JP7037811B2 - Purification method of spheroid formation promoter - Google Patents
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Description
本発明は、スフェロイド形成促進作用のあるタンパク質の濃縮方法および精製方法に関し、特に、魚等から得られる当該タンパク質の臭気成分その他の夾雑物を除去して濃縮する方法および精製する方法に関する。 The present invention relates to a method for concentrating and purifying a protein having a spheroid formation promoting action, and more particularly to a method for removing and concentrating an odorous component and other impurities of the protein obtained from fish and the like.
本願発明者は、先に、ノロゲンゲを始めとし、特定の魚種の皮下組織等にスフェロイドの形成を促進するタンパク質が存在することを発見し、これを契機とし、スフェロイド形成促進剤に関する発明をなした（特許文献１）。
この促進剤は、簡便かつ低廉に得ることができ、かつ、動物細胞の培地中に添加するだけでスフェロイドが形成されることから、ｉＰＳ細胞を始めとした多能性幹細胞の三次元培養、その他の再生医療に重要な役割を果たすことが期待される。
The inventor of the present application first discovered that there is a protein that promotes the formation of spheroids in the subcutaneous tissue of a specific fish species, including Bothrocara hollandi, and took this opportunity to invent an invention relating to a spheroid formation promoter. (Patent Document 1).
Since this accelerator can be obtained easily and inexpensively and spheroids are formed only by adding it to the medium of animal cells, three-dimensional culture of pluripotent stem cells such as iPS cells, etc. It is expected to play an important role in regenerative medicine.
しかしながら、特許文献１で得られる促進剤は、特有の魚臭を有しており、臭気成分その他の夾雑物を除去し、より利用しやすく精製度を高めることが求められていた。
また、特許文献１で得られる促進剤は、凍結して提供した場合に融解の際に夾雑物の存在により沈殿が生じやすく、その防止のために緩慢凍結剤や緩衝剤を添加する必要があるものの、それ自体が培養系に影響を及ぼすこともあり、この点からも夾雑物の除去が望まれていた。
However, the accelerator obtained in Patent Document 1 has a peculiar fishy odor, and is required to remove odorous components and other impurities to make it easier to use and increase the degree of purification.
Further, when the accelerator obtained in Patent Document 1 is provided frozen, precipitation is likely to occur due to the presence of impurities during thawing, and it is necessary to add a slow freezing agent or a buffering agent to prevent this. However, it may affect the culture system by itself, and it has been desired to remove impurities from this point as well.
本発明は上記に鑑みてなされたものであって、スフェロイド形成促進剤の濃縮方法および精製方法を提供することを目的とする。 The present invention has been made in view of the above, and an object of the present invention is to provide a method for concentrating and purifying a spheroid formation promoter.
請求項１に記載の方法は、粘膜質の体表皮を有するゲンゲ、カレイ、エイ、若しくは、ニギスの表皮から筋肉組織までの部位より採取された体液由来の、または、頭足類のうちイカ若しくはタコの胴部にある体液由来または肝臓から採取された体液由来の、スフェロイド形成促進活性を有するタンパク質原料の、スフェロイド形成促進剤としての精製方法であって、ケイ酸アルミニウムまたは天然アロフェンの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、次に、タンパク質の吸着された粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法である。
The method according to claim 1 is derived from a body fluid collected from a site from the epidermis of genge, curry, ray, or nigis having a mucous body epidermis to muscle tissue, or head and foot. A method for purifying a protein raw material having a spheroid formation-promoting activity, which is derived from a body fluid in the body of a squid or an octopus or a body fluid collected from the liver, as a spheroid formation-promoting agent, and is made of aluminum silicate or natural. The allophen powder and the protein raw material are mixed to leave impurities on the liquid side and adsorb and separate the protein having spheroid formation promoting activity on the powder side, and then separate the powder with the adsorbed powder of the protein. , A method for purifying a spheroid formation promoter, which comprises mixing an acidic aqueous solution in which an acidic amino acid is dissolved and eluting a protein on the aqueous solution side.
請求項１にかかる発明によれば、原料から臭気成分その他の夾雑物を分離し、目的物であるスフェロイド形成促進活性を有するタンパク質を粉末側に担持ないし吸着させることができ、変性や失活することなく、スフェロイド形成促進剤としてタンパク質を分離精製することができる。また、濾過その他の工程で水分も適宜飛ばすことができ、効率的な濃縮を実現可能とする。
According to the first aspect of the present invention, an odorous component and other impurities can be separated from a raw material, and a protein having a spheroid formation promoting activity, which is a target substance, can be supported or adsorbed on the powder side, and is denatured or deactivated. The protein can be separated and purified as a spheroid formation promoter without any problem. In addition, water can be appropriately removed by filtration and other steps , enabling efficient concentration.
