KR20190013296A - Method for obtaining bordetella pertussis related protein comprising processes of freezing and thawing - Google Patents

Abstract

The present invention relates to a method for obtaining a Bordetella pertussis-derived PRN protein, comprising a freezing and thawing process capable of effectively increasing an extraction level of PRN protein. In the present invention, it was found that when a pellet was frozen before treatment with urea and then slowly thawed at a cold temperature, the extraction level of PRN protein was effectively increased compared to experiment groups under other conditions. In addition, even when the method was applied to mass-scale production, the PRN extraction level was effectively increased. Therefore, a pretreatment process for Bordetella pertussis comprising the freezing and thawing process can be effectively used for mass-production of PRN for Bordetella pertussis vaccines.

Description

METHOD FOR OBTAINING BORDETELLA PERTUSSIS RELATED PROTEIN COMPRISING PROCESSES OF FREEZING AND THAWING [0002]

The present invention relates to a method for purifying PRN protein derived from pertussis bacteria including a freezing and thawing process.

Whooping cough is an acute respiratory disease characterized mainly by infants and cough over 2 weeks. Bordetella pertussis , a gram-negative short aerobic bacterium, has been reported as a cause of pertussis. Pertussis bacteria use humans as their only host and are mainly infected through the respiratory tract. In addition, the pertussis bacteria live in the respiratory tract mucosa and cause diseases in the human body. The 1930s cell pertussis vaccine was developed and proved to be effective against pertussis. In addition, the pertussis vaccine was used in combination with the tetanus and diphtheria inactivated vaccine of the 1940s, but side effects (seizures, edema, fever, etc.) of the whole-cell pertussis vaccine were reported, and the safety of the pertussis vaccine Development was required.

In the 1950s, the pathogenesis of Pertussis toxin (PT), filamentous hamagglutinin (FHA), Pertactin (PRN) and fimbriae (FIM) were reported as antigens. Since then, the development of the acellular pertussis vaccine has been progressed. In the 1980s, the purified pertussis vaccine was first developed and inoculated in Japan.

Proteins such as PT, FHA, and PRN must be purified to produce a non-cellulolytic pertussis vaccine. Conventionally, the antigen was simultaneously purified by repeating the ammonium sulfate precipitation method and the density gradient centrifugation method, but this method has a disadvantage in that it has many impurities and it is difficult to control the purification process. Another method is to purify each antigen individually by combining physical and chemical methods. Korean Laid-Open Publication No. 2015-0124973 discloses a cell-free pertussis vaccine composition comprising PT, FHA, and FIM types 2 and 3.

On the other hand, the method of producing pertussis vaccine, which is produced by mixing three or four components, has a problem in that, when one component is inferior in productivity, the entire production period is increased.

Therefore, a method for efficient mass production of PRN protein has been sought to efficiently produce pertussis vaccine.

KR 10-2015-0124973 A

Accordingly, the present inventors have made efforts to secure a condition capable of increasing the amount of PRN protein extracted from pertussis bacteria. When a sample containing pertussis bacteria undergoes cold-shock and thawing under specific conditions, The present inventors have completed the present invention by confirming that the amount of PRN protein extracted from bacteria is increased.

Accordingly, the present invention provides a method for obtaining a PRN protein capable of increasing the amount of PRN protein extracted from pertussis bacteria.

In order to achieve the above object, the present invention provides a method for producing a pertussis microorganism, comprising: 1) freezing a sample containing pertussis bacteria; 2) thawing the frozen sample; 3) crushing the thawed sample; And 4) purifying the fragmented sample.

The method for obtaining the PRN protein of the pertussis bacteria according to the present invention is to maximize the extraction amount of the PRN protein by freezing the sample containing the pertussis bacteria and thawing at the refrigeration temperature. When PRN was obtained by purifying a sample pretreated under the above conditions, the amount of PRN protein extracted was significantly increased compared with the extraction method without such pretreatment. In addition, the method for obtaining the PRN protein may be useful for mass production of PRN protein for producing a pertussis vaccine.

