CN111194321A

CN111194321A - Method for obtaining protein derived from Bordetella pertussis including freezing and thawing processes

Info

Publication number: CN111194321A
Application number: CN201880048447.2A
Authority: CN
Inventors: 安东浩; 崔起燮; 文在勋; 全亨珍; 朴钟冠; 金惠连; 崔普渼
Original assignee: Green Cross Corp Korea
Current assignee: GC Biopharma Corp
Priority date: 2017-08-01
Filing date: 2018-07-23
Publication date: 2020-05-22
Anticipated expiration: 2038-07-23
Also published as: AU2018310071A1; BR112020002232A2; JP7186215B2; AU2018310071B2; CN111194321B; WO2019027168A2; JP2020529989A; MX2020001304A; WO2019027168A3; WO2019027168A9; KR20190013296A; KR102426041B1

Abstract

The present invention relates to a method for obtaining a PRN protein derived from Bacillus pertussis, which comprises freezing and thawing processes capable of effectively improving the extraction level of the PRN protein. In the present invention, it was found that when the pellets were frozen before treatment with urea and then slowly thawed at low temperature, the extraction level of PRN protein was effectively increased compared to the experimental group under other conditions. Additionally, even when the method is applied to large-scale production, the level of PRN extraction is effectively improved. Therefore, the pretreatment method of B. pertussis including freezing and thawing process can be effectively used for mass production of PRN for pertussis vaccine.

Description

Method for obtaining protein derived from Bordetella pertussis including freezing and thawing processes

Technical Field

The present invention relates to a method for purifying Bordetella pertussis (Bordetella pertussis) derived PRN protein, comprising a freezing and thawing process.

Background

Pertussis is an acute respiratory disease, occurring primarily in infants, characterized by a cough of 2 weeks or more. Bordetella pertussis is a gram-negative, aerobic bacterium brevibacterium, and has been reported to cause pertussis. Bordetella pertussis uses human as the only host, mainly through respiratory tract infections. In addition, bordetella pertussis lives on the respiratory mucosa and causes diseases in the human body. In the 1930's, a cellular pertussis vaccine was developed that proved to have a prophylactic effect on pertussis. In addition, in the 1940's, pertussis vaccines were used in combination with tetanus and diphtheria inactivated quadruple vaccines. However, adverse reactions (convulsions, redness, fever, etc.) of whole-cell pertussis vaccines (whole-cell pertussis vaccine) have also been reported. Therefore, there is a need to develop a safe pertussis vaccine.

In 1950 s, the pathogenesis of bordetella pertussis was studied, and components such as Pertussis Toxin (PT), Filamentous Hemagglutinin (FHA), Pertactin (PRN), and Fimbriae (FIM) were reported as antigens. Later, the development of acellular (acellular) pertussis vaccines involving the isolation and purification of these proteins is underway. After the 1980 s, purified pertussis vaccines were first developed and vaccinated in japan.

Proteins (e.g. PT, FHA and PRN) must be purified to prepare acellular pertussis vaccines. Traditionally, antigens were purified simultaneously by repeated ammonium sulfate precipitation and density gradient centrifugation. However, this method has disadvantages in that many impurities are generated and it is difficult to control the purification process. Another approach is to purify each antigen separately using a combination of physical and chemical methods. Korean laid-open patent publication No. 2015-0124973 discloses a decellularized pertussis vaccine composition comprising PT, FHA, and

FIM types

2 and 3.

Meanwhile, in the production method of a pertussis vaccine comprising mixing three or four components, there is a problem in that even a decrease in the productivity of one component may increase the entire production cycle.

Thus, a method for efficiently producing PRN protein in large quantities is being sought for efficiently producing pertussis vaccines.

Disclosure of Invention

Technical problem

In view of this, the inventors have made an effort to find conditions capable of increasing the extraction amount of bordetella pertussis PRN protein, and have determined a case where a sample containing bordetella pertussis is subjected to a freezing (cold-shock) and thawing step under specific conditions, and the extraction amount of bordetella pertussis PRN protein is increased, thereby completing the present invention.

It is therefore an object of the present invention to provide a method for obtaining a PRN protein, which method can lead to an increase in the extraction of the PRN protein from b.