なお、体液由来のタンパク質原料は、ゲンゲ、カレイ、エイ、若しくは、ニギスの場合にあっては表皮から筋肉組織までの部位を、イカ若しくはタコの胴部または肝臓の場合にあっては当該部位を、それぞれ圧搾もしくは遠心分離して得られる原料、または、当該部位をそれぞれ凍結融解によるドリップとして得られる原料をいい、いずれも、適宜メンブランフィルターによる濾過工程やエーテル処理等による脱脂工程を経たものであってもよい。滅菌処理をおこなってもよい。得られるタンパク質原料には水分（液体）が含まれ、この意味で、タンパク質原料とはタンパク質原料液と表現してもよい。
また、原料とは、濃縮のターゲットとする、スフェロイド形成促進作用ないし活性を有するタンパク質、の他にニオイ成分その他の夾雑物が含まれていることを表現するものである。
濃縮とは、ここでは分離と表現することもできる。なお、粉末は水に溶解せず分散する
だけであるので、所定時間の撹拌後に濾過等により粉末部分を分離すれば、夾雑物が除去され、かつ、原料に比して水分も除去された（すなわち濃縮された）タンパク質が得られることとなる。
なお、請求項において混和とは、混ぜて攪拌することも当然に含まれるものとする。
酸性アミノ酸としては、グルタミン酸やアスパラギン酸を挙げることができる。溶解させる酸性水溶液としては、塩酸溶液を挙げることができる。なお、タンパク質を溶出させた後は、そのまま保存したり、ｐＨを調整しタンパク質を沈殿分離したりするなどして、適宜、スフェロイドを形成させる使用環境に合わせた性状（溶液状、ゲル状、粉末状）に調整すればよい。
The protein raw material derived from body fluid is the part from the epidermis to the muscle tissue in the case of Genge, flatfish, ray, or Nigisu, and the part in the case of the body or liver of squid or octopus. , Each of which refers to a raw material obtained by pressing or centrifuging, or a raw material obtained as a drip by freezing and thawing the relevant part, both of which have undergone a filtration step with a membrane filter or a degreasing step by ether treatment or the like as appropriate. You may. It may be sterilized. The obtained protein raw material contains water (liquid), and in this sense, the protein raw material may be expressed as a protein raw material liquid .
Further , the raw material expresses that it contains a protein having a spheroid formation promoting action or activity, which is a target for concentration, and an odor component and other impurities.
Concentration can also be expressed here as separation. Since the powder does not dissolve in water but only disperses, if the powder portion is separated by filtration or the like after stirring for a predetermined time, impurities are removed and water is also removed as compared with the raw material (). That is, a concentrated protein is obtained.
In the claims, the term "miscibility" naturally includes mixing and stirring.
Examples of the acidic amino acid include glutamic acid and aspartic acid. Examples of the acidic aqueous solution to be dissolved include a hydrochloric acid solution. After elution of the protein, it can be stored as it is, or the protein can be precipitated and separated by adjusting the pH to form spheroids as appropriate. Properties (solution, gel, powder) suitable for the usage environment. It may be adjusted to the shape).
請求項２に記載の方法は、粘膜質の体表皮を有するゲンゲ、カレイ、エイ、若しくは、ニギスの表皮から筋肉組織までの部位より採取された体液由来の、または、頭足類のうちイカ若しくはタコの胴部にある体液由来または肝臓から採取された体液由来の、スフェロイド形成促進活性を有するタンパク質原料の、スフェロイド形成促進剤としての精製方法であって、ケイ酸アルミニウムまたは天然アロフェンの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、次に、タンパク質の吸着された粉末に対し、緩衝液または界面活性剤を添加した緩衝液により一回以上洗浄し、その後、粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法である。
The method according to claim 2 is derived from a body fluid collected from a site from the epidermis of genge, curry, ray, or nigis having a mucous body epidermis to muscle tissue , or head and foot. A method for purifying a protein raw material having a spheroid formation-promoting activity, which is derived from a body fluid in the body of a squid or an octopus or a body fluid collected from the liver, as a spheroid formation-promoting agent, and is made of aluminum silicate or natural. The allophen powder and the protein raw material are mixed to leave impurities on the liquid side and adsorb and separate the protein having spheroid formation promoting activity on the powder side, and then to the powder on which the protein is adsorbed. On the other hand, it is characterized by washing once or more with a buffer solution or a buffer solution to which a surfactant is added , and then mixing the powder with an acidic aqueous solution in which an acidic amino acid is dissolved to elute the protein on the aqueous solution side. This is a method for purifying a spheroid formation-promoting agent.
請求項２にかかる発明によれば、いわゆる洗い込みにより、粉末側に付着している夾雑物を除去し、スフェロイド形成促進剤の純度を上げることができる。 According to the second aspect of the present invention, by so-called washing, impurities adhering to the powder side can be removed and the purity of the spheroid formation promoter can be increased.