FIG. 1 shows a flow chart of a PRN purification process.
Figure 2 is a flow chart of the pellet preparation, thawing and urea treatment processes in detail.
Figure 3 shows the results of treatment of urea with each pellet with different manufacturing and storage conditions.
FIG. 4A shows differences in the amount of protein extracted when the PRN protein was obtained by the methods of Comparative Examples 1 to 5 and Examples 1 to 3 using the SDS-PAGE technique.
FIG. 4B shows the difference in the amount of protein extracted when the PRN protein was obtained by the methods of Comparative Examples 6 to 10, Example 4, and Example 5 using the SDS-PAGE technique.
FIG. 4C shows differences in the amount of protein extracted when the PRN protein was obtained by the methods of Comparative Examples 11 to 16 and Example 6 using the SDS-PAGE technique.
FIG. 5A shows the difference in the amount of PRN protein extracted when the PRN protein was obtained by the methods of Comparative Examples 1 to 5 and Examples 1 to 3 using the Western blotting technique.
5B shows the difference in the amount of PRN protein extracted when the PRN protein was obtained by the methods of Comparative Examples 6 to 10 and 4 and 5 using the Western blot technique.
FIG. 5c shows differences in the amount of PRN protein extracted when the PRN protein was obtained by the methods of Comparative Examples 11 to 16 and Example 6 using the Western blot technique.
FIG. 6 shows the amounts of PRN protein extracted at the time of obtaining PRN proteins by the methods of Comparative Examples 1 to 7 and Examples 1 to 3 in which slurry and pellet storage conditions were different.
7 shows the extraction amount of the PRN protein when the PRN protein was obtained by the methods of Comparative Examples 15 to 17 and Example 7 in which the pellet storage conditions were different.
Fig. 8 shows the difference in the amount of PRN protein extracted when the thawing conditions of the pellet were different.

In one aspect, the present invention provides a method for producing a pertussis-containing microorganism, comprising: 1) freezing a sample containing pertussis bacteria; 2) thawing the frozen sample; 3) crushing the thawed sample; And 4) purifying the crushed sample. The present invention also provides a method for obtaining the pertussis bacteria-derived PRN protein.

First, a step of freezing a sample containing pertussis bacteria is performed.

In the present invention, "pertussis bacterium" is also referred to as Bordetella pertussis pertussis bacterium. PRN, which is one of the major proteins of pertussis, is an abbreviation of pertactin and a toxic factor of pertussis causing pertussis. In addition, PRN is an outer membrane protein adhered to tracheal epithelial cells, which is obtained from pertussis bacteria and is one of the important components of the pertussis vaccine.

In the present specification, the "sample containing pertussis bacteria" may be a culture medium in which pertussis bacteria are cultured, a slurry separated from the culture medium through continuous centrifugation, or a pellet obtained from the slurry through high-speed centrifugation, Preferably pellets. At this time, the pellets can be obtained by directly centrifuging the obtained slurry. However, the obtained slurry can be centrifuged after 1 to 5 days to obtain pellets. At this time, the slurry can be stored at 0 캜 to 25 캜, but is not limited thereto.

In one embodiment of the present invention, the pertussis strain is cultured in a modified stainer scholte (MSS) medium at a temperature of 35 DEG C, the cell culture is subjected to continuous centrifugation at room temperature for 2 hours, and the slurry obtained therefrom is heated at 2 to 8 DEG C At a speed of 7,000 rpm for 50 minutes.

Second, the frozen sample is thawed.

