KR101825996B1 - Methods for Preparing Human Papillomavirus Virus-Like Particles - Google Patents

Methods for Preparing Human Papillomavirus Virus-Like Particles Download PDF

Info

Publication number
KR101825996B1
KR101825996B1 KR1020150142107A KR20150142107A KR101825996B1 KR 101825996 B1 KR101825996 B1 KR 101825996B1 KR 1020150142107 A KR1020150142107 A KR 1020150142107A KR 20150142107 A KR20150142107 A KR 20150142107A KR 101825996 B1 KR101825996 B1 KR 101825996B1
Authority
KR
South Korea
Prior art keywords
hpv
protein
concentration
nacl
salt
Prior art date
Application number
KR1020150142107A
Other languages
Korean (ko)
Other versions
KR20160080060A (en
Inventor
조양제
김광성
Original Assignee
아이진 주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 아이진 주식회사 filed Critical 아이진 주식회사
Publication of KR20160080060A publication Critical patent/KR20160080060A/en
Application granted granted Critical
Publication of KR101825996B1 publication Critical patent/KR101825996B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]

Abstract

The present invention provides a method for producing L1 virus-like particles (VLPs) of HPV. The present invention relates to a method of removing impurities by firstly removing impurities through ammonium sulfate precipitation and then secondarily removing impurities by dialysis in a solution containing 0.35-0.60 M of a salt, Purity HPV L1 protein can be produced.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for producing virus-like particles of human papilloma virus,

The present invention relates to a method for producing L1 virus-like particles (VLPs) of HPV.

Human Papillomavirus (HPV) is known to be present in more than 80 species. About 30 cases of HPV infection are caused by sexual contact, and about half of them are related to cervical cancer. In addition, the incidence of cancer among women with HPV types 16 and 18, especially HPV, is 500 times higher than that of uninfected women, and they are known to malignant transformation into cervical cancer after infection through the genital epithelium (Hausen, Biochemica Biophysica Acta 1288: 55-78, 1996).

The presently developed preventive vaccine against cervical cancer has been developed using high-risk HPV-like particles (VLPs). VLP is an L1 protein (about 55 kDa), the main capsid protein of HPV, and has a property of self-aligning to VLPs without any other viral gene product, which is similar to that of natural HPV virion. In addition, the immunity is long-lasting, and it is type-specific to the genotype, so that it can be expected to have a high efficiency as a vaccine candidate.

Attempts have been made to produce VLPs of HPV using the yeast expression system and the following method for purifying VLPs of HPV produced by Saccharomyces cerevisiae is known . Korean Patent No. 10-0959145 discloses a method in which a transforming yeast expressing an HPV L1 protein is cultured and then lysed and ammonium sulfate is added to the yeast lysate to precipitate the protein, A method for purifying HPV L1 protein by sequentially performing size-exclusion chromatography and cation-exchange chromatography on protein precipitates is disclosed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-0959145 (2010.05.13)

The present inventors have conducted studies on the purification conditions capable of increasing the production yield in the industrial scale production and purification of HPV L1 protein using a yeast expression system. As a result, ammonium sulfate precipitation was performed on the yeast product containing L1 protein, and the resulting precipitate was treated with a salt of 0.35-0.60 M to remove impurities and then subjected to chromatography to obtain HPV L1 protein The purification efficiency of the present invention can be greatly improved, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for producing L1 virus-like particles of HPV.

Another object of the present invention is to provide Ll virus-like particles of the HPV virus produced by the above method.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing L1 virus-like particles (VLPs) of HPV (HPV) comprising the steps of:

(a) culturing a transforming yeast expressing the L1 protein of HPV;

(b) performing ammonium sulfate precipitation on the product of the transformed yeast cultured in step (a) to obtain a precipitate;

(c) treating the precipitate obtained by ammonium sulfate precipitation with a salt of 0.35-0.60 M, incubating and removing the formed insoluble protein; And

(d) Chromatography of the product of step (c) to obtain L1 protein.

The present inventors have conducted studies on the purification conditions capable of increasing the production yield in the industrial scale production and purification of HPV L1 protein using a yeast expression system. As a result, ammonium sulfate precipitation was performed on the yeast product containing L1 protein, and the resulting precipitate was treated with a salt of 0.35-0.60 M to remove impurities and then subjected to chromatography to obtain HPV L1 protein The purification efficiency of the present invention can be greatly improved.

Hereinafter, each step of the present invention will be described in detail.

(a) Culturing of transforming yeast expressing HPV L1 protein

In step (a) of the present invention, a transforming yeast expressing the L1 protein of HPV is cultured.

Cells that can be used to express the HPV L1 protein in the present invention are yeast, and examples thereof include Saccharomyces cerevisiae , Saccharomyces pastorianus , Saccharomyces sp . Saccharomyces sp. , Schizosaccharomyces pombe , and baker's yeast.

According to one embodiment of the present invention, the transgenic yeast of the present invention is Saccharomyces cerevisiae .

The transforming yeast expressing the HPV L1 protein refers to a yeast cell transformed with an expression vector that successfully expresses the HPV L1 protein. The expression vector may comprise a transcription or translation regulatory element, other marker genes, as known in the art.

