JP3912827B2 - Protein and production method thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein and a method for producing the same. More specifically, the present invention relates to a novel protein having epithelial cell adhesion activity derived from a bacterium belonging to the genus Lactobacillus and a cell surface protein extracted from the bacterium belonging to the genus Lactobacillus. It relates to a manufacturing method. The protein of the present invention is very useful as a culture substrate for efficiently adhering epithelial cells to a culture vessel and allowing them to proliferate.
[0002]
[Prior art]
Currently, cultured cells are widely used in biological and medical research. When cell culture is performed, particularly when a new cell line is established or when cryopreserved cells are newly cultured, adhesion of the cells to the culture container is important. In particular, epithelial cells are anchorage-dependent cells that can only proliferate after adhering to the surface of the culture vessel. It is known that it is low, cell growth is delayed, and culture is not performed efficiently.
Therefore, conventionally, in order to improve the adhesion of cells, particularly epithelial cells, to the culture container, it is common practice to coat the surface of the culture container with a cell adhesion protein such as collagen or gelatin derived from animal tissue. Containers with coatings are commercially available. Collagen is a cell-adhesive protein that is contained in animal connective tissue and has a role of adhering epithelial cells and connective tissue, and is divided into several types such as type I to type V.
[0003]
[Problems to be solved by the invention]
However, at present, there is no large-scale preparation method using genetic engineering techniques for collagen, and it must be separated and purified from animal tissues that are relatively difficult to obtain. large. In addition, commercially available collagen products are very expensive and are not generally used for cell culture on a daily basis.
As other cell adhesion proteins, vitronectin, laminin, fibronectin and the like are known, but all are derived from animal tissues, are more expensive than collagen, and are not generally used for cell culture. It was.
[0004]
Therefore, in order to solve the above-mentioned problems of the prior art, the present inventors have conducted intensive research for the purpose of providing a novel protein that has cell adhesion and can be produced easily and inexpensively. Piled up. As a result, the inventors have found that a novel protein having cell adhesion activity exists in the cell surface protein of bacteria belonging to the genus Lactobacillus, and furthermore, found a method for producing it easily and inexpensively, and completed the present invention. .
[0005]
[Means for Solving the Problems]
That is, the present invention cultivates Lactobacillus acidophilus SBT2062 strain (FERM P-10730 ) having the ability to produce a protein having an adhesion activity to epithelial cells, collects the cultured cells, and wash the collected cells. The washed cells are prepared by adding an alkali metal salt solution to the washed cells, or by adding a surfactant-containing solution to the membrane fraction precipitated by crushing the washed cells. extracting the protein was dialyzed crude extract of the resulting surface layer protein, to recover the surface layer protein as a precipitate, the recovered precipitate is dried, is prepared by obtaining a surface layer protein, molecular weight from about 33 kDa A protein having the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing , derived from the bacterium and having an adhesion activity to epithelial cells.
The present invention is also an antibody against the protein.
The present invention also cultivates Lactobacillus acidophilus SBT2062 strain (FERM P-10730 ) having the ability to produce a protein having an adhesion activity to epithelial cells, collects the cultured cells, and wash the collected cells. The surface protein is prepared by preparing a washed microbial cell and adding an alkali metal salt solution to the washed microbial cell, or by adding a surfactant-containing solution to a membrane fraction obtained by crushing and precipitating the washed microbial cell. The method for producing a protein according to the present invention is characterized in that a crude protein extract of the obtained surface protein is dialyzed, the surface protein is recovered as a precipitate, and the recovered precipitate is dried to obtain a surface protein. .
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The bacteria belonging to the genus Lactobacillus that can be used in the present invention are not particularly limited, but are more preferably selected from bacterial species and strains that colonize the intestines of animals from which cultured cells are derived.
The bacteria belonging to the genus Lactobacillus that can be used for culturing human cells are not particularly limited, but preferred examples include Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus crispatus) or Lactobacillus johnsonii.