Solution to the problem

In order to achieve the above object, the present invention provides a method for obtaining a bordetella pertussis-derived PRN protein, comprising the steps of: 1) freezing a sample containing bordetella pertussis; 2) thawing the frozen sample; 3) fragmenting (disrupting) the thawed sample; 4) purifying the disrupted sample.

Advantageous effects of the invention

The method for obtaining PRN protein of bordetella pertussis according to the present invention includes freezing a sample containing bordetella pertussis and thawing the sample at a refrigeration temperature, thereby maximizing the extraction amount of PRN protein. In the case where the PRN protein is obtained by purifying a sample pretreated under the above conditions, the extraction amount of the PRN protein significantly increases as compared to an extraction method without such pretreatment. In addition, the method of obtaining PRN protein can also be effectively used for PRN protein that is mass-produced for the production of pertussis vaccine.

Brief Description of Drawings

Fig. 1 shows a flow diagram of the PRN purification process.

Figure 2 shows a detailed flow diagram of the pellet (pellet) preparation, thawing and treatment with urea.

Figure 3 shows the results obtained by urea treatment of each pellet obtained under different preparation and storage conditions.

Fig. 4A shows the difference in the extraction amount of PNR protein in the case where PRN protein was obtained by each method of comparative examples 1 to 5 and examples 1 to 3 using the SDS-PAGE technique.

Fig. 4B shows the difference in the extraction amount of PNR protein in the case where PRN protein was obtained by each method of comparative examples 6 to 10 and examples 4 and 5 using the SDS-PAGE technique.

Fig. 4C shows the difference in the extraction amount of PNR protein in the case where PRN protein was obtained by each method of comparative examples 11 to 16 and example 6 using the SDS-PAGE technique.

Fig. 5A shows the difference in the extraction amount of PNR protein in the case where PRN protein was obtained by each method in comparative examples 1 to 5 and examples 1 to 3 using the Western blotting technique (Western blotting).

Fig. 5B shows the difference in the extraction amount of PNR protein in the case where PRN protein was obtained by each method in comparative examples 6 to 10 and examples 4 and 5 using the Western blotting technique (Western blotting).

Fig. 5C shows the difference in the extraction amount of PNR protein in the case where PRN protein was obtained by each of the methods in comparative examples 11 to 16 and example 6 using the Western blotting technique (Western blotting).

Fig. 6 shows the extraction amounts of PRN protein obtained by each of the methods in comparative examples 1 to 7 and examples 1 to 3, with different slurry (slurry) and pellet storage conditions.

Fig. 7 shows the extraction amounts of PRN protein obtained by each of the methods in comparative examples 15 to 17 and example 7, with different pellet storage conditions.

Fig. 8 shows the difference in the extraction amount of PNR protein in the case of using different pellet thawing conditions.

Detailed Description

Best Mode for Carrying Out The Invention

In one aspect of the invention, there is provided a method of obtaining a bordetella pertussis-derived PRN protein, comprising the steps of: 1) freezing a sample containing bordetella pertussis; 2) thawing the frozen sample; 3) disrupting the thawed sample; 4) purifying the disrupted sample.

First, a step of freezing a sample containing bordetella pertussis was performed.

In the present invention, "Bordetella pertussis" also called Bordetella parapertussis, is a gram-negative coccobacillus, amastigotis and spores as small as about 0.3-1 μm. PRN is one of the major proteins of bordetella pertussis, is an abbreviation for pertactin, and is a causative agent of bordetella pertussis. In addition, PRN, an outer membrane protein that adheres to tracheal epithelial cells, is obtained from bordetella pertussis and is one of the important components of a pertussis vaccine.

In the present specification, the "sample containing bordetella pertussis" may be a culture obtained by culturing bordetella pertussis, a slurry (slurry) separated from the culture by continuous centrifugation, or a pellet obtained from the slurry by high-speed centrifugation, preferably the pellet. Here, the obtained slurry may be immediately centrifuged to obtain pellets. However, the obtained slurry may be centrifuged after 1 to 5 days to obtain pellets. Here, the slurry may be stored at 0 to 25 ℃. However, the present invention is not limited thereto.