The HPV L1 protein expressing transformed yeast of the present invention can be easily prepared using methods known in the art, and these methods are described in U.S. Pat. Pat. Nos. US 7250170, US 6613557, US 5888516, US 5871998, US 5618536, US5437951, and the contents of these patent documents are incorporated herein by reference.

The transformed yeast can be cultured by a method used in the art for the production of HPV L1 protein using yeast. For example, the transformed yeast can be cultured in a medium supplemented with a carbon source of at least one of glucose and galactose as a carbon source. Exemplary media used in the transformation yeast culture include YPDG medium (yeast extract, peptone, glucose, Galactose). In addition, the transformed yeast can be cultured in a medium supplemented with a carbon source for less than 96 hours (e.g., 95 hours or less, 10-95 hours, 15-95 hours, 20-95 hours, or 24-95 hours).

According to one embodiment of the present invention, the L1 protein of the HPV is selected from the group consisting of HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 35, HPV type 39 , HPV type 45, HPV type 51, HPV type 52, HPV type 56, HPV type 58 and HPV type 68. According to a more specific embodiment, the L1 protein of the HPV is HPV type 16 or HPV type 18.

(b) Acquisition of ammonium sulfate precipitate

In step (b) of the present invention, the product of the transformed yeast cultured in step (a) (including the L1 protein) is treated with ammonium sulfate to obtain a precipitate. For example, in step (b), the transformed yeast cultured in step (a) may be lysed, and the yeast lysate may be treated with ammonium sulfate to obtain a precipitate.

As a method of dissolving the cultured yeast cells, there can be used any method for obtaining a cell lysate known in the art such as, for example, sonication, disintegration with glass beads, and the like.

Thereafter, the yeast product is treated with ammonium sulfate (added) to precipitate the expressed protein, and the impurities are removed by centrifugation or the like.

According to one embodiment of the present invention, the concentration of ammonium sulfate treated in step (b) is 20-60 wt%. According to a more specific embodiment, the treatment concentration of the ammonium sulfate is 40-50% by weight or 42-48% by weight.

According to one embodiment of the present invention, the precipitate (protein pellets) obtained by performing the ammonium sulfate precipitation in step (b) is incubated in a solution containing a salt (e.g. NaCl).

According to one more specific embodiment, the precipitate obtained by ammonium sulfate precipitation is diluted to a concentration of 25-65 mg / mL by adding a buffer solution and then diluted to a concentration of 0.5 M or more (e.g., 0.5-0.9 M, 0.6-0.9 M or 0.7-0.9 M) and incubated for a period of time and then incubated for at least 12 hours (e.g., 18-20 hours). The temperature of the incubation may be 3-5 DEG C (e.g., 4 DEG C).

(c) Treatment of a salt of 0.35-0.60 M to remove impurities

In step (c) of the present invention, the ammonium sulfate precipitate is treated with a salt of 0.35-0.60 M, and the salt-treated product is incubated to form an insoluble protein, which is an impurity. Thereafter, the purification efficiency of the HPV L1 protein is greatly improved by removing the formed insoluble protein.

As described in the following examples, the salt treatment of 0.35-0.60 M in step (c) is a step contributing greatly to the improvement of the purification efficiency of the HPV L1 protein (see Example 3). That is, if the insoluble protein produced in this step is removed by centrifugation, the purity of the VLP eluted in the subsequent chromatography step can be greatly increased.

According to one embodiment of the present invention, in step (c), the ammonium sulfate precipitation-containing solution incubated for a certain period of time in the presence of the salt (for example, NaCl) is dialyzed in a buffer containing 0.35-0.60 M of salt Followed by incubation to remove the impurities by insolubilizing them. The buffer solution containing the 0.35-0.60 M salt may include a nonionic surfactant. For example, the nonionic surfactant may be included at a concentration of 0.001-0.1%.

According to one embodiment of the present invention, the concentration of the ammonium sulfate precipitate to be treated with the salt of 0.35-0.60 M is 25-65 mg / mL. The concentration of this ammonium sulfate precipitate can be controlled by mixing the buffer solution with the ammonium sulfate precipitate.

According to one more specific embodiment, the concentration of the ammonium sulfate precipitate is 28-62 mg / mL, 28-60 mg / mL, 28-55 mg / mL, 28-50, 30-62 mg / mL, mg / mL, 30-55 mg / mL, or 30-50 mg / mL.

According to one embodiment, the salt is NaCl, KCl or Na 2 CO 3.

According to one embodiment of the present invention, the concentration of the salt to be treated in step (c) is 0.35-0.60 M, 0.35-0.55 M, 0.35-0.50 M, or 0.35-0.45 M; 0.40-0.60 M, 0.40-0.55 M, 0.40-0.50 M, or 0.40-0.45 M; 0.45-0.60 M, 0.45-0.55 M, or 0.45-0.50 M; Or 0.50-0.60 M or 0.50-0.55 M.