In the step of freezing the sample containing the pertussis strain, the freezing temperature is -1 ° C, -2 ° C, -5 ° C, -10 ° C, -20 ° C, -30 ° C, -40 ° C, -50 ° C, -70 ° C, -80 ° C and -90 ° C and may be -1 ° C to -90 ° C, -5 ° C to -85 ° C, -15 ° C to -75 ° C, -30 ° C to -60 ° C . The freezing time may be 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 20 hours, 30 hours, 40 hours, and may be from 0.5 hours to 40 hours, 1 hour to 30 hours, 2 hours to 20 hours, 5 hours To 10 hours.

In addition, the frozen sample is stored in a refrigerator and thawed slowly. At this time, the refrigeration temperature for thawing may be 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 18 ° C, 5 ° C to 15 ° C, 7 ° C to 10 ° C. The refrigeration time may be 0.1 hour, 1 hour, 5 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, and 0.1 hour to 60 hours, 1 hour to 55 hours, 5 hours to 50 hours , And 12 hours to 48 hours.

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

At this time, the step of freezing and thawing may be repeated one or more times.

Third, the step of shredding the thawed sample is performed.

The thawed sample is subjected to a crushing step. At this time, the crushing may be performed through, for example, urea treatment, heat treatment, guanidine treatment, or iodine treatment, but not limited thereto, various crushing methods known in the art can be applied.

When the urea is treated to break up the sample, the urea concentration may be 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, M, 1 M to 8 M, 3 M to 7 M, 4 M to 5 M. The urea treatment time may be 5 minutes, 10 minutes, 20 minutes, 50 minutes, 80 minutes, 120 minutes, 180 minutes, 240 minutes, 360 minutes, 5 minutes to 360 minutes, 20 minutes to 240 minutes, .

In one embodiment of the present invention, the defrosted pellet was treated with a 5.0 M urea buffer for 4 times the weight of the pellet and stirred for 3 hours to break the outer membrane of the pertussis bacteria in the pellet.

Fourth, the step of purifying the crushed sample is carried out.

Cell lysates or cell cultures can be centrifuged and unnecessary cell debris and the like can be removed by various kinds of column chromatography or the like. The column chromatography may be ion exchange chromatography, hydrophobic interaction chromatography, gel exclusion chromatography, gel filtration chromatography, HPLC, reverse phase-HPLC, affinity chromatography, or the like.

In an embodiment of the present invention, ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and gel filtration chromatography (HIC) are used in purifying the pulverized pellets. , GFC) was performed to obtain a PRN protein.

As used herein, the term "ion exchange chromatography" can utilize anion exchange resins in which anions or cations are covalently bonded to a stationary phase called exchange resins. The solute ions of the opposite charge in the mobile phase are attracted to the stationary phase by electrostatic attraction. Ion exchange chromatography is based on the adsorption of an ion or an electrically charged compound to an ion exchanger by electrostatic force to form an equilibrium.

In one embodiment of the present invention, an anion exchange resin was used in an ion exchange chromatography column, a Tris-HCl solution as an equilibrium buffer, a Tris-HCl solution containing a NaCl solution as a washing buffer, and a NaCl solution as an elution buffer The purified Tris-HCl solution was used to elute the PRN protein.

As used herein, the term "hydrophobic interaction chromatography" is a method of separating using a hydrophobic interaction between a molecule and a molecule having a hydrophobic functional group. The support is hydrophilic and modifies the inactive agarose to have hydrophobic functionality. Alkylamines are reacted with BrCN-activated agarose to make a lot of modified agarose. Hydrophobic interaction chromatography is widely used for protein purification. It is also possible to reduce the ionic strength or increase the pH for the elution of the proteins in hydrophobic interaction chromatography and to reduce the polarity of aliphatic amines, alcohols or nonionic detergents (for example Tween- 20, Triton X-100, etc.) may also be used.