In one embodiment of the present invention, pellets are prepared by culturing a bordetella pertussis strain in a modified schalt's disease (MSS) (modifidtainer scholte) medium at a temperature of 35 ℃ and subjecting the cell culture to continuous centrifugation at room temperature for 2 hours and centrifugation at room temperature, and the slurry thus obtained is subjected to high-speed centrifugation at a speed of 7,000rpm at 2 ℃ to 8 ℃ for 50 minutes.

Second, a step of thawing the frozen sample is performed.

In the step of freezing the sample containing the bordetella pertussis strain, the freezing may be performed at a temperature of-1 ℃, -2 ℃, -5 ℃, -10 ℃, -20 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -70 ℃, -80 ℃ or-90 ℃, or at a temperature of-1 ℃ to-90 ℃, -5 ℃ to-85 ℃, -15 ℃ to-75 ℃, or-30 ℃ to-60 ℃. Freezing may be performed for 0.5 hour, 1 hour, 2 hours, 5 hours, 10 hours, 20 hours, 30 hours, or 40 hours, or for 0.5 hour to 40 hours, 1 hour to 30 hours, 2 hours to 20 hours, or 5 hours to 10 hours.

In addition, frozen samples were slowly thawed by refrigerated storage. Here, the thawing can be performed at a temperature of 1 deg.C, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 7 deg.C, 10 deg.C, 15 deg.C, 20 deg.C or 25 deg.C, or at a temperature of 1 deg.C to 25 deg.C, 2 deg.C to 18 deg.C, 5 deg.C to 15 deg.C or 7 deg.C to 10 deg.C. Thawing may be performed by refrigeration for 0.1 hour, 1 hour, 5 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 60 hours, or 0.1 hour to 60 hours, 1 hour to 55 hours, 5 hours to 50 hours, or 12 to 48 hours.

In one embodiment of the present invention, the pellets are stored frozen at a temperature of-30 ℃ or less for 2 hours, and the frozen pellets are thawed at room temperature or a refrigeration temperature of 1 ℃ to 7 ℃.

Here, the freezing step and the thawing step may be repeatedly performed one or more times.

Thirdly, a step of crushing and thawing the sample is performed.

The thawed sample is subjected to a disruption step. Here, the crushing may be performed by, for example, urea treatment, heat treatment, guanidine treatment and iodine treatment. However, the crushing method is not limited, and various crushing methods known in the art may be applied.

In the case of crushing the sample by urea treatment, it may be carried out with a urea concentration of 0.1M, 0.5M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, or 10M, or a urea concentration of 0.1M to 10M, 1M to 8M, 3M to 7M, or 4M to 5M. The treatment with urea may be carried out for 5 minutes, 10 minutes, 20 minutes, 50 minutes, 80 minutes, 120 minutes, 180 minutes, 240 minutes or 360 minutes, or 5 minutes to 360 minutes, 20 minutes to 240 minutes or 50 minutes to 180 minutes.

In one embodiment of the present invention, the outer membrane of bordetella pertussis was broken in the pellets by placing the thawed pellets in 5.0M urea buffer 4 times the weight of the pellets and stirring for 3 hours.

Fourth, a step of purifying the disrupted sample is performed.

After centrifugation of the cell lysate (lysate) or cell culture, unnecessary cell debris (cell debris) can be removed using various column chromatography methods and the like. Column chromatography may include ion exchange chromatography, hydrophobic interaction chromatography, gel exclusion chromatography, gel filtration chromatography, HPLC, reverse phase HPLC, affinity chromatography, and the like.

In one embodiment of the present invention, in the step of purifying the crushed pellets, the PRN protein is obtained by performing ion exchange chromatography (IEX) (ion exchange chromatography), Hydrophobic Interaction Chromatography (HIC) (hydrobicinctional chromatography) and Gel Filtration Chromatography (GFC) (gel filtration chromatography).

As used herein, "ion exchange chromatography" may use an ion exchange resin in which an anion or cation is covalently bonded to a stationary phase as an exchange resin. Solute ions of opposite charge in the mobile phase are attracted to the stationary phase by electrostatic attraction. Ion exchange chromatography is based on an equilibrium achieved by adsorption of ions or charged compounds onto an ion exchanger (ionexchanger) by electrostatic forces.