According to one embodiment of the present invention, the salt to be treated in step (c) is NaCl and the treatment concentration of NaCl is 0.35-0.60 M or 0.35-0.55 M; 0.40-0.60 M or 0.40-0.55 M; Or 0.45-0.60 M or 0.45-0.55 M.

According to another embodiment of the present invention, the salt to be treated in step (c) is KCl and the treatment concentration of KCl is 0.35-0.60 M or 0.40-0.60 M; 0.35-0.55 M; Or 0.55-0.60 M

to be.

According to another embodiment of the present invention, the salt to be treated in step (c) is Na 2 CO 3 and the treatment concentration of Na 2 CO 3 is 0.35-0.60 M or 0.40-0.60 M; 0.35-0.55 M or 0.40-0.55 M; 0.35-0.50 M or 0.40-0.50 M; Or 0.35-0.45 M.

According to one embodiment of the invention, the incubation temperature in step (c) is 25-34 占 폚. According to a more specific embodiment, the incubation temperature is 26-34 ° C, 27-34 ° C, 28-34 ° C, 29-34 ° C or 29.5-34 ° C, according to another embodiment 25-33.5 ° C, 26 -33.5 占 폚, 27-33.5 占 폚, 28-33.5 占 폚, 29-33.5 占 폚, 29.5-33.5 占 폚, or 30-33 占 폚.

According to one embodiment of the invention, the incubation time in step (c) is 21-27 hours. According to a more specific embodiment, the incubation time is 21-26 hours or 22-26 hours.

(d) Chromatography

In step (d) of the present invention, the solution from which the impurities have been removed is subjected to chromatography to obtain the L1 protein. When the solution in which impurities are removed through the above-described steps (a) to (c) is purified through chromatography, residual impurities can be very effectively removed.

According to one embodiment of the present invention, the chromatography is affinity chromatography or cation exchange chromatography. According to one more specific embodiment, the affinity chromatography is heparin chromatography.

For example, when performing heparin chromatography, the heparin resin is first equilibrated with a suitable binding buffer before applying the result of step (c) to the heparin resin column. The binding buffer preferably comprises NaCl and a non-ionic surfactant (e.g., Tweeen 80), and NaCl is preferred to have a low concentration (0.1-0.4 M). Then, the sample is applied to heparin chromatography, and the protein bound to the resin is eluted. The elution method is preferably a NaCl linear gradient, more preferably a 0.33-0.66 M NaCl linear gradient, and eluting the desired L1 protein in a 0.66-2 M NaCl linear gradient.

As another example, when carrying out cation exchange chromatography, the resin is first equilibrated with a suitable binding buffer prior to applying the resulting 0.35-0.60 M salt to remove the impurities from the column. The binding buffer preferably comprises NaCl and a non-ionic surfactant (e.g., Tweeen 80), with NaCl being preferred at low concentrations (0.3-0.6 M). The sample is then subjected to cation exchange chromatography and then the protein bound to the resin is eluted. The elution method is preferably a NaCI step concentration gradient, more preferably eluting the desired L1 protein using an elution buffer containing 0.6 M, 0.7 M, 0.8 M and 1 M NaCl.

Cation exchange chromatography can be carried out using various resins and is preferably carried out using a cation exchanger with sulfo, sulfoalkyl (e.g. sulfomethyl, sulfoethyl and sulfopropyl), phosphate or phosphate alkyl functionalities And most preferably with a cation exchange agent to which a phosphate functional group is attached.

In the present invention, the L1 protein fraction obtained through the above-mentioned chromatography process can be concentrated by a membrane filter. For example, chromatographic fractions can be concentrated using a membrane that can cut off molecular weights of 50-100 kDa. VLPs having an average size of about 50 nm have a larger size than other proteins, so they can not pass through the membrane and are concentrated. On the other hand, most residual impurities can be removed through the membrane.

According to one embodiment of the present invention, when the chromatography of step (d) is heparin chromatography, the salt to be treated in step (c) is NaCl and the treatment concentration of NaCl is 0.35-0.60 M, 0.40-0.60 M, 0.45-0.60 M or 0.45-0.55 M.

According to another embodiment of the present invention, when the chromatography of step (d) is heparin chromatography, the salt to be treated in step (c) is KCl and the treatment concentration of KCl is 0.35-0.60 M, 0.40-0.60 M , 0.45-0.60 M, 0.50-0.60 M, or 0.55-0.60 M.

According to another embodiment of the present invention, and when the chromatography of step (d) the heparin chromatography, the salt to be treated in step (c) is Na 2 CO 3, concentrations of Na 2 CO 3 was 0.35- 0.60 M or 0.40-0.60 M; 0.35-0.45 M; Or 0.55-0.60 M.

According to another embodiment of the present invention, when the chromatography of step (d) is cation exchange chromatography, the salt to be treated in step (c) is NaCl and the treatment concentration of NaCl is 0.35-0.60 M, 0.60 M or 0.45-0.55 M.

According to another embodiment of the present invention, when the chromatography of step (d) is cation exchange chromatography, the salt to be treated in step (c) is KCl and the treatment concentration of KCl is 0.35-0.60 M, 0.35- 0.55 M, 0.35-0.50 M, or 0.35-0.45 M.