緩衝液および界面活性剤は、タンパク質が液側に溶出しないのであれば特に限定されず、また、粉末側に付着ないし残存している夾雑物を粉末から解離できるのであれば種々のものを採用できる。たとえば、トリス（トリスヒドロキシメチルアミノメタン）緩衝液を挙げることができる。トリス緩衝液のｐＨの例としては８を挙げることができ、他の緩衝液も、ｐＨ＝７～９程度のもの、たとえば、ＨＥＰＥＳ（ヒドロキシエチルピペラジンエタンスルホン酸）、ＭＯＰＳ（３－モルホリノプロパンスルホン酸）などを挙げることができる。界面活性剤としてはＴｗｅｅｎ２０（ポリオキシエチレンソルビタンモノラウラート）を挙げることができるが、スフェロイド形成促進活性を有するタンパク質の活性を損なわないのであれば特に限定されない。すなわち、タンパク質と粉末との結合力は比較的強固であるので、臭気除去の効率性を目安として緩衝液および界面活性剤を適宜選択すればよい。 The buffer solution and the surfactant are not particularly limited as long as the protein does not elute to the liquid side, and various substances can be adopted as long as the contaminants adhering to or remaining on the powder side can be dissociated from the powder. .. For example, Tris (Tris hydroxymethylaminomethane) buffer can be mentioned. 8 can be mentioned as an example of the pH of the Tris buffer, and other buffers also have a pH of about 7 to 9, for example, HEPES (hydroxyethylpiperazine ethanesulfonic acid), MOPS (3-morpholinopropanesulfon). Acid) and the like. Examples of the surfactant include Tween 20 (polyoxyethylene sorbitan monolaurate), but the surfactant is not particularly limited as long as it does not impair the activity of the protein having spheroid formation promoting activity. That is, since the binding force between the protein and the powder is relatively strong, the buffer solution and the surfactant may be appropriately selected with the efficiency of odor removal as a guide.
請求項３に記載の方法は、粘膜質の体表皮を有するゲンゲ、カレイ、エイ、若しくは、ニギスの表皮から筋肉組織までの部位より採取された体液由来の、または、頭足類のうちイカ若しくはタコの胴部にある体液由来または肝臓から採取された体液由来の、スフェロイド形成促進活性を有するタンパク質原料の、スフェロイド形成促進剤としての精製方法であって、酸化アルミニウムまたはハイドロタルサイトの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、次に、タンパク質の吸着された粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法である。
The method according to claim 3 is derived from a body fluid collected from a site from the epidermis to muscle tissue of Genge, curry, ray, or nigis having a mucous body epidermis , or head and foot. A method for purifying a protein raw material having a spheroid formation-promoting activity, which is derived from a body fluid in the body of a squid or an octopus or collected from a liver, as a spheroid formation-promoting agent, such as aluminum oxide or hydrotalsai. The powder of the protein and the protein raw material are mixed to leave impurities on the liquid side and adsorb and separate the protein having spheroid formation promoting activity on the powder side. It is a method for purifying a spheroid formation promoter, which comprises mixing an acidic aqueous solution in which an acidic amino acid is dissolved and elution of a protein on the aqueous solution side.
請求項３にかかる発明によれば、原料から臭気成分その他の夾雑物を分離し、目的物であるスフェロイド形成促進活性を有するタンパク質を粉末側に担持させることができ、変性や失活することなく、スフェロイド形成促進剤としてタンパク質を分離精製することができる。また、濾過その他の工程で水分も適宜飛ばすことができ、効率的な濃縮を実現可能とする。
According to the third aspect of the present invention, the odorous component and other impurities can be separated from the raw material, and the target protein having spheroid formation promoting activity can be supported on the powder side without denaturation or deactivation. , Protein can be separated and purified as a spheroid formation promoter. In addition, water can be appropriately removed by filtration and other steps , enabling efficient concentration.
請求項４に記載の方法は、粘膜質の体表皮を有するゲンゲ、カレイ、エイ、若しくは、ニギスの表皮から筋肉組織までの部位より採取された体液由来の、または、頭足類のうちイカ若しくはタコの胴部にある体液由来または肝臓から採取された体液由来の、スフェロイド形成促進活性を有するタンパク質原料の、スフェロイド形成促進剤としての精製方法であって、酸化アルミニウムまたはハイドロタルサイトの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、次に、タンパク質の吸着された粉末に対し、緩衝液または界面活性剤を添加した緩衝液により一回以上洗浄し、その後、粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法である。
The method according to claim 4 is derived from a body fluid collected from a site from the epidermis of genge, curry, ray, or nigis having a mucous body epidermis to muscle tissue , or head and foot. A method for purifying a protein raw material having a spheroid formation-promoting activity, which is derived from a body fluid in the body of a squid or an octopus or a body fluid collected from the liver, as a spheroid formation-promoting agent, such as aluminum oxide or hydrotalsai. G. Powder and the protein raw material are mixed to leave impurities on the liquid side and adsorb and separate the protein having spheroid formation promoting activity on the powder side, and then to the powder on which the protein is adsorbed. , Washed once or more with a buffer or a buffer to which a surfactant is added , and then the powder and an acidic aqueous solution in which an acidic amino acid is dissolved are mixed to elute the protein on the aqueous solution side. This is a method for purifying a spheroid formation promoter.
請求項４にかかる発明によれば、いわゆる洗い込みにより、粉末側に付着している夾雑物を除去し、スフェロイド形成促進剤の純度を上げることができる。
According to the invention of claim 4 , by so-called washing, impurities adhering to the powder side can be removed and the purity of the spheroid formation promoter can be increased.
本発明によれば、臭気成分その他の夾雑物を分離し、精製度の高まったスフェロイド形成促進剤を提供することができる。また、臭気成分その他の夾雑物を分離し、高濃度のスフェロイド形成促進剤を提供することができる。 According to the present invention, it is possible to provide a spheroid formation promoter having an increased degree of purification by separating odorous components and other impurities. In addition, it is possible to separate odorous components and other impurities to provide a high-concentration spheroid formation promoter.