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

The term "gel filtration chromatography" as used herein refers to the use of molecular sieves on a stationary phase, which are hydrophilic as sephadex, polyacrylamide or agarose gel, so that they can absorb water and swell. When the size of the sample molecule is larger than the maximum pore of the swollen gel, the molecule does not pass through the gel particles, and therefore, it exits the column through the space of the stationary phase particle. Smaller molecules enter the open pores of the gel particles, but at different rates depending on their size and shape. Thus, the molecules are eluted in decreasing order of size. In gel chromatography, the size, viscosity, ionic strength, and flow rate of the sample are considered.

In one embodiment of the present invention, PRN protein was eluted using sodium phosphate containing NaCl solution as the equilibrium buffer.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  One. PRN  Obtaining step

The process of obtaining the PRN is a process of purifying and removing impurities from the PRN protein by pellet preparation and thawing, urea treatment, column chromatography (IEX, HIX, and SEC), concentration and buffer exchange (UF / DF) Respectively. A flow chart of the PRN obtaining process is shown in Fig.

Example  1.1. Culture of pertussis bacteria

Pertussis (National Institute of Health) was cultivated in MSS medium at 35 ℃ for 4 days. The culture period was 1 day for seed culture, 1 day for primary enrichment culture, 1 day for secondary enrichment culture, .

Example  1.2. Pellets  Produce

The cell culture broth was separated by continuous centrifugation at a flow rate of 100 L / h and 9,500 rpm at room temperature for 2 hours, and then separated into a supernatant and a slurry. The separated slurry was subjected to high-speed centrifugation at 7,000 rpm at 5 ° C ± 3 ° C for 50 minutes to prepare pellets. In the following example, when the slurry was stored for 3 days, it was stored at a refrigeration temperature of 5 ° C for 3 days, and then the pellet was prepared by high-speed centrifugation.

Example  1.3. Frozen and thawed

The prepared pellets were stored at -30 ° C or lower for 2 hours and then stored at room temperature (RT) for 1 day to dissolve the pellets. Thereafter, the procedures of Examples 1.4 to 1.7 below were carried out to obtain a PRN protein. Experiments were conducted in the same manner as in Examples 1 to 6 and Comparative Examples 1 to 16 of Table 1, except that the storage conditions of the slurry, the freezing and thawing conditions were different, and then the procedures of Examples 1.4 to 1.7 were carried out To obtain a PRN protein.

Experiment number Comparative Example  One Example  One Comparative Example  2 Example  2 Example  3 Comparative Example  3 Comparative Example  4 Comparative Example  5 Comparative Example  6 Comparative Example  7 Comparative Example  8 Slurry 0 day
keep Pellets
keep
Condition cold-
shock X O X O O X X X X X X keep
Condition X At room temperature (RT) Cold room Frozen 1
(F1, -30 ° C) Frozen 2
(F2, -70 < 0 > C) keep
Days X One One One 3 One 3 One 3 One 3 Experiment number Comparative Example  9 Example  4 Comparative Example  10 Example  5 Example  6 Comparative Example  11 Comparative Example  12 Comparative Example  13 Comparative Example  14 Comparative Example  15 Comparative Example  16 3 days of slurry
keep Pellets
keep
Condition cold-
shock X O X O O X X X X X X keep
Condition X At room temperature (RT) Cold room Frozen 1
(F1, -30 ° C) Frozen 2
(F2, -70 < 0 > C) keep
Days X One One One 3 One 3 One 3 One 3

Example  1.4. Urea  process

5.0 M urea buffer was added to the thawed pellet by 4 times the weight of the pellet, and the mixture was stirred at a speed of 280 rpm for 3 hours using a magnetic bar. The urea treatment solution stirred for 3 hours was subjected to high-speed centrifugation at a rate of 7,000 rpm (12230 RCF) at a temperature of 5 캜 3 캜 for 1.5 hours. After centrifugation, the cell pieces were removed and the supernatant was collected. The recovered supernatant was filtered with a 0.22 mu m filter. The detailed process flow charts of pellet preparation and thawing and urea treatment of Examples 1.2 and 1.3 are shown in FIG.