According to another embodiment of the present invention, when the chromatography of step (d) is cation exchange chromatography, the salt to be treated in step (c) is Na 2 CO 3 and the treatment concentration of Na 2 CO 3 is 0.35 -0.60 M, 0.35-0.55 M, 0.35-0.50 M, or 0.35-0.45 M.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a method for producing L1 virus-like particle (VLP) of HPV.

(Ii) In the present invention, the impurities are firstly removed through ammonium sulfate precipitation and then subjected to a single chromatography process by a method of removing impurities by dialysis in a solution containing a salt of 0.35-0.60 M And HPV L1 protein can be produced with high purity.

(Iii) The HPV antigen produced according to the production method of the present invention is suitable for use as a raw material for not only a vaccine but also an HPV antibody diagnostic agent requiring high stability.

Fig. 1 is a diagram related to Example 1, showing the removal efficiency of insoluble proteins according to the type of size-up buffer and the incubation temperature in the purification of HPV type 16 L1 protein.
Fig. 2 is a diagram related to Example 1, showing the removal efficiency of insoluble proteins according to the type of size-up buffer and incubation time in the purification of HPV type 16 L1 protein.
FIG. 3 is a diagram related to Example 1 showing the purification pattern of each fraction when incubated at 35 DEG C in the step of removing insoluble proteins in the purification of HPV type 16 L1 protein. FIG.
FIG. 4 is a diagram related to Example 2 showing the yield change of purification of HPV type 16 L1 protein according to ammonium sulfate precipitation concentration. FIG.
FIG. 5 is a diagram related to Example 3, showing the result of performing purification by cation exchange chromatography after performing NaCl concentration at 0.15-0.6 M in the size-up step.
FIG. 6 is a diagram related to Example 3, showing the purification yield purified by cation exchange chromatography after performing the NaCl concentration at 0.15-0.6 M in the size-up step.
FIG. 7 is a diagram related to Example 3, showing the results of performing purification by cation exchange chromatography after performing a KCl concentration of 0.15-0.6 M in the size-up step.
FIG. 8 is a diagram related to Example 3, showing the yield of purification purified by cation exchange chromatography after performing a KCl concentration of 0.15-0.6 M in the size-up step. FIG.
FIG. 9 is a diagram related to Example 3, showing the result of performing purification by cation exchange chromatography after performing Na 2 CO 3 concentration at 0.15-0.6 M in the size-up step.
FIG. 10 is a diagram related to Example 3, showing the purification yields purified by cation exchange chromatography after performing Na 2 CO 3 concentration at 0.15-0.6 M in the size-up step.
FIG. 11 is a diagram related to Example 3, showing the result of performing purification by affinity chromatography after performing NaCl concentration at 0.15-0.6 M in the size-up step.
FIG. 12 is a diagram related to Example 3 showing the purification yield purified by affinity chromatography after performing the NaCl concentration at 0.15-0.6 M in the size-up step. FIG.
FIG. 13 is a diagram related to Example 3, showing the result of performing purification by affinity chromatography after performing a KCl concentration of 0.15-0.6 M in the size-up step.
FIG. 14 is a diagram related to Example 3, showing the purification yield purified by affinity chromatography after performing a KCl concentration of 0.15-0.6 M in the size-up step.
FIG. 15 is a diagram related to Example 3, showing the result of performing purification by affinity chromatography after performing Na 2 CO 3 concentration at 0.15-0.6 M in the size-up step.
FIG. 16 is a diagram related to Example 3, showing the purification yield purified by affinity chromatography after performing Na 2 CO 3 concentration at 0.15-0.6 M in the size-up step.
17 is a diagram related to Example 3 showing the results of cation exchange chromatography purification on HPV type 16 and type 18 L1 proteins. 1 lane: 0.5 M NaCl, 2 lanes: 0.4 M KCl, 3 lanes: 0.4 M Na 2 CO 3 .
Fig. 18 is a diagram related to Example 3, showing affinity chromatography purification results for HPV type 16 protein. Fig. 1 lane: 0.5 M NaCl, 2 lanes: 0.4 M KCl, 3 lanes: 0.4 M Na 2 CO 3 .
Fig. 19 is a diagram related to Example 4, showing the results of evaluating the stability of HPV antigen according to the kind and concentration of a salt in the size-up step and the reaction temperature. 1 lane: 0.5 M NaCl, 2 lanes: 0.4 M KCl, 3 lanes: 0.4 M Na 2 CO 3 , 4 lanes: 0.15 M NaCl.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1. Temperature and time setting for efficient removal of insoluble protein

1-1. Experimental Method

In order to set the effective temperature and time range of insoluble protein removal, which is a step for removing impurities, in the production process of L1 protein of HPV type 16 and type 18, the following experiment was conducted.