以下、本発明の実施の形態を図面を参照しながら詳細に説明する。以降では、原料（すなわち精製前のスフェロイド形成促進剤）のうち、スフェロイド形成促進活性を有するタンパク質を目的タンパク質と称し、それ以外を夾雑物と称することとする。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Hereinafter, among the raw materials (that is, the spheroid formation promoting agent before purification), the protein having the spheroid formation promoting activity will be referred to as a target protein, and the others will be referred to as impurities.
＜吸着による濃縮＞
原料は、山陰沖で採取したノロゲンゲの魚皮と筋肉の間の間隙物質（粘液部分）をピペットで採取したものを、０．２２μｍのメンブランフィルターで濾過滅菌して用いた。以降ではこれを原料液と称することとする。
<Concentration by adsorption>
As the raw material, the gap substance (mucus part) between the fish skin and the muscle of Bothrocara hollandi collected off the coast of San'in was collected with a pipette and sterilized by filtration with a 0.22 μm membrane filter. Hereinafter, this will be referred to as a raw material liquid.
次に、目的タンパク質または夾雑物の吸着を目的として、水に溶けない次の候補物質を検討することとした。 Next, we decided to investigate the following candidate substances that are insoluble in water for the purpose of adsorbing the target protein or contaminants.
候補物質：
ａ．ケイ酸アルミニウム（ナカライテスク（株）製：品名または型番＝０１７－３２）
ｂ．活性炭（和光純薬工業（株）製：品名または型番＝０３７－０２１１５）
ｃ．シリカゲル６０（ナカライテスク（株）製：品名または型番＝３０７－３１）
ｄ．酸化アルミニウム（メルク（株）製：品名または型番＝１０９０．０５）
ｅ．珪藻土（ナカライテスク（株）製：品名または型番＝０４５－００８７５）
ｆ．ガラスウール（島久薬品（株）製：品名または型番＝ＧＳＲ２９０）
ｇ．アンバーライト（オルガノ（株）製：品名または型番＝ＩＲＣ７４８）
ｈ．キチン（和光純薬工業（株）製：品名または型番＝０３４－１３６３５）
ｉ．ＤＥＡＥセルロース（和光純薬工業（株）製：品名または型番＝０４９－２３６１５）
ｊ．アルミニウム（和光純薬工業（株）製：品名または型番＝０１４－０１７８５）
ｋ．ケイ素（和光純薬工業（株）製：品名または型番＝１９１－０５５８２）
ｌ．水酸化アルミニウム（和光純薬工業（株）製：品名または型番＝０１４－０１９２５）
ｍ．水酸化マグネシウム（協和化学工業（株）製：品名または型番＝ＫＩＳＵＭＡ ５Ｑ－Ｓ）
ｎ．ハイドロタルサイト１（協和化学工業（株）製：品名または型番＝ＤＨＴ－４Ｈ：分子式＝Ｍｇ６Ａｌ２（ＯＨ）１６ＣＯ３・４Ｈ２Ｏ）
ｏ．ハイドロタルサイト２（協和化学工業（株）製：品名または型番＝ＤＨＴ－６：分子式＝Ｍｇ４．５Ａｌ２（ＯＨ）１３ＣＯ３・３．５Ｈ２Ｏ）
ｐ．天然アロフェン１（北溟工業（有）製：品名または型番＝ミソイル：大山ミソ土由来）
ｑ．天然アロフェン２（品名または型番＝鹿沼土由来アロフェン：鹿沼土由来）
ｒ．精製アロフェン（品川化成（株）製：品名または型番＝セカードＰ１）
ｓ．雲母焼成品（ナーク研究所（株）製：品名または型番＝Ｎ－１）
ｔ．ガラス発泡体（（株）鳥取再資源化研究所製：品名または型番＝ＰＷ－３５０）
ｕ．モンモリロナイト（クニミネ工業（株）製：品名または型番＝クニピアＦ）
Candidate substance:
a. Aluminum silicate (manufactured by Nacalai Tesque Co., Ltd .: product name or model number = 017-32)
b. Activated carbon (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 037-02115)
c. Silica gel 60 (manufactured by Nacalai Tesque Co., Ltd .: product name or model number = 307-31)
d. Aluminum oxide (manufactured by Merck Co., Ltd .: product name or model number = 1090.05)
e. Diatomaceous earth (manufactured by Nacalai Tesque Co., Ltd .: product name or model number = 045-00875)
f. Glass wool (manufactured by Shimahisa Pharmaceutical Co., Ltd .: product name or model number = GSR290)
g. Amber Light (manufactured by Organo Co., Ltd .: product name or model number = IRC748)
h. Chitin (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 034-13635)
i. DEAE Cellulose (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 049-23615)
j. Aluminum (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 014-01785)
k. Silicon (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 191-05582)
l. Aluminum hydroxide (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 014-01925)
m. Magnesium hydroxide (manufactured by Kyowa Chemical Industry Co., Ltd .: product name or model number = KISUMA 5Q-S)
n. Hydrotalcite 1 (manufactured by Kyowa Chemical Industry Co., Ltd .: product name or model number = DHT-4H: molecular formula = Mg 6 Al 2 (OH) 16 CO 3.4H 2 O)
o. Hydrotalcite 2 (manufactured by Kyowa Chemical Industry Co., Ltd .: product name or model number = DHT-6: molecular formula = Mg 4.5 Al 2 (OH) 13 CO 3.3.5H 2 O)
p. Natural allophane 1 (manufactured by Hokurin Kogyo Co., Ltd .: product name or model number = misoil: derived from Oyama miso soil)
q. Natural Allophane 2 (Product name or model number = Allophane derived from Kanuma soil: Allophane derived from Kanuma soil)
r. Purified allophane (manufactured by Shinagawa Kasei Co., Ltd .: product name or model number = Secard P1)
s. Fired mica product (manufactured by Nark Research Institute Co., Ltd .: product name or model number = N-1)
t. Glass foam (manufactured by Tottori Recycling Research Institute Co., Ltd .: product name or model number = PW-350)
u. Montmorillonite (manufactured by Kunimine Kogyo Co., Ltd .: product name or model number = Kunipia F)
候補物質で粉末状でないものは粉末にし、原料液１００重量部に対していずれも１０重量部添加し、４℃で２４時間振とうしながら混和した。 The candidate substances that were not in powder form were powdered, 10 parts by weight was added to 100 parts by weight of the raw material solution, and the mixture was mixed at 4 ° C. for 24 hours with shaking.