In one embodiment of the invention, the PRN protein is eluted by using an anion exchange resin in an ion exchange chromatography column and using Tris-HCl solution as an equilibration buffer, Tris-HCl with NaCl solution as a wash buffer and Tris-HCl with NaCl solution as an elution buffer.

As used herein, the term "hydrophobic interaction chromatography" refers to a separation method using hydrophobic interactions between a substrate (matrix) having hydrophobic functional groups and molecules. The matrix can be made hydrophobic by modifying hydrophilic and non-reactive agarose. Modified agarose obtained by reacting alkylamine with BrCN-activated agarose is widely used. Hydrophobic interaction chromatography is widely used for the pure separation of proteins. Furthermore, in hydrophobic interaction chromatography, for protein elution, an aliphatic amine (aliphatic amine) which may decrease the ionic strength or increase the pH value and which may decrease the polarity, an alcohol or a non-ionic detergent (e.g., tween 20 and Triton X-100) may be used.

In one embodiment of the invention, the PRN protein is eluted by using a Tris-HCl solution containing a NaCl solution as the equilibration buffer, a Tris-HCl solution containing a NaCl solution as the wash buffer and a Tris-HCl solution containing a NaCl solution as the elution buffer.

As used herein, "gel filtration chromatography" uses a molecular sieve in the stationary phase, which is a hydrophilic Sephadex, polyacrylamide or agarose gel, and is therefore capable of absorbing water and thereby swelling. In the case where the sample molecules are larger in size than the largest pores of the swollen gel, the molecules do not pass through the gel particles and therefore exit the column through the spaces in the stationary phase particles. Smaller molecules enter the open pores of the gel particles and pass through at different rates depending on their size and shape. Thus, the molecules elute in order of decreasing size. In gel chromatography, sample size, viscosity, ionic strength, flow rate, etc. are taken into account.

In one embodiment of the invention, the PRN protein is eluted using sodium phosphate containing NaCl solution as equilibration buffer.

Modes for carrying out the invention

Further, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 procedure for obtaining PRN

The process of obtaining the PRN proceeds as follows: pellet preparation and thawing, treatment with urea, column chromatography (IEX, HIC and SEC), concentration and buffer exchange (UF/DF) and purification of impurity-free PRN protein. A flowchart of the process of obtaining a PRN is shown in fig. 1.

Example 1.1 culture of Bordetella pertussis

Bordetella pertussis (Korea national Institute of Health) was cultured in MSS medium at a temperature of 35 ℃ for 4 days. During the culture period, specifically, seed culture was performed for 1 day, primary enrichment culture was performed for 1 day, secondary enrichment culture was performed for 1 day, and main culture (main culture) was performed for 1 day.

Example 1.2 pellet preparation

The cell culture was separated into a culture supernatant and a slurry by continuous centrifugation at 100L/h and a flow rate of 9,500rpm for 2 hours at room temperature. The separated slurry was centrifuged at 7,000rpm at 5 ℃. + -. 3 ℃ for 50 minutes at high speed to prepare pellets. In the following examples, in the case of storing the slurry for 3 days, the slurry was stored at a refrigeration temperature of 5 ℃ for 3 days, and then subjected to high-speed centrifugation to prepare pellets.

Example 1.3 freezing and thawing

The prepared pellets were stored at-30 ℃ or less for 2 hours and then thawed by storing at Room Temperature (RT) for 1 day. Subsequently, PRN protein was obtained by performing the following steps of examples 1.4 to 1.7. In addition, as in examples 1 to 6 and comparative examples 1 to 16 shown in table 1 below, experiments were performed under conditions where the storage of the slurry, freezing and thawing conditions were different, and then PRN protein was obtained by performing the steps of examples 1.4 to 1.7 below.

TABLE 1

Example 1.4 treatment with Urea

To the thawed pellets was added 5.0M urea buffer in an amount of 4 times the weight of the pellets. The mixture was stirred with a magnetic bar at 280rpm for 3 hours. The urea treatment solution, which had been stirred for 3 hours, was subjected to high-speed centrifugation at 7,000rpm (12230RCF) at 5 ℃. + -. 3 ℃ for 1.5 hours. After centrifugation, cell debris was removed and the supernatant was collected. The collected supernatant was filtered through a 0.22 μm filter. The detailed process flow for pellet preparation and thawing and treatment with urea of examples 1.2 and 1.3 is shown in figure 2.