Among these, Lactobacillus acidophilus, especially Lactobacillus acidophilus SBT2062 strain is preferable. The Lactobacillus acidophilus SBT2062 strain was established as FERM P-10730 at the Institute of Microbial Technology of the Ministry of International Trade and Industry, Institute of Industrial Science (currently the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry). Deposited on the day.
Moreover, Lactobacillus gasseri, especially Lactobacillus gasseri SBT2055 strain is preferable. This Lactobacillus gasseri SBT2055 strain was deposited on March 27, 1996 as FERM P-15535 at the Institute of Biotechnology, Ministry of International Trade and Industry.
Moreover, Lactobacillus crispatas, especially Lactobacillus crispatas JCM5808 strain is preferable.
Further, Lactobacillus johnsonii, particularly Lactobacillus johnsonii SBT1839 strain is preferred. This Lactobacillus johnsonii SBT1839 strain was deposited on March 27, 1996 as FERM-P15534 at the Institute of Biotechnology, Ministry of International Trade and Industry.
[0007]
Further, the cells to which the protein of the present invention is attached are not particularly limited, but are highly useful for epithelial cells in which adhesion is particularly important in culture, and can be used for most epithelial cells. is there. In addition, since the bacteria belonging to the genus Lactobacillus live in the intestines of animals, the protein of the present invention is effective for culturing intestinal epithelial cells.
In addition, the protein having cell adhesion activity of the present invention includes not only the active fraction but also the active fraction, such as a product obtained by separating only the surface protein of the cell from the cell belonging to the genus Lactobacillus. Etc. are also included. That is, the protein having cell adhesion activity of the present invention includes both a cell surface protein extracted from a bacterial cell belonging to the genus Lactobacillus and a protein obtained by purifying it.
[0008]
The protein of the present invention can be produced, for example, by the following method.
First, bacteria belonging to the genus Lactobacillus are cultured overnight in a medium, and the cells are collected by centrifugation. Preferred strains or strains of bacteria belonging to the genus Lactobacillus are as described above. Examples of the medium include MRS medium, BL medium, BLA medium, GPV medium, and the like. The culture is preferably performed at 37 ° C. for about 16 hours. Centrifugation is preferably performed at 5000 rpm for about 15 minutes.
Next, after the collected cells are suspended in ion-exchanged water, the cells are repeatedly collected by centrifugation to prepare washed cells. Centrifugation in this case is preferably performed at 5000 rpm for about 15 minutes, and is preferably repeated 3 to 4 times.
[0009]
Next, the surface layer protein is extracted from the washed cells thus obtained. For the extraction of the surface layer protein, a known method such as a method using an alkali metal salt or a method using a surfactant after crushing bacterial cells and then obtaining a membrane fraction can be used.
Among these, in the case of the method using an alkali metal salt, a high concentration alkali metal salt solution of 1M or more is prepared in advance, and the extraction is performed by vigorously stirring while adding ice to the washed cells. Is preferred. The alkali metal salt is not particularly limited, and examples thereof include lithium chloride, sodium chloride, and potassium chloride.
Next, this is centrifuged at about 7000 rpm for about 20 minutes to obtain a crude extract of the surface protein of bacteria belonging to the genus Lactobacillus in the supernatant.
Next, the obtained crude extract is dialyzed against ion-exchanged water to precipitate the surface layer protein as a precipitate and remove low-molecular impurities. For dialysis, a dialysis membrane having a molecular weight fraction of 6000 to 8000 is preferably used.
Next, the deposited surface protein is recovered by centrifugation, washed by centrifugation several times with ion-exchanged water, and then freeze-dried to obtain a dried preparation of the protein of the present invention.