Yeast cells ( Saccharomyces cerevisiae ; KCCM11036P and KCCM11037P, respectively) transformed with the YEGα-HPV16L1-ROS and YEGα-HPV18L1-ROS vectors into which the recombinant HPV type 16 L1 and HPV type 18 L1 were introduced, The flask was inoculated onto synthetic complete medium SD-ura without uracil and cultured with shaking at 30 캜. YPDG medium was used to express HPV type 16 L1 and type 18 L1 protein from the GAL10 promoter. 1% yeast extract (Duchefa Biochem, Netherlands) and 2% peptone (Duchefa Biochem, Netherlands) were added to all media and glucose and galactose were added. The transformed yeast strain was inoculated into YPDG medium and cultured at 30 ° C using a fermentor. The incubation time was set to be not longer than 96 hours.

The yeast cells were mixed with buffer (20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80), and the cells were disrupted with a cell disruptor. The disruptant was centrifuged to recover the supernatant . To the recovered supernatant was added 45% by weight of ammonium sulfate and precipitated. The supernatant was discarded by centrifugation at high speed (24,000 g), and only the precipitate was recovered. The recovered precipitate was measured for protein concentration by BCA assay, diluted with 1X PBS-T to a concentration of 30 mg / mL of ammonium salt precipitate, and NaCl was added to the diluted solution to a final concentration of 0.8M. After stirring for about 30 minutes, the mixture was allowed to stand at 4 DEG C and incubated for 18 hours.

(0.15 M NaCl, 0.5 M NaCl, 0.4 M KCl, 0.4 KCl) added with three kinds of salts (NaCl, KCl, Na 2 CO 3 ) were added to the dialysis membrane after dividing the suspension into four groups M Na 2 CO 3 ) size-up buffer (PBS + 0.01% Tween 80). After completion of the dialysis, each of the size-up buffer solutions was added to dilute the protein to a concentration of 10 mg / mL or less, incubated in an incubator at a temperature of 20-35 DEG C (+/- 2 DEG C), and incubated for 24 hours. After 24 hours, the resultant was centrifuged at high speed (24,000 g) to collect the supernatant, and the turbidity of the recovered supernatant was measured using a spectrophotometer (OD = 600 nm).

The precipitate samples were dialyzed in the size-up buffer to which the four kinds of salts were added and allowed to stand for 16-26 hours (2 hours apart) or for 48 hours in an incubator at a temperature of 30 占 폚 (占 2 占 폚) The time zone indicating the turbidity was confirmed.

1-2. Experiment result

The process of removing insoluble proteins is a method of precipitating impurities at room temperature to increase the purity at the time of purification, which means that the lower the OD value of the supernatant after the incubation, the more impurities are removed.

As shown in FIG. 1, although the value of turbidity varied depending on the salt type of the size-up buffer, overall, the higher the incubation temperature, the lower the OD 600 nm value. As a result, the higher the temperature, , But it was judged that there would be a problem in protein structure at too high temperature. As a result of further analysis, the purification yields of the purified HPV type 16 L1 protein by incubating at 30 ° C and 35 ° C for 24 hours to remove insoluble proteins were compared, respectively. As a result, the yield of purification at 30 ° C was about 2.65 mg / L, but the purification yield at about 35 DEG C was about 1.24 mg / L and the purity was also low. From these results, it was confirmed that the efficiency of removing the insoluble protein at a temperature higher than the proper range was low, and the effective temperature range for purification was about 25-34 ° C.

As shown in Fig. 2, the incubation time of the insoluble protein was measured at intervals of 2 hours and 48 hours from 16 to 26 hours under the respective salt conditions. As a result, the insoluble protein precipitated over time, It was confirmed that turbidity was increased and maintained at a constant level. The turbidity of the suspension means that impurities are induced and the purity is increased. However, since incubation for too long may cause the target protein to precipitate, the range of incubation time suitable for the removal of the insoluble protein may be 21-26 hours.

Example 2. Comparison of purification yield according to the concentration of ammonium sulfate PPT

2-1. Experimental Method

The yeast cells of Example 1 were mixed with a buffer solution (20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80), and the cells were disrupted using a cell disruptor. , And the supernatant was recovered. 45% by weight of ammonium sulfate was added to the recovered supernatant to precipitate, followed by centrifugation at high speed (24,000 g), discarding the supernatant, and collecting only the precipitate. The precipitate collected was diluted with 10 mg / mL-60 mg / mL (10 mg / mL) concentration of ammonium salt precipitate by measuring the protein concentration by BCA assay and adding 1X PBS-T. NaCl was added to the precipitate of each concentration at a final concentration of 0.8 M, stirred for about 30 minutes, allowed to stand at 4 ° C, and incubated for 18 hours. After incubation with NaCl, the suspension of each concentration was put into a dialysis membrane and dialyzed with a size-up buffer. After completion of the dialysis, the size-up buffer of Example 1 was added to dilute the protein to a concentration of 10 mg / mL or less, incubated in an incubator at 30 ° C (± 2 ° C), and incubated for 24 hours. After 24 hours, the resultant was centrifuged at high speed (24,000 g), and the supernatant was recovered. The recovered supernatant was dialyzed at 4 ° C with binding buffer (PBS + 0.35 M NaCl pH 7.2, 0.01% Tween 80) Respectively. After dialysis, the supernatant was filtered by centrifugation at high speed (24,000 g). The filtrate was purified by cation exchange chromatography. Specifically, a 1.6 cm X 5 cm Poly-Prep column (GE healthcare lifescience, USA) filled with P-11 cellulose phosphate resin (Whatman, UK) was padded with binding buffer (PBS + 0.5 M NaCl) Maintaining equilibrium state. The dialyzed solution was passed through a P-11 column and bound, and the binding buffer was washed by flowing 5 times the resin volume. After washing, the elution buffer containing 0.6 M, 0.7 M, 0.8 M and 1 M NaCl was added to the binding buffer in an amount of 4-5 ml each, to elute the L1 protein). The fraction in which the target band was identified was classified to calculate the purification yield after quantification of BCA.