Example 1.5 purification on IEX column

Distilled Water (DW) was passed through a column packed with IEX resin at a rate of 100cm/hr at 3 Column Volumes (CV) to remove the stock solution. Then, a 1N NaOH solution was passed therethrough at 40cm/hr for 1 hour to conduct washing. Tris-HCl solution as equilibration buffer was flowed through it in 3 column volumes to reach equilibrium. After equilibration was complete, the concentrate and buffer exchange 1 treatment solution were adsorbed by IEX chromatography. The equilibration buffer was flowed through the column in 3 column volumes to remove the solution remaining in the column. Subsequently, a Tris-HCl solution containing a NaCl solution, i.e., a washing solution, was flowed therethrough in 5 column volumes to perform washing. After the washing was completed, Tris-HCl solution containing NaCl solution, i.e. elution buffer, was flowed through it in 5 column volumes to elute PRN protein.

Example 1.6 purification on HIC column

Distilled water was passed through the chromatographic column packed with the HIC resin at a rate of 100cm/hr for 3 column volumes to remove the storage solution. Then, a 1N NaOH solution was passed therethrough at 40cm/hr for 1 hour to conduct washing. Tris-HCl solution containing NaCl solution as the equilibration buffer was passed through it in 3 column volumes to reach equilibrium. After equilibration was complete, a solution obtained by passing the eluate obtained from the purification process on the IEX column through the HIC column with 3.6M NaCl in a ratio of 1: 1 ratio of the mixture. The equilibration buffer was flowed through the column in 3 column volumes to remove the solution remaining in the column. Subsequently, a Tris-HCl solution containing a NaCl solution, i.e., a washing solution, was flowed therethrough in 5 column volumes to perform washing. After the washing was completed, Tris-HCl solution containing NaCl solution, i.e. elution buffer, was flowed through it in 5 column volumes to elute PRN protein.

Example 1.7 purification on a GFC column

Distilled water was passed through a column packed with GFC resin at a rate of 15cm/hr at 3 column volumes to remove the storage solution. Then, a 1N NaOH solution was passed therethrough at 15cm/hr for 1 hour to conduct washing. Sodium phosphate solution containing NaCl as an equilibration buffer was flowed through it in 3 column volumes to reach equilibration. After equilibration is complete, the concentrate and buffer exchange 2 treatment solution are passed through a GFC column to elute the PRN protein.

Experimental example 1(Experimental samples) the difference in PRN extraction depending on pellet preparation and storage conditions was determined

To determine the conditions effective to increase PRN protein extraction, various pellet preparation and storage condition experiments were performed in obtaining the PRN of example 1. Pellet preparation and storage conditions as shown in table 1 in example 1.3, experimental procedures and results depending on the respective conditions are shown in the following experimental examples 1.1 to 1.5.

Experimental example 1.1. color change of supernatant after Urea treatment depends on storage conditions of slurry and pellets

Each pellet obtained by using different preparation and storage conditions as shown in table 1 of example 1.3 was treated with urea buffer and stirred. Then, the resultant was centrifuged. The supernatant obtained by the centrifugal separation was filtered with a filter, and the color difference of the supernatant was recognized according to each storage condition. The above procedure is described in detail in example 1.4 and the experimental results are shown in fig. 3.

As shown in fig. 3, under the conditions in which the pellets were stored frozen and then refrigerated (examples 2 and 3 and examples 5 and 6), the supernatant showed a deep yellow color. This means that after cryopreservation and thawing, many pellets break up in the case of treatment with urea.

Experimental example 1.2 determination of differences in protein extraction amount according to pellet storage conditions

SDS-PAGE and western blot techniques were used to identify differences in PRN protein extraction depending on pellet storage conditions. Here, In the western blotting technique, Guinea Pig (guineapig) anti-PRN (Young In Frontier) and biotin-labeled Guinea Pig anti-PRN (Young In Frontier) were used as antibodies. These results are shown in fig. 4 and 5.

As shown in fig. 4 and 5, under the conditions of freeze-preservation and then cold-preservation of the pellets (examples 1 to 6), a large amount of PRN protein was obtained.