[0010]
In the case of a method using a surfactant after crushing bacterial cells and using a surfactant, for example, first, the N-acetylmuramidase solution is added to the washed bacterial cells prepared as described above. (50 μg / ml) is added and the mixture is allowed to stand at 37 ° C. for 30 minutes to lyse and protoplast the cell wall. Next, after collecting this by centrifugation at 3000 rpm for 20 minutes, the cells are ruptured by adding distilled water. The disruption of the cells can also be performed by physical treatment such as ultrasonic treatment or French press. The disrupted bacterial solution is centrifuged at 5000 rpm for 10 minutes to remove undisrupted bacterial cells that precipitate, and then the membrane fraction is precipitated by centrifugation at 15000 rpm for 30 minutes. Surface protein is extracted from the precipitated membrane fraction using a surfactant. The surfactant used at this time is not particularly limited, but 0.5-2% of CHAP or octyl glucoside is desirable in view of subsequent removal by dialysis.
Next, the obtained crude extract is dialyzed against ion-exchanged water to precipitate the surface layer protein as a precipitate and remove low-molecular impurities. For dialysis, a dialysis membrane having a molecular weight fraction of 6000 to 8000 is preferably used.
Next, the deposited surface protein is recovered by centrifugation, washed by centrifugation several times with ion-exchanged water, and then freeze-dried to obtain a dried preparation of the protein of the present invention.
[0011]
Further, the cell surface protein of the present invention thus extracted can be further purified by a conventional purification method such as SDS-PAGE to obtain an active fraction of the protein of the present invention.
[0012]
The protein of the present invention produced in this way is dissolved in distilled water, and a commercially available polystyrene dish, a plate or other culture container is coated by a conventional method, thereby easily obtaining a substrate for cell culture. Can do. The concentration when the protein of the present invention is coated on the culture vessel is preferably 0.1 to 10 mg of the protein of the present invention per 1 ml of the coating solution (usually 5M lithium chloride solution).
[0013]
The protein of the present invention can be identified by, for example, the following method.
First, epithelial cells are cultured in a cell culture medium until they reach confluence, and the cells are peeled off by trypsin-EDTA treatment, then centrifuged and washed with phosphate buffered saline (PBS), and again. Suspend in PBS. On the other hand, the protein of the present invention is subjected to SDS-polyacrylamide gel electrophoresis to separate the proteins by molecular weight, and then transferred to a nitrocellulose membrane using a blotting apparatus to transfer 1% bovine serum albumin (BSA). Block overnight at 4 ° C in PBS. When the epithelial cells suspended in PBS are layered on the membrane after blocking and gently shaken at 37 ° C. for 1 hour, the epithelial cells adhere to a protein having cell adhesion activity. Subsequently, after the membrane was washed with PBS, the cells adhered with 3% paraformaldehyde solution were fixed, immersed in 40% amide black solution, and then decolorized with methanol, only the protein to which the cells adhered was stained, Thereby, it can be identified.
[0014]
In addition, proof that the protein of the present invention thus identified acts as a cell adhesion factor can be performed using an antibody.
The antibody can be prepared using the purified cell adhesion protein by performing SDS-polyacrylamide gel electrophoresis of the protein of the present invention, staining with Coomassie brilliant blue solution, and then cutting out the gel. .
The antibody can be prepared using a known method. A suspension of intestinal cells and the antibody obtained as described above are added to a cell culture container coated with the protein of the present invention. In addition, a cell culture vessel to which no antibody is added is prepared as a control. If the cell adhesiveness of the cell culture container to which the antibody has been added decreases, it can be considered that the antibody first binds to the cell adhesive protein in the surface protein of the present invention and the cell adhesiveness is lost. It is proved that the protein plays a role of cell adhesion in the culture vessel.
[0015]
The protein of the present invention can be purified by known column chromatography such as ion exchange and gel filtration, but can be more easily purified by using an antibody against the protein of the present invention.