Example  1.5. IEX column  refine

Distilled water (DW) was poured into the column filled with IEX resin with 3 CV at 100 cm / hr to remove the storage solution, followed by washing with 1 N NaOH solution at 40 cm / hr for 1 hour . The equilibrium buffer, Tris-HCl solution, was flowed with 3 CVs to equilibrate. After the equilibration was completed, the 1 solution of concentration and buffer exchange was flowed into the IEX column to adsorb it, and the equilibrium buffer was purged with 3 CV to remove the solution remaining in the column. Then, a Tris-HCl solution containing a NaCl solution as a washing buffer was purged with 5 CV to perform washing. After the washing was completed, the Tris-HCl solution containing the NaCl solution as the elution buffer was flowed with 5 CV to elute the PRN protein.

Example  1.6. HIC column  refine

The column was filled with HIC resin and distilled water was flowed at 100 cm / hr and 3 CV to remove the stock solution, and 1N NaOH solution was flowed at 40 cm / hr for 1 hour. A Tris-HCl solution containing an equilibrium buffer, NaCl solution, was poured into 3 CVs to equilibrate. After the equilibrium was completed, a 1: 1 mixture of the eluate obtained from the IEX column purification step and 3.6 M NaCl was adsorbed on the HIC column, and the equilibrium buffer was poured into 3 CV to remove the remaining solution in the column. Then, a Tris-HCl solution containing a NaCl solution as a washing buffer was purged with 5 CV to perform washing. After the washing was completed, the Tris-HCl solution containing the NaCl solution as the elution buffer was flowed with 5 CV to elute the PRN protein.

Example  1.7. GFC column  refine

The column was filled with GFC resin and distilled water was flowed at 15 cm / hr and 3 CV to remove the storage solution. The column was washed with 1N NaOH solution at 15 cm / hr for 1 hour. Sodium phosphate containing an equilibrium buffer, NaCl solution, was poured into 3 CVs to equilibrate. After the equilibrium was completed, the concentrated and buffer exchange 2 process liquid was flowed through the GFC column to elute the PRN protein.

Experimental Example  One. Pellets  Depending on manufacturing and storage conditions PRN  Confirm difference in extraction volume

In order to confirm the condition for effectively increasing the extraction amount of the PRN protein, the experiment was carried out by varying the preparation and storage conditions of the pellet in the PRN obtaining step of Example 1 above. The preparation and storage conditions of the pellets are shown in Table 1 of Example 1.3, and the experimental procedures and results according to each condition are shown in Experimental Examples 1.1 to 1.5.

Experimental Example  1.1. Slurry and Pellets  By storage condition Urea  Depending on the treatment Supernatant  Color change

As shown in Table 1 of Example 1.3, the pellet prepared and stored at different conditions was treated with urea buffer, stirred, and centrifuged. The supernatant obtained by centrifugation was filtered with a filter, and the color difference of the supernatant was confirmed according to each storage condition. The above procedure was specifically described in Example 1.4 and the results of the experiment are shown in FIG.

As shown in Fig. 3, the pellets were shown frozen and stored in a refrigerator (Examples 2 and 3, and Examples 5 and 6). This means that many pellets will break when urea is treated after freezing and thawing.

Experimental Example  1.2. Pellets  Confirm difference of protein extraction amount according to storage condition

SDS-PAGE and Western blot techniques were used to confirm the difference in the amount of PRN protein extracted according to the storage conditions of the pellet. At this time, Guinea Pig anti-PRN and Biotin-labeled Guinea Pig anti-PRN were used as antibodies in Western blotting. This is shown in Fig. 4 and Fig.

As shown in Figs. 4 and 5, a large amount of PRN protein was obtained under the conditions of cryopreservation of the pellet and storage at room temperature or refrigeration (Examples 1 to 6).