Experimental example 1.3 extraction amount of PRN protein was determined according to storage conditions of slurry and pellet

Enzyme-linked immunosorbent assay (ELISA) techniques were used to determine differences in PRN protein extraction depending on slurry and pellet storage conditions. The results are shown in FIG. 6. Here, the respective pellet storage conditions of item 1 to item 11 at the bottom of the graph in fig. 6 are as follows, and more detailed conditions are shown in the following table 2:

1: treating the granules with urea immediately after preparation;

2: frozen for storage and thawed at room temperature for 1 day;

3: thawing for 1 day at room temperature without freezing;

4: freezing for storage and thawing in cold storage for 1 day;

5: frozen storage and thawing under refrigeration for 3 days;

6: unfreezing for 1 day without freezing and under refrigeration;

7: unfreezing for 3 days without freezing and under refrigeration;

8: freezing (-30 deg.C) and storing for 1 day;

9: freezing (-30 deg.C) and storing for 3 days;

10: freezing at (-70 deg.C) for 1 day; and

11: frozen (-70 ℃) for 3 days.

TABLE 2

	Storing the slurry for 0 day	Slurry, store for 3 days
			1. Treatment immediately after pellet preparation	Comparative example 1	Comparative example 9
2. Stored frozen and then stored at room temperature for 1 day	Example 1	Example 4
			3. Storing at room temperature for 1 day	Comparative example 2	Comparative example 10
4. Freezing and then cold-storing for 1 day	Example 2	Example 5
			5. Freezing and then cold-storing for 3 days	Example 3	Example 6
6. Refrigerating for 1 day	Comparative example 3	Comparative example 11
			7. Refrigerating for 3 days	Comparative example 4	Comparative example 12
8. Freezing at (-30 deg.C) for 1, 1 day	Comparative example 5	Comparative example 13
			9. Freezing at (-30 deg.C) for 1, 3 days	Comparative example 6	Comparative example 14
10. Freezing at (-70 deg.C) for 2, 1 days	Comparative example 7	Comparative example 15
			11. Freezing at (-70 deg.C) for 2, 3 days	Comparative example 8	Comparative example 16

As shown in fig. 6, pellets prepared after storage for 0 days in a slurry state, the extraction amount of PRN protein increased with time upon cryopreservation (by comparison of examples 2 and 3); further, when pellets prepared after storage in a slurry state for 3 days were stored under refrigeration, the extraction amount of PRN protein decreased with the passage of time (see comparison between example 5 and example 6). In addition, the cryopreserved pellets showed an increase in extraction amount of PRN protein compared to pellets that were not cryopreserved (comparison of example 1 with comparative example 2, comparison of example 2 with comparative example 3, and comparison of example 3 with comparative example 4). This means that in order to increase the extraction of PRN protein, it is more efficient to immediately granulate (pelletize) and store the pellet than to store the slurry as such, and the freezing step results in a large increase in extraction of PRN protein.

Experimental example 1.4 determination of the differences in PRN protein extraction depending on pellet thawing

In fig. 6, of the experimental groups stored frozen (examples 1 and 4, examples 2 and 5, examples 3 and 6, comparative examples 5 and 13, comparative examples 6 and 14, comparative examples 7 and 15, and comparative examples 8 and 16), the experimental groups stored frozen and thawed (examples 1 and 4, examples 2 and 5, and examples 3 and 6) showed a significant increase in the extraction amount of PRN protein. Therefore, further experiments were performed to identify whether the amount of PRN protein extracted varies depending on thawing. Here, pellet thawing was carried out at a refrigeration temperature of 5 ℃ for 48 hours. The experimental results are shown in table 3 and fig. 7.