2-2. Experiment result

As shown in FIG. 4, when the ammonium salt precipitate concentration was 30 mg / mL, the purification yield was the highest at about 2.6 mg / L, followed by the ammonium salt When the concentration of the precipitate was 40 mg / mL, the yield was about 2.2 mg / L. These results suggest that the concentration of ammonium salt precipitate with high purification yield of HPV antigen is in the range of 25 mg / mL-65 mg / mL.

Example 3: Setting the type and concentration of the optimum salt in the size-up step

3-1. Experimental Method

In the HPV L1 protein production step, the size-up step is a step of removing impurities through incubation using a salt. In order to select the kind of salt suitable for HPV VLP antigen purification and to confirm the degree of removal of impurities by the concentration of the salt, The experiment was carried out. Table 1 shows the kinds of common salts.

Cation / anion NO 3- Cl - S 2- SO 4 2- CO 3 2- Na + NaNO 3 NaCl Na 2 S Na 2 SO 4 Na 2 CO 3 K + KNO 3 KCl K 2 S K 2 SO 4 K 2 CO 3 NH 4+ NH 4 NO 3 NH 4 Cl (NH 4) 2 S (4 NH) 2 SO 4 (NH 4) 2 CO 3 Mg 2+ Mg (NO 3) 2 MgCl 2 MgS MgSO 4 MgCO 3 Ba 2+ Ba (NO 3) 2 BaCl 2 BaS BaSO 4 BaCO 3 Ca 2+ Ca (NO 3) 2 CaCl 2 CaS CaSO 4 CaCO 3 Pb 3+ Pb (NO 3 ) 2 PbCl 2 PbS PbSO 4 PbCO 3 Ag + AgNO 3 AgCl Ag 2 S Ag 2 SO 4 Ag 2 CO 3

The yeast cells of Example 1 were mixed with a buffer solution (20 mM sodium phosphate, pH 7.2, 100 mM NaCl, 1.7 mM EDTA, 0.01% Tween 80), and the cells were disrupted using a cell disruptor. Respectively. 45% by weight ammonium sulfate was added to the recovered supernatant, centrifuged at high speed (24,000 g), the supernatant was discarded and only the precipitate was recovered (the precipitate was made into a suspension by adding a small amount of 1X PBS-T buffer). The precipitate was assayed for protein concentration by BCA assay and 1 × PBS-T was added to dilute the ammonium salt precipitate to a concentration of 30 mg / mL or less. NaCl was slowly added to the diluted precipitate with a final concentration of 0.8M. After stirring for about 30 minutes, the mixture was allowed to stand at 4 DEG C and incubated for 18 hours. After incubation with NaCl, the suspension was placed in the dialysis membrane and the components in Table 2 were dialyzed in the size-up buffer composition. After completion of the dialysis, each of the size-up buffer solutions was added to dilute the protein to a concentration of 10 mg / mL or less, followed by incubation at 30 ° C (± 2 ° C) in an incubator for 24 hours.

NaCl KCl Na 2 CO 3 MgCl 2 0.15 M NaCl 0.15M KCl 0.15M Na 2 CO 3 0.15 M MgCl 2 0.3 M NaCl 0.3 M KCl 0.3 M Na 2 CO 3 0.3 M MgCl 2 0.4 M NaCl 0.4 M KCl 0.4 M Na 2 CO 3 0.4M MgCl 2 0.5 M NaCl 0.5 M KCl 0.5 M Na 2 CO 3 0.5 M MgCl 2 0.6 M NaCl 0.6M KCl 0.6 M Na 2 CO 3 0.6M MgCl 2

After 24 hours, the resultant was centrifuged at high speed (24000 g), and the supernatant was recovered. The recovered supernatant was dialyzed at 4 ° C with binding buffer (PBS + 0.35 M NaCl pH 7.2, 0.01% Tween 80) . Dialyzed with binding buffer, centrifuged at high speed (24000 g) and the supernatant was filtered. The filtrate was purified by cation exchange chromatography as in Example 2, and the fraction of high purity was collected and the yield was calculated and analyzed. In order to confirm that the same result is also obtained in other types of resins, purification is carried out using affinity chromatography based on a salt condition showing the highest yield among the respective salt types in cation exchange chromatography Experiments were performed additionally.