In this case, the antibody may be a polyclonal antibody or a monoclonal antibody. First, an antibody column was prepared by immobilizing the obtained antibody on a protein immobilization support such as cyanogen bromide-activated Sepharose 4B, and the above surface protein dissolved in PBS was added to this column. To load. After washing the column, the target purified protein can be obtained by eluting the protein bound to the antibody with an appropriate eluent. The purified protein thus obtained can be used as a cell culture substrate in the same manner as in the case of the surface protein, and a high cell adhesion effect can be obtained with a smaller amount of protein than the surface protein. Is possible. However, surface proteins are sufficient for general cell culture, and are economically advantageous.
[0016]
When intestinal epithelial cells are inoculated to the protein of the present invention thus produced, the cells can be efficiently adhered and proliferated, and the culture period can be shortened, as compared with conventional culture substrates. . In particular, primary cultured cells have a significantly increased adhesion rate and are useful for establishing cell lines.
[0017]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, these are for the purpose of illustration only and this invention is not limited to these.
[0018]
Example 1
(Production of the protein of the present invention)
The pre-cultured Lactobacillus acidophilus SBT2062 strain was inoculated 3% in 5 L of MRS medium and cultured at 37 ° C. for 24 hours. The cultured cells were collected by centrifugation at 7000 rpm for 20 minutes, and the cells were centrifuged and washed with ion-exchanged water. To the obtained washed cells, 3 times the amount of 5M lithium chloride solution was added, and the surface protein was extracted by vigorously stirring for 30 minutes while cooling with ice. Subsequently, centrifugation was performed to separate the microbial cell lysate to obtain a surface layer protein extract, which was dialyzed against ion-exchanged water using a dialysis membrane (molecular weight fraction of 6,000 or less). The precipitated surface protein was collected by centrifugation and freeze-dried to obtain a freeze-dried preparation of the surface protein.
[0019]
Test example 1
(Confirmation of cell adhesion activity)
The freeze-dried sample (compared product) obtained from the freeze-dried sample (dialysis precipitation fraction) obtained in Example 1 and the protein solubilized in the supernatant (dialysis-soluble fraction), respectively, It was dissolved in a 5M lithium chloride solution to prepare solutions of various concentrations from 0.001 to 10 mg / ml, and 100 μL was layered on a 96-well microplate. Each surface protein was coated by allowing to stand at 4 ° C. overnight, and then washed twice with PBS.
On the other hand, cells HCT-8 derived from the small intestine were cultured in RPMI 1640 medium until they reached confluence, and the cells were detached from the culture vessel by trypsin-EDTA treatment, and then washed with PBS at 1000 rpm for 5 minutes. Was repeated three times and suspended in PBS. This cell suspension was overlaid on the above plate and allowed to stand at 37 ° C. for 1 hour, and then the plate was washed. The number of adherent cells was observed with a microscope and measured with hexosaminidase activity. The results are shown in FIG.
As is clear from FIG. 1, a stronger cell adhesion activity was observed in the dialysis precipitate fraction than in the dialysis soluble fraction.
[0020]
Test example 2
(Identification of active fraction and molecular weight measurement)
2.5 μg of a surface protein dry preparation of Lactobacillus acidophilus SBT2062 strain obtained in Example 1 was dissolved in 200 μL of a sample buffer, and 7 μL of the sample was subjected to SDS-polyacrylamide gel electrophoresis. Proteins separated according to molecular weight are transferred to a nitrocellulose membrane using a blotting apparatus (TEFCO), and are then added at 4 ° C. in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). Blocked overnight. On the other hand, cells HCT-8 derived from the small intestine were cultured in RPMI 1640 medium until they reached confluence, and the cells were detached from the culture vessel by trypsin-EDTA treatment, and then washed with PBS at 1000 rpm for 5 minutes. Repeatedly and suspended in PBS. This cell suspension was layered on the membrane after blocking, and the cells were allowed to adhere by gently shaking at 37 ° C. for 1 hour. Subsequently, the membrane was washed with PBS, the adhered cells were fixed with a 3% paraformaldehyde solution, immersed in a 40% amide black solution, and decolorized with methanol to stain the adhered cells. . Using the same procedure, Lactobacillus gasseri, Lactobacillus crispatus and Lactobacillus johnsonii were also tested as strains of other bacteria belonging to the genus Lactobacillus. The results are shown in FIG.