Experimental Example  1.3. Slurry and Pellets  Depending on storage conditions PRN  Identify protein extraction

Using ELISA (enzyme-linked immunosorbent assay), the difference in the amount of PRN protein extracted by slurry and pellet storage conditions was confirmed. This is shown in Fig. In this case, the pellet storage conditions of the pellets 1 to 11 at the bottom of the graph in FIG. 6 are as follows, and more specific conditions are shown in Table 2 below:

1: Urea treatment immediately after pellet preparation;

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

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

4: Thaw for 1 day in freezer storage and refrigeration;

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

6: Thaw for 1 day in chilled without refrigeration;

7: Thaw for 3 days in the refrigerator without freezing;

8: 1 day storage at freezing (-30 ℃);

9: kept frozen (-30 ℃) for 3 days;

10: stored for 1 day in freezing (-70 ° C); And

11: Store in freezing (-70 ℃) for 3 days.

Slurry 0 days storage Slurry 3 days storage One. Pellets  Process immediately after creation Comparative Example 1 Comparative Example 9 2. Frozen storage ( C.S ) After RT 1 day Example 1 Example 4 3. RT 1 day storage Comparative Example 2 Comparative Example 10 4. Frozen storage ( C.S ) 1 day after refrigerated storage Example 2 Example 5 5. Frozen storage ( C.S ) 3 days after refrigerated storage Example 3 Example 6 6. Keep one day refrigerated Comparative Example 3 Comparative Example 11 7. Keep refrigerated for 3 days Comparative Example 4 Comparative Example 12 8. Frozen 1  (-30 ° C) 1 day storage Comparative Example 5 Comparative Example 13 9. Frozen 1  (-30 ℃) 3 days storage Comparative Example 6 Comparative Example 14 10. Frozen 2  (-70 ° C) 1 day storage Comparative Example 7 Comparative Example 15 11. Frozen 2  (-70 ° C) 3 days storage Comparative Example 8 Comparative Example 16

As shown in FIG. 6, the pellets prepared after storage for 0 days in a slurry state showed an increase in the amount of PRN protein extracted over time in the freezing state (compared with Examples 2 and 3), and the pellets were stored for 3 days in a slurry state The pellet produced afterwards showed a decrease in the amount of PRN protein extracted over time in the freezing state (compare Examples 5 and 6). In addition, the frozen pellets showed an increase in the amount of PRN protein extracted compared to pellets that were not cryopreserved (comparison of Example 1 and Comparative Example 2, comparison of Example 2 and Comparative Example 3, and comparison of Example 3 and Comparative Example 4). In order to increase the amount of PRN protein extracted, it is effective to store pellet rather than to store it in a slurry state. This means that the refrigeration step greatly increases the amount of PRN protein extracted.

Experimental Example  1.4. Pellet  Depending on thawing condition PRN  Confirm difference in protein extraction amount

6 and Comparative Example 14, Comparative Example 6 and Comparative Example 14, which were stored in the cryopreserved experimental group (Example 1 and Example 4, Example 2 and Example 5, Example 3 and Example 6, Comparative Example 5 and Comparative Example 13 (Example 1 and Example 4, Example 2 and Example 5, and Example 3 and Example 6), which were subjected to a thawing process after cryopreservation in Example 7 and Comparative Example 15, and Comparative Example 8 and Comparative Example 16 ) Significantly increased the amount of PRN protein extracted. Therefore, further experiments were carried out to confirm the difference in the amount of PRN protein extracted depending on the thawing condition. At this time, thawing of the pellet was carried out at a refrigeration temperature of 5 캜 for 48 hours. The experimental results are shown in Table 3 and FIG.