続いて、遠心分離により、候補物質と溶液とを分離した。総てについて溶液側に臭気成分が残っていたことから、候補物質側に目的タンパク質が吸着されているか否かを溶液側にスフェロイド形成促進活性あるかにより判定した。すなわち、溶液をヒト肝がん細胞ＨｅｐＧ２の播種時に、培養液である１０％ＦＢＳ／ＤＭＥＭに対して５％容量添加し、培養７２時間後の細胞がスフェロイド化している際には溶液側に、スフェロイド化していない場合には候補物質側に、活性物質があると判定した。 Subsequently, the candidate substance and the solution were separated by centrifugation. Since the odorous component remained on the solution side in all cases, it was determined whether or not the target protein was adsorbed on the candidate substance side based on whether or not the solution side had spheroid formation promoting activity. That is, when the human liver cancer cell HepG2 was seeded, 5% of the solution was added to 10% FBS / DMEM, which was the culture solution, and when the cells were spheroidized after 72 hours of culture, the solution was placed on the solution side. When it was not spheroidized, it was determined that the candidate substance had an active substance.
確認試験の結果を表２に示す。
以上の結果から、目的タンパク質の吸着剤として以下が好適であることがわかった。
ケイ酸アルミニウム、酸化アルミニウム、珪藻土、ハイドロタルサイト、アロフェン、モンモリロナイト
From the above results, it was found that the following are suitable as the adsorbent for the target protein.
Aluminum silicate, aluminum oxide, diatomaceous earth, hydrotalcite, allophane, montmorillonite
なお、動物細胞を培養する場合、目的タンパク質を粉末状の吸着剤に担持（吸着）させたままで使用し、粉末形状を足がかりとして、三次元培養が誘発ないし促進される態様もあり得る。
以上から、上記の吸着剤（粉末状）とタンパク質原料（原料液）とを混和・撹拌すれば、吸着剤側に目的タンパク質が吸着し、水溶液側に夾雑物が残るので、目的タンパク質が分離濃縮されたこととなり、結果、スフェロイド形成促進剤の濃縮技術が構築できたということができる。
When culturing animal cells, the target protein may be used while being supported (adsorbed) on a powdery adsorbent, and the powder shape may be used as a foothold to induce or promote three-dimensional culture.
From the above, if the above adsorbent (powder) and the protein raw material (raw material liquid) are mixed and stirred, the target protein is adsorbed on the adsorbent side and impurities remain on the aqueous solution side, so that the target protein is separated and concentrated. As a result, it can be said that a technology for concentrating spheroid formation promoters has been established.
なお、以下でも説明するように、目的タンパク質を担持した吸着剤を、緩衝液または界面活性剤を添加した緩衝液で洗浄することにより、わずかに残っている臭気成分その他の夾雑物を除去し、精製度をより高めるようにしても良い。 As will be described below, the adsorbent carrying the target protein is washed with a buffer solution or a buffer solution to which a surfactant is added to remove a small amount of residual odorous components and other impurities. The degree of purification may be increased.
＜吸着剤からの目的タンパク質の分離精製（溶出）＞
次に、目的タンパク質の吸着剤からの分離を検討することとした。ここでは、目的タンパク質をケイ酸アルミニウムに吸着させた試料を用いて、分離精製条件を検討した。
<Separation and purification (elution) of target protein from adsorbent>
Next, it was decided to examine the separation of the target protein from the adsorbent. Here, the separation and purification conditions were examined using a sample in which the target protein was adsorbed on aluminum silicate.
溶出液候補を以下のようにして予備試験をおこなった。
・予備試験１：緩衝液（リン酸、クエン酸、または、グリシン）＋金属キレート剤（ＥＤＴＡまたはＥＧＴＡ）による溶出検討
・予備試験２：緩衝液（リン酸、クエン酸、または、グリシン）＋金属キレート剤（ＥＤＴＡまたはＥＧＴＡ）＋塩（ＫＣｌ）による溶出検討
・予備試験３：緩衝液（リン酸、クエン酸、または、グリシン）＋塩（ＫＣｌ）の濃度勾配による溶出検討
・予備試験４：糖（グルコースまたはスクロース）による溶出検討
・予備試験５：緩衝液（トリス）＋塩（ＮａＣｌ）＋界面活性剤（ＰＥＧ）＋アミノ酸（グルタミン酸）＋塩酸（ＨＣｌ）による溶出検討。なお、グルタミン酸は酸性アミノ酸であって酸性でないと溶解しないため塩酸を添加してある。
A preliminary test was conducted on the eluate candidates as follows.