As shown in FIG. 2, it was revealed that cell adhesion proteins exist in all these strains belonging to the genus Lactobacillus. For example, the Lactobacillus acidophilus SBT2062 strain has a protein that strongly adheres to cells in the vicinity of a molecular weight of 33 kDa. This protein was named LA5.
[0021]
Test example 3
(Cell adhesion test)
According to the method of Test Example 2, a cell adhesion test of the protein of the present invention derived from Lactobacillus acidophilus was performed on duodenum-derived Hutu-80, colon-derived Caco-2, and stomach-derived KATOIII epithelial cells. . The results are shown in FIG.
As is clear from FIG. 3, the protein (LA5) of the present invention was stained as a cell-adhesive protein regardless of which cell was used. That is, it was confirmed that the protein (LA5) of the present invention has adhesiveness to many epithelial cells and is effective for culturing epithelial cells.
[0022]
Example 2
(Preparation of polyclonal antibody)
2.5 μm of a dry preparation of the protein of the present invention was dissolved in a 200 μm sample buffer, and 7 μL of the sample was subjected to SDS-polyacrylamide gel electrophoresis. After staining with Coomassie Brilliant Blue solution, the protein of the present invention (LA5) was cut out and purified by eluting it electrically. Balb / c mice were immunized with the purified protein of the present invention (LA5). Immunization was performed 4 times every other week, and the initial immunization was performed by mixing with Freund's complete adjuvant (FCA), and subsequent immunization with Freund's incomplete adjuvant (FIA). After the final immunization, MethA cells causing ascites type cancer were injected into the peritoneal cavity of the mice. One week later, ascites collected in the abdominal cavity was collected to obtain a polyclonal antibody against the protein of the present invention (LA5).
[0023]
Test example 4
(Cell adhesion test of the protein of the present invention using a polyclonal antibody)
Using the polyclonal antibody against the protein of the present invention obtained in Example 2, the cell adhesion test of the protein of the present invention was performed.
That is, the adhesion test of HCT-8 cells was performed according to the method of Test Example 1 using a microplate coated with the surface protein (LA5) of the present invention. At that time, the polyclonal antibody against the protein (LA5) obtained in Example 2 was added to the cell suspension so as to obtain a dilution ratio of 3 to 50 times, and was layered on the plate. The results are shown in FIG.
For comparison, a similar cell adhesion test was also performed when an antibody (ascites) from a normal mouse that was not immunized was added. The results are shown in FIG.
Furthermore, the same cell adhesion test was conducted when a polyclonal antibody against all Lactobacillus acidophilus cells was added. The results are shown in FIG.
As is apparent from FIGS. 4 to 6, when an antibody against the protein of the present invention (LA5) is added, it is compared with a case where a normal mouse antibody is added or a polyclonal antibody against all strains of Lactobacillus acidophilus is added. As a result, the adhesion of the cells was greatly reduced. From this, it was confirmed that the protein of the present invention is a protein having cell adhesion activity in a culture vessel.
[0024]
Test Example 5
(Determining the N-terminal amino acid sequence)
The N-terminal amino acid sequence of the protein having a molecular weight of 33 kD derived from Lactobacillus acidophilus SBT2062 strain obtained in Example 1 was analyzed.