Specifically, the affinity chromatography was maintained at equilibrium by pouring the binding buffer (PBST containing 0.33 M NaCl) into the column packed with heparin sepharose resin (GE healthcare life science, USA) at 10 times the volume of the resin . The dialyzed solution was passed through a heparin column, and eluted buffer containing 2 M NaCl was washed with a linear concentration gradient of 0.33 M NaCl to 0.5 M NaCl at a flow rate of 50 times the resin volume. After washing, the elution buffer containing 2 M NaCl was eluted with a linear concentration gradient from 0.5 M NaCl to 1.5 M NaCl at 20 times the resin volume. The fraction in which the target band was identified was classified to calculate the purification yield after quantification of BCA. For affinity chromatography, 1 mL Heparin column (GE, USA) was used.

3-2. Experiment result

The purification yield was calculated by different kinds and concentrations of salt using cation exchange chromatography. As a result, the purification was not carried out under all salt concentration conditions in MgCl 2 , and in the other NaCl, KCl and Na 2 CO 3 , The results were as follows. Further affinity chromatography performed additionally showed a high yield at a high salt concentration of 0.3 M or more as in cation exchange chromatography (Figs. 11 to 16).

Based on the above results, samples were quantitatively analyzed by BCA assay for the highest yield of each salt type, and 0.5 μg of the sample was applied to two 12% acrylamide gels at 150 V, 150 mA for 1.5 hours And electrophoresed. To identify the developed samples, one sheet was subjected to silver staining and the other sheet was subjected to Western blotting using a commercially available monoclonal antibody that specifically reacted with HPV 16 and 18 antigens.

As a result, as shown in Fig. 17, antibody reactivity at both the high purity and the appropriate size was confirmed in each of the buffer conditions in which the cation exchange chromatography was performed (1 lane: 0.5 M NaCl, 2 lanes: 0.4 M KCl, lane 3: 0.4 M Na 2 CO 3) . As a result of further affinity chromatography, the antibody reactivity at high purity and suitable size was confirmed as shown in Fig. 18 (1 lane: 0.5 M NaCl, 2 lanes: 0.6 M KCl, 3 lanes: 0.4 M Na 2 CO 3 ) .

These results indicate that the salts that have good effects in producing HPV type 16 and 18 L1 VLP antigens are NaCl, KCl, and Na 2 CO 3 , and the concentration range of these salts for optimal insoluble protein removal is 0.35-0.60 M, respectively.

4. Stability evaluation according to salt type and concentration at the size-up stage

4-1. Experimental Method

The stability of the antigen was evaluated by varying the reaction temperature and storage time conditions for the antigen produced according to the type and concentration of the salt in the size - up step. For this, the size-up process was performed according to the kind and concentration of the salt, and the produced HPV type 16 L1 VLP antigen was prepared (0.15 M NaCl, 0.5 M NaCl, 0.4 M KCl, 0.4 M Na 2 CO 3 ). Antigen stability was evaluated by setting the reaction temperature of the antigen to 40 占 폚 and 60 占 폚, and the reaction time to 12, 24 and 36 hours. The antigen was dispensed into new tubes in the amount of 150 占 퐇 in accordance with the conditions shown in Table 3 below. Antigen samples were collected over time and the status of the antigen was confirmed by Western blot. Western blot was quantitated by BCA assay and 0.6 ㎍ was electrophoresed on a 12% acrylamide gel at 150 V, 150 mA for 1.5 hours and transferred to nitrocellulose membrane. The transferred sample was reacted with a commercially available monoclonal antibody that specifically reacted with HPV 16 and 18 antigens, and then color development was confirmed.

Figure 112015098151701-pat00001

4-2. Experiment result

As shown in FIG. 19, the antigen sample was confirmed by Western blotting according to the temperature, and it was confirmed that the antigenic stability was maintained at 40 ° C and 60 ° C. In view of the severe temperature conditions of 40 ° C and 60 ° C, the above results support that the HPV antigen produced by the production method of the present invention can maintain its stability as an antigen even at normal refrigeration and freezing temperatures.

As shown in FIG. 19, the stability as an antigen was higher than 0.4 M KCl and 0.4 M Na 2 CO 3 and 0.5 M NaCl, respectively, depending on the type and concentration of the salt, as compared with the salt concentration of 0.15 M NaCl. These results indicate that the HPV antigen prepared according to the production method of the present invention is superior not only in terms of purification yield but also in stability as compared with the HPV antigen prepared with 0.15 M NaCl salt, It is more suitable for use as a raw material of the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A method for producing L1 virus-like particles (VLP) of HPV (HPV) comprising the steps of:
(a) culturing a transforming yeast expressing the L1 protein of HPV;
(b) performing ammonium sulfate precipitation on the product of the transformed yeast cultured in step (a) to obtain a precipitate;
(c) then subjected to ammonium sulfate precipitation to a treatment of 0.35-0.60 M NaCl, KCl or Na 2 CO 3 to the obtained precipitate, and the incubation, removing the formed water-insoluble protein; And
(d) Chromatography of the product of step (c) to obtain L1 protein.
The method of claim 1, wherein the concentration of ammonium sulfate treated in step (b) is 40-50 wt%.
The method of claim 1, wherein the method further comprises the step of incubating the precipitate obtained by performing the ammonium sulfate precipitation between steps (b) and (c) in a solution containing the salt.
The method of claim 1, wherein the precipitate obtained by performing the ammonium sulfate precipitation of step (c) has a concentration of 25-65 mg / mL.
delete The method of claim 1, wherein the concentration of the salt to be treated in step (c) is 0.35-0.55 M.
2. The method of claim 1 wherein step (c) is incubated at a temperature of 25-34 < 0 > C.
2. The method of claim 1, wherein step (c) comprises incubating for 21-27 hours.
The method according to claim 1, wherein the HPV is selected from the group consisting of HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 35, HPV type 39, Type 51, HPV type 52, HPV type 56, HPV type 58 and HPV type 68.
KR1020150142107A 2014-12-26 2015-10-12 Methods for Preparing Human Papillomavirus Virus-Like Particles KR101825996B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20140190902 2014-12-26
KR1020140190902 2014-12-26