Condition number PRN Conc
(쨉 g / mL) Storage details Urea  Before processing Pellets  condition Comparative Example  15 39.95 Storage of slurry for 3 days, storage of pellet at -70 ℃ for 1 day frozen Comparative Example  16 34.68 3 days for slurry, 3 days for pellet -70 ℃ frozen Comparative Example  17 32.31 Slurry 3 days storage, pellet 10 days -70 ℃ storage frozen Example  7 99.94 Slurry 3 days storage, pellet 10 days -70 ℃ storage Thawing (5 ℃, 48 hours)

As shown in Table 3 and FIG. 7, when the pellet before urea treatment was thawed, the amount of PRN protein extracted was about three times higher than that in the case of freezing. In all three experimental groups treated with urea in frozen pellet, the PRN protein was low. This means that thawing of the pellet before urea treatment has a great influence on the extraction of PRN protein. Thus, in order to effectively increase the extraction amount of the PRN protein, it was found that the pellet had to be thawed before the urea treatment.

Experimental Example  1.5. Pellet  Depending on thawing conditions PRN  Confirm difference in protein extraction amount

The pellets were stored frozen (2 days), at room temperature (1 hour) and at 37 ° C (15 minutes), respectively, in order to determine the difference in the amount of PRN protein extracted according to the thawing method of the pellet. Respectively. This is shown in FIG.

As shown in FIG. 8, when the extract was slowly thawed in the refrigerator, the extraction amount of the PRN protein increased, rather than rapidly thawing at a high temperature during thawing. In addition, the extract of PRN protein increased about two times when it was thawed for two days in the refrigerator after thawing at 37 ℃ for 15 min. In order to effectively increase the amount of PRN protein extracted, it was found that the pellet should be slowly thawed from the refrigerator after freezing.

Experimental Example  2. Confirm Scale up possibility

In order to confirm whether the conditions for effectively increasing the extraction amount of the PRN protein in Experimental Examples 1.1 to 1.5 were applicable even at 50 L, the slurry was immediately prepared as pellets without being stored, and stored for 1 day in freezing (-30 ° C.) (5 < 0 > C) for two days. Then, 533 g of the pellet was recovered, treated with 1,800 mL of 5.0 M urea buffer, and stirred for 3 hours using a magnetic bar. The stirred urea treatment solution was centrifuged at a rate of 6,300 rpm for 1.5 hours. After centrifugation, cell debris was removed and 1,650 mL of supernatant was collected and filtered with a filter. Then, the procedure of Examples 1.5 to 1.7 was carried out. The results are shown in Table 4.

Total volume (mL) PRN  Concentration (/ / mL) gun PRN  Content (mg) PRN 176.3 701.98 123.76

As shown in Table 4, it can be seen that the conditions for effectively increasing the extraction amount of PRN protein in Experiments 1.1 to 1.5 can be applied even at 50 L, and about 300 to 400 mg of PRN protein is obtained at 200 L scale I can see that it can be done.

Claims (11)

1) freezing a sample containing pertussis bacteria;
2) thawing the frozen sample;
3) crushing the thawed sample; And
4) purifying the above-mentioned crushed sample. The method according to claim 1,
Wherein the sample containing the pertussis bacteria is a pellet obtained by centrifuging the culture of the pertussis bacterial culture. The method according to claim 1,
The method for obtaining a PRN protein according to claim 1, wherein the freezing temperature of 1) is -5 ° C to -85 ° C. The method according to claim 1,
Wherein the freezing time of the above 1) is 20 hours to 30 hours. The method according to claim 1,
And the thawing of 2) is carried out in a cold state. 6. The method of claim 5,
Wherein the refrigeration temperature is 1 占 폚 to 10 占 폚. 6. The method of claim 5,
Wherein the refrigeration time is 36 to 48 hours. The method according to claim 1,
The method for obtaining a PRN protein according to claim 3, wherein the disruption of 3) is carried out through 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 urea concentration is from 3M to 7M. 9. The method of claim 8,
Wherein the urea treatment time is from 150 to 210 minutes. The method according to claim 1,
The purification of the above 4) is performed by performing ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and gel filtration chromatography (GFC) ≪ / RTI >

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