That is, the surface layer protein extracted with 5M lithium chloride in Example 1 was prepared using SDS-PAGE (TEFCO-CELL TC-16; manufactured by TEFCO and 491 PREP CELL; BIO-RAD) using a 10% gel. The 33 kD protein, which is the active fraction, was eluted and collected. The collected purified protein was again subjected to SDS-PAGE for analysis (10% gel, C-808; manufactured by TEFCO), and then subjected to a PVDF membrane (in a CAPS buffer (pH 11) under conditions of 50 V and 60 minutes. 03056; manufactured by TEFCO). The protein (equivalent to 4 μg) on the obtained membrane was stained with Coomassie brilliant blue dissolved in 40% methanol, decolorized with 40% methanol, and the stained band was cut out and air-dried. The cut-out band was determined for the N-terminal amino acid sequence using a protein sequencer (476A; manufactured by PerkinElmer) according to the protocol. A result is shown to sequence number 1 of a sequence table.
[0025]
【The invention's effect】
According to the present invention, there is provided a novel protein having cell adhesion activity derived from a bacterium belonging to the genus Lactobacillus, and a method for producing the protein comprising extracting a cell surface protein from a bacterium belonging to the genus Lactobacillus Is done.
The novel protein of the present invention is very useful as a culture substrate for efficiently attaching and proliferating cells, particularly epithelial cells, to a culture vessel.
Therefore, the novel protein of the present invention can be used in a wide range of fields such as medicine production, inspection, biochemical reagents, biology, medicine, etc. It is highly expected that this will be used in research.
[Brief description of the drawings]
FIG. 1 shows the cell adhesion of a precipitate fraction (invention) and a soluble fraction (comparison) after dialysis of Lactobacillus acidophilus surface protein.
[Explanation of symbols]
○: Dialysis precipitate fraction ●: Dialysis soluble fraction FIG. 2 shows the molecular weight in SDS-PAGE of cell adhesion proteins obtained from each bacterial species belonging to the genus Lactobacillus.
[Explanation of symbols]
Lane 1: Molecular weight marker Lane 2: Cell surface protein Lane 3: Cell adhesion protein FIG. 3 shows cell adhesion of the protein of the present invention to various epithelial cells.
[Explanation of symbols]
Lane 1: Molecular weight marker Lane 2: Cell surface protein Lane 3: Cell adhesion protein FIG. 4 shows the cell adhesion inhibitory effect of Lactobacillus acidophilus by a polyclonal antibody against the protein of the present invention.
FIG. 5 shows a cell adhesion inhibitory effect of Lactobacillus acidophilus by a normal mouse antibody.
FIG. 6 shows the cell adhesion inhibitory effect of Lactobacillus acidophilus by a polyclonal antibody against whole Lactobacillus acidophilus cells.
[Sequence Listing]

Claims (3)

Lactobacillus acidophilus SBT2062 strain (FERM P-10730 ) having the ability to produce a protein having an adhesion activity to epithelial cells is cultured, the cultured cells are collected, the collected cells are washed and washed. Prepare and extract surface protein by adding an alkali metal salt solution to the washed cells or by adding a surfactant-containing solution to the membrane fraction that has been crushed and precipitated from the washed cells. The resulting crude extract of the surface protein is dialyzed, the surface protein is recovered as a precipitate, and the recovered precipitate is dried to obtain a surface protein .
The molecular weight is about 33 kDa ,
Having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing;
A protein derived from the aforementioned fungus and having an adhesion activity to epithelial cells.   An antibody against the protein of claim 1.   Lactobacillus acidophilus SBT2062 strain (FERM P-10730) having the ability to produce a protein having an adhesion activity to epithelial cells is cultured, the cultured cells are collected, the collected cells are washed and washed. Prepare and extract surface protein by adding an alkali metal salt solution to the washed cells or by adding a surfactant-containing solution to the membrane fraction that has been crushed and precipitated from the washed cells. The method for producing a protein according to claim 1, wherein the obtained crude protein of surface layer is dialyzed, the surface layer protein is recovered as a precipitate, and the recovered precipitate is dried to obtain a surface layer protein.

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