Publications (2)

Publication Number Publication Date
KR20160080060A KR20160080060A (en) 2016-07-07
KR101825996B1 true KR101825996B1 (en) 2018-02-09

Family

ID=56150907

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020150142107A KR101825996B1 (en) 2014-12-26 2015-10-12 Methods for Preparing Human Papillomavirus Virus-Like Particles

Country Status (4)

Country Link
KR (1) KR101825996B1 (en)
EA (1) EA201790944A1 (en)
RU (1) RU2677336C2 (en)
WO (1) WO2016104923A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100959145B1 (en) 2008-03-21 2010-05-25 중앙대학교 산학협력단 Process for Producing and Purifying Human Papillomavirus Virus-Like Particles

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2189882C (en) * 1994-05-16 2005-09-20 Kathrin U. Jansen Papillomavirus vaccines
US5821087A (en) * 1995-03-30 1998-10-13 Merck & Co., Inc. Production of recombinant human papillomavirus type II protein utilizing papillomavirus 6/11 hybrid DNA
AU742409B2 (en) * 1997-09-05 2002-01-03 Medimmune, Llc In vitro method for disassembly/reassembly of papillomavirus virus-like particles (VLPs)
RU2356943C2 (en) * 2003-03-24 2009-05-27 Мерк Энд Ко., Инк. Nucleic acid molecule encoding hpv 31 l1, expression vector, host cell, virus-like particle and method of its obtainment, vaccine, pharmaceutical composition and methods involving them
US9428555B2 (en) * 2007-04-29 2016-08-30 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Truncated L1 protein of Human Papillomavirus type 16
CN104288763B (en) * 2009-06-19 2018-03-06 艾金株式会社 Vaccine for cervical cancer
KR101182663B1 (en) * 2010-06-29 2012-09-18 주식회사 유라코퍼레이션 Process box in one body nametag
KR101559622B1 (en) * 2012-07-30 2015-10-13 중앙대학교 산학협력단 An Efficient Method for Purifying Virus-Like Particles of Human Papillomavirus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100959145B1 (en) 2008-03-21 2010-05-25 중앙대학교 산학협력단 Process for Producing and Purifying Human Papillomavirus Virus-Like Particles

Also Published As

Publication number Publication date
KR20160080060A (en) 2016-07-07
RU2015149330A (en) 2018-11-30
WO2016104923A1 (en) 2016-06-30
RU2015149330A3 (en) 2018-11-30
RU2677336C2 (en) 2019-01-16
EA201790944A1 (en) 2017-11-30

Similar Documents

Publication Publication Date Title
US10792353B2 (en) Virus like particle purification
AU774437B2 (en) Method for obtaining a purified viral preparation
MX2013014773A (en) Methods of purification of native or mutant forms of diphtheria toxin.
CN107567496B (en) Method for sterile purification of relevant viruses
CZ2001570A3 (en) Purification process of particles similar human papillomavirus
JP2016531128A (en) Novel adsorption composition and use thereof
US10987416B2 (en) Purification of recombinant EV71 virus-like particle and method for preparing vaccine thereof
Effio et al. High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli
KR101825996B1 (en) Methods for Preparing Human Papillomavirus Virus-Like Particles
US20110184154A1 (en) Cell broth clarification and host cell protein removal
CN101942496B (en) Method for preparing virus analogs of nervous necrosis viruses
KR101942626B1 (en) Method for enhancing purification yield of nodavirus capsid protein
JP4491588B2 (en) Purification method of killer protein
KR20160063984A (en) Porous cellulose particles having amino group-containing ion-exchange group and butyl group-containing hydrophobic group, and chromatography media containing the same, and method for purifying virus-like particles of hepatitis b virus
CN104558166B (en) The method for reducing non-specific antibody in anti-GCRV polyclonal antibody
CN113528544B (en) Gene for coding soluble HPV23L1 protein and construction and application of recombinant plasmid thereof
AU2013242822B2 (en) Virus like particle purification
CN110981946A (en) Solution for large-scale production of foot-and-mouth disease virus-like particle antigen and purification and assembly method
CN116536306A (en) Kit for in vitro transcription synthesis of mRNA capable of reducing or inhibiting formation of dsRNA and using method thereof
AU2016269506A1 (en) Virus like particle purification

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right