-Preliminary test 1: Buffer solution (phosphate, citric acid, or glycine) + elution study with metal chelating agent (EDTA or EGTA) -Preliminary test 2: Buffer solution (phosphate, citric acid, or glycine) + metal Elution study with chelating agent (EDTA or EGTA) + salt (KCl) / Preliminary test 3: Elution study with buffer solution (phosphate, citric acid, or glycine) + salt (KCl) concentration gradient / Preliminary test 4: Sugar Elution study with (glucose or sucrose) -Preliminary test 5: Elution study with buffer solution (Tris) + salt (NaCl) + surfactant (PEG) + amino acid (glutamic acid) + hydrochloric acid (HCl). Since glutamic acid is an acidic amino acid and does not dissolve unless it is acidic, hydrochloric acid is added.
以上の予備試験において、試料を各液に浸して室温で１時間振とうし、遠心分離により分離した液についてタンパク質の溶出量の確認をおこなうとともに、タンパク質の溶出がみとめられたものについては、滅菌後にスフェロイド形成がされるかの細胞培養試験をおこなった。その結果、予備試験１～４では、タンパク質の溶出が認められず、予備試験５のみにタンパク質の溶出および溶出タンパク質の活性が確認できた。 In the above preliminary test, the sample was immersed in each solution and shaken at room temperature for 1 hour to confirm the amount of protein eluted in the solution separated by centrifugation, and sterilized in the case where protein elution was found. A cell culture test was conducted to see if spheroid formation would occur later. As a result, protein elution was not observed in preliminary tests 1 to 4, and protein elution and eluted protein activity could be confirmed only in preliminary test 5.
続いて予備試験５に基づき必須の構成の検討を同様におこなったところ、緩衝液＋グルタミン酸＋ＨＣｌの組み合わせであれば溶出および活性の維持があることがわかった。 Subsequently, when the essential composition was similarly examined based on the preliminary test 5, it was found that the combination of buffer solution + glutamic acid + HCl had elution and maintenance of activity.
続いてアミノ酸について検討した。
・予備試験５－１：緩衝液（トリス）＋酸性アミノ酸塩（グルタミン酸ナトリウム）
・予備試験５－２．緩衝液（トリス）＋塩基性アミノ酸（アルギニンまたはヒスチジン）
・予備試験５－３．緩衝液（トリス）＋グリシン（最も単純な形を持つアミノ酸として検討）
Subsequently, amino acids were examined.
・ Preliminary test 5-1: Buffer solution (Tris) + acidic amino acid salt (monosodium glutamate)
・ Preliminary test 5-2. Buffer solution (Tris) + basic amino acid (arginine or histidine)
・ Preliminary test 5-3. Buffer solution (Tris) + glycine (considered as an amino acid with the simplest form)
以上の予備試験の結果、いずれについてもタンパク質の溶出が認められなかった。
すなわち、ケイ酸アルミニウムに吸着させた試料から目的タンパク質を溶出させるには、グルタミン酸が溶ける程度のｐＨに調整した緩衝液を用いればよいことが確認できた。
また、酸性アミノ酸として別途アスパラギン酸を用いた場合でも溶出および活性が確認できた。
緩衝液は特に限定されないが、トリスのほかＨＥＰＥＳ、ＭＯＰＳなどを挙げることができる。
As a result of the above preliminary tests, no protein elution was observed in any of them.
That is, it was confirmed that in order to elute the target protein from the sample adsorbed on aluminum silicate, a buffer solution adjusted to a pH at which glutamic acid can be dissolved should be used.
In addition, elution and activity could be confirmed even when aspartic acid was separately used as the acidic amino acid.
The buffer solution is not particularly limited, and examples thereof include HEPES and MOPS in addition to Tris.
なお、スフェロイド形成促進活性を確認した試験条件をしめす。
被抽出試料は、ケイ酸アルミニウム粉末に吸着させたものを用いた。この試料０．１ｇに対して溶出液１ｍｌを添加し、室温で一時間振とう処理した。溶出液の組成は１Ｍ塩酸に、グルタミン酸０．５Ｍを溶解し、更に、トリス（和光純薬工業社製：品名または型番＝２０７－０６２７５）を最終濃度が０．１Ｍになるように添加したものである。
次に、遠心分離によりケイ酸アルミニウムから溶出液を分離し、これを培養皿表面にコーティングして、肝臓ガン細胞ＨｅｐＧ２細胞を７２時間培養した。なお、分離前の溶出液をコーティングしたものと、原料液をコーティングして培養したものとの比較もおこなった。
The test conditions for confirming the spheroid formation promoting activity are shown.
As the sample to be extracted, a sample adsorbed on aluminum silicate powder was used. 1 ml of the eluate was added to 0.1 g of this sample, and the mixture was shaken at room temperature for 1 hour. The composition of the eluate is that 0.5 M of glutamic acid is dissolved in 1 M hydrochloric acid, and Tris (manufactured by Wako Pure Chemical Industries, Ltd .: product name or model number = 207-06275) is added so that the final concentration becomes 0.1 M. Is.