TABLE 3

Condition numbering	PRN concentration (μ g/mL)	Specific storage conditions	State of the pellets before Urea treatment
				Comparative example 15	39.95	Slurry, store for 3 days; pellets were stored at-70 ℃ for 1 day	Freezing
Comparative example 16	34.68	Slurry, store for 3 days; pellets were stored at-70 ℃ for 3 days	Freezing
				Comparative example 17	32.31	Slurry, store for 3 days; pellets were stored at-70 ℃ for 10 days	Freezing
Example 7	99.94	Slurry, store for 3 days; pellets were stored at-70 ℃ for 10 days	Unfreezing (5 ℃, 48 hours)

As shown in table 3 and fig. 7, in the case where the pellets were thawed before treatment with urea, an increase of about 3 times in the extraction amount of PRN protein was observed compared to the case of frozen pellets. In all three experimental groups in which urea treatment of frozen pellets was performed, the extraction of PRN protein was low. This means that the thawing of the pellets has a great influence on the extraction of PRN protein before the treatment with urea. From these results, it was found that in order to effectively increase the extraction amount of PRN protein, the pellets had to be thawed before treatment with urea.

Experimental example 1.5 determination of the difference in PRN protein extraction amount depending on pellet thawing conditions

To determine the difference in the extraction amount of PRN protein depending on the pellet thawing method, the extraction amount of PRN protein was examined in the following cases: after frozen storage of the pellets, they were refrigerated (2 days), at room temperature (1 hour) and 37 deg.C (15 minutes). These results are shown in fig. 8.

As shown in fig. 8, when thawing was performed, an increase in the extraction amount of PRN protein was observed in the case of slow thawing in cold storage, compared to the case of fast thawing at high temperature. In addition, an increase of about 2-fold in the extraction amount of PRN protein was observed in the case of thawing in a refrigerator for 2 days, compared to the case of thawing at 37 ℃ for 15 minutes. From these results, it was found that in order to effectively increase the extraction amount of PRN protein, the pellet had to be slowly refrigerated and thawed after freezing.

Experimental example 2 determination of the possibility of amplification (scale-up)

To determine whether the conditions identified in examples 1.1 to 1.5 can be effectively applied at a rate of even 50L and under which the amount of PRN protein extracted can be effectively increased, the slurry was immediately granulated without storage, and the resulting pellets were stored frozen (-30 ℃) for 1 day and then thawed in cold storage (5 ℃) for 2 days. Subsequently, 533g of pellets were collected, treated with 1800mL of 5.0M urea buffer, and stirred using a magnetic bar for 3 hours. The stirred urea-treated solution was centrifuged at 6,300rpm for 1.5 hours. After centrifugation, cell debris was removed; 1650mL of supernatant was collected and filtered through a filter. Subsequently, the procedure of examples 1.5 to 1.7 was carried out. The results are shown in Table 4.

TABLE 4

	Total volume (mL)	PRN concentration (μ g/mL)	Total PRN amount (mg)
				PRN	176.3	701.98	123.76

As shown in Table 4, it was found that under the conditions specified in Experimental examples 1.1 to 1.5, the amount of extraction of PRN protein could be effectively increased even on the 50-L scale, and 300mg to 400mg of PRN protein could be obtained on the 200-L scale.

Claims

1. A method of obtaining a bordetella pertussis-derived PRN protein, comprising the steps of:

1) freezing a sample containing bordetella pertussis;

2) thawing the frozen sample;

3) disrupting the thawed sample; and

4) purifying the disrupted sample.

2. The method according to claim 1, wherein the sample containing bordetella pertussis is a pellet obtained by centrifugation of a bordetella pertussis culture.

3. The method of claim 1, wherein, in step 1), the freezing is performed at a temperature of-5 ℃ to-85 ℃.

4. The method according to claim 1, wherein, in step 1), the freezing is performed for 20 to 30 hours.

5. The method according to claim 1, wherein in step 2) the thawing is performed in refrigeration.

6. The method of claim 5, wherein the thawing is performed at a temperature of 1 ℃ to 10 ℃ under refrigeration.

7. The method of claim 5, wherein the thawing is performed by cold storage for 36 to 48 hours.

8. The method according to claim 1, wherein, in step 3), the destruction is performed by any one selected from the group consisting of urea treatment, heat treatment, guanidine treatment and iodine treatment.

9. The method of claim 8, wherein the treatment with urea is performed at a urea concentration of 3M to 7M.

10. The method of claim 8, wherein the treatment with urea is carried out for 150 to 210 minutes.

11. The method according to claim 1, wherein, in step 4), the purification is performed by ion exchange chromatography (IEX), Hydrophobic Interaction Chromatography (HIC) and Gel Filtration Chromatography (GFC).