protein (mg) Total active (U) Trypsin activity
(U / mg) Tablets (times) Yield (%) Supernatant 500 1275 2.55 1.0 100 pH, temperature 27 550 20.06 7.9 43 Affinity 7 502 71.71 28.1 39

Claims (12)

(a) suspending a transformed plant cell producing a recombinant protein; And
(b) culturing the cell culture solution of step (a) at a pH of 2.5 to 4.5 at 50 to 60 ° C to remove amylase. The method for purifying recombinant proteins according to claim 1, wherein the culturing of step (b) is performed for 2 to 4 hours. The method for purifying recombinant proteins according to claim 1, wherein the plant cell is a rice cell. The method for purifying recombinant proteins according to claim 1, wherein the production of the recombinant protein is carried out under the condition of no sugar under the control of rice amylase 3D promoter. The method of claim 1, wherein the recombinant protein is recombinant trypsin. (a) suspending a transformed plant cell producing a recombinant protein;
(b) culturing the cell culture solution of step (a) at a pH of 2.5 to 4.5 at 50 to 60 ° C to remove amylase;
(c) concentrating the culture medium in step (b) and then dialyzing the culture medium; And
(d) performing affinity chromatography with the dialysis solution of step (c). The method of claim 6, wherein the plant cell is a rice cell. The method for purifying recombinant proteins according to claim 6, wherein the production of the recombinant protein is carried out under the condition of no sugar under the control of rice amylase 3D promoter. 7. The method of claim 6, wherein the recombinant protein is recombinant trypsin. [7] The method of claim 6, wherein the culturing of step (b) is performed for 2 to 4 hours. 7. The method of claim 6, wherein the affinity chromatography is benzamidine affinity chromatography. 12. A recombinant protein purified by the method of any one of claims 1 to 11.

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