Next, the eluate was separated from aluminum silicate by centrifugation, coated on the surface of a culture dish, and liver cancer cells HepG2 cells were cultured for 72 hours. A comparison was also made between the one coated with the eluate before separation and the one coated with the raw material solution and cultured.
培養結果を図１に示す。写真から明らかなように、精製液は原料液と同様にスフェロイド形態に培養されることを確認した。 The culture results are shown in FIG. As is clear from the photograph, it was confirmed that the purified liquid was cultured in the spheroid form in the same manner as the raw material liquid.
溶出液によるコーティングのほかに、例えば、限外ろ過や透析などで溶出液のｐＨを中性域に調整したものを細胞培養液に添加してもよい（これを精製液と称する）。精製液を用いた培養試験の例としては、細胞培養液ＤＭＥＭ－１０％ＦＢＳ１０ｍｌに精製液０．５ｍｌ～１ｍｌを添加し、肝臓ガン細胞ＨｅｐＧ２細胞を７２時間培養する例を挙げることができる。 In addition to coating with the eluate, for example, an eluate whose pH is adjusted to the neutral range by ultrafiltration or dialysis may be added to the cell culture medium (this is referred to as a purified solution). As an example of the culture test using the purified solution, 0.5 ml to 1 ml of the purified solution is added to 10 ml of the cell culture solution DMEM-10% FBS, and the liver cancer cells HepG2 cells are cultured for 72 hours.
以上は、ケイ酸アルミニウムの場合である。次に、実施例１で目的タンパク質を吸着した他の吸着剤についても同様に溶出試験をおこなった。溶出液の組成および溶出方法は、上記したものにならった。結果を表３に示す。 The above is the case of aluminum silicate. Next, the elution test was similarly performed on the other adsorbents that adsorbed the target protein in Example 1. The composition of the eluate and the method of elution were as described above. The results are shown in Table 3.
以上の検討結果から、吸着剤としてケイ酸アルミニウムまたは天然アロフェンの粉末と原料液とを混和することで、粉末側に目的タンパク質を選択的に吸着させることができ、また、この粉末をさらに、グルタミン酸やアスパラギン酸などの酸性アミノ酸を溶解させた酸性水溶液に晒して水溶液側に目的タンパク質を溶出させることができる。すなわち目的タンパク質すなわちスフェロイド形成促進剤の精製技術が構築できたということができる。 From the above examination results, by mixing the powder of aluminum silicate or natural allophen as an adsorbent with the raw material solution, the target protein can be selectively adsorbed on the powder side, and this powder can be further adsorbed with glutamic acid. The target protein can be eluted on the aqueous solution side by exposing it to an acidic aqueous solution in which acidic amino acids such as or aspartic acid are dissolved. That is, it can be said that a purification technique for the target protein, that is, a spheroid formation promoter, has been established.
なお、吸着後には、緩衝液または界面活性剤を添加した緩衝液で洗浄することにより、わずかに残っている臭気成分その他の夾雑物を除去し、精製度をより高めるようにしても良い。実際、この工程を経ることにより吸着剤が無臭となることを確認した。
緩衝液の例としては、ＨＥＰＥＳ、ＭＯＰＳなどを挙げることができる。
また、界面活性剤としてはＴｗｅｅｎ２０、Ｔｗｅｅｎ８０などを挙げることができる。
After adsorption, washing with a buffer solution or a buffer solution to which a surfactant is added may be used to remove a small amount of residual odorous components and other contaminants to further increase the degree of purification. In fact, it was confirmed that the adsorbent became odorless by going through this step.
Examples of the buffer solution include HEPES, MOPS and the like.
Further, examples of the surfactant include Tween 20 and Tween 80.
以上は、ノロゲンゲを用いた例であるが、カレイ、エイ、ニギスを用いた場合も、イカ若しくはタコの胴部を用いた場合も、同様に精製可能である。 The above is an example using Bothrocara hollandi, but it can be similarly purified by using flatfish, ray, nigis, or by using the body of squid or octopus.
一般的にタンパク質の精製は、分子量、荷電、親和性などのタンパク質の各種性質を利用して夾雑物を除去していくのであるが、容易に精製できるタンパク質は稀であり、仮に精製できるとしても実際には特殊な技術や設備を要し複雑に精製法を組み合わる必要があり、価格の上昇を招来しやすい。
本発明は、以上説明したように、簡便に、吸着剤への吸着→洗浄→吸着剤からの分離（溶出）を行うことができ、かつ、強酸性雰囲気下に晒されても、スフェロイド形成促進活性を損なわず、極めて実用的な精製方法であるといえる。
In general, protein purification uses various properties of proteins such as molecular weight, charge, and affinity to remove impurities, but proteins that can be easily purified are rare, and even if they can be purified, they are rare. In reality, special technology and equipment are required, and it is necessary to combine purification methods in a complicated manner, which tends to lead to an increase in price.
As described above, the present invention can easily adsorb to the adsorbent → wash → separate (elute) from the adsorbent, and promote spheroid formation even when exposed to a strongly acidic atmosphere. It can be said that it is an extremely practical purification method without impairing its activity.
溶出液に目的タンパク質を溶出させた後は、そのまま保存していてもよく、適宜、スフェロイドを形成させる使用環境に合わせて安定化処理や精製処理をおこなうようにして、適用範囲を広げるようにし、利便性をたかめてもよい。 After the target protein is eluted in the eluate, it may be stored as it is, and the scope of application may be expanded by appropriately performing stabilization treatment or purification treatment according to the usage environment in which spheroids are formed. You may increase the convenience.
Claims (4)
ケイ酸アルミニウムまたは天然アロフェンの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、
次に、タンパク質の吸着された粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法。 From body fluids collected from the epidermis to muscular tissue of Genge, curry, ray, or nigis, which has a mucous body epidermis, or on the body of a squid or octopus among cephalopods. A method for purifying a protein raw material having spheroid formation-promoting activity, which is derived from a certain body fluid or collected from a body fluid, as a spheroid formation-promoting agent.
The powder of aluminum silicate or natural allophane is mixed with the protein raw material to allow impurities to remain on the liquid side and to adsorb and separate the protein having spheroid formation promoting activity on the powder side.
Next, a method for purifying a spheroid formation promoter, which comprises mixing a powder on which a protein is adsorbed and an acidic aqueous solution in which an acidic amino acid is dissolved to elute the protein on the aqueous solution side.
ケイ酸アルミニウムまたは天然アロフェンの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、
次に、タンパク質の吸着された粉末に対し、緩衝液または界面活性剤を添加した緩衝液により一回以上洗浄し、
その後、粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法。 From body fluids collected from the epidermis to muscular tissue of Genge, curry, ray, or nigis, which has a mucous body epidermis, or on the body of a squid or octopus among cephalopods. A method for purifying a protein raw material having spheroid formation-promoting activity, which is derived from a certain body fluid or collected from a body fluid, as a spheroid formation-promoting agent.
The powder of aluminum silicate or natural allophane is mixed with the protein raw material to allow impurities to remain on the liquid side and to adsorb and separate the protein having spheroid formation promoting activity on the powder side.
Next, the powder on which the protein is adsorbed is washed at least once with a buffer solution or a buffer solution containing a surfactant.
Then, a method for purifying a spheroid formation promoter, which comprises mixing a powder and an acidic aqueous solution in which an acidic amino acid is dissolved to elute a protein on the aqueous solution side.
酸化アルミニウムまたはハイドロタルサイトの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、
次に、タンパク質の吸着された粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法。 From body fluids collected from the epidermis to muscular tissue of Genge, curry, ray, or nigis, which has a mucous body epidermis, or on the body of a squid or octopus among cephalopods. A method for purifying a protein raw material having spheroid formation-promoting activity, which is derived from a certain body fluid or collected from a body fluid, as a spheroid formation-promoting agent.
The powder of aluminum oxide or hydrotalcite and the protein raw material are mixed to leave impurities on the liquid side and adsorb and separate the protein having spheroid formation promoting activity on the powder side.
Next, a method for purifying a spheroid formation promoter, which comprises mixing a powder on which a protein is adsorbed and an acidic aqueous solution in which an acidic amino acid is dissolved to elute the protein on the aqueous solution side.
酸化アルミニウムまたはハイドロタルサイトの粉末と前記タンパク質原料とを混和して、液体側に夾雑物を存置させるとともに粉末側にスフェロイド形成促進活性を有するタンパク質を吸着させて分離し、
次に、タンパク質の吸着された粉末に対し、緩衝液または界面活性剤を添加した緩衝液により一回以上洗浄し、
その後、粉末と、酸性アミノ酸を溶解させた酸性水溶液と、を混和し、水溶液側にタンパク質を溶出させることを特徴とするスフェロイド形成促進剤の精製方法。
From body fluids collected from the epidermis to muscular tissue of Genge, curry, ray, or nigis, which has a mucous body epidermis, or on the body of a squid or octopus among cephalopods. A method for purifying a protein raw material having spheroid formation-promoting activity, which is derived from a certain body fluid or collected from a body fluid, as a spheroid formation-promoting agent.
The powder of aluminum oxide or hydrotalcite and the protein raw material are mixed to leave impurities on the liquid side and adsorb and separate the protein having spheroid formation promoting activity on the powder side.
Next, the powder on which the protein is adsorbed is washed at least once with a buffer solution or a buffer solution containing a surfactant.
Then, a method for purifying a spheroid formation promoter, which comprises mixing a powder and an acidic aqueous solution in which an acidic amino acid is dissolved to elute a protein on the aqueous solution side.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003042236A1 (en) | 2001-11-13 | 2003-05-22 | The Nisshin Oillio, Ltd. | Process for producing concentrated/purified protein using clay mineral composition |
| JP2004505615A (en) | 2000-06-23 | 2004-02-26 | ワーナー−ランバート・カンパニー、リミテッド、ライアビリティ、カンパニー | Methods for isolation and purification of proteins, resulting proteins |
| JP2008069118A (en) | 2006-09-15 | 2008-03-27 | Hiroshima Univ | Sweat antigen adsorbent |
| JP2010538299A (en) | 2007-09-06 | 2010-12-09 | ウオーターズ・テクノロジーズ・コーポレイシヨン | Isolation method of functional polymer |
| JP2011062129A (en) | 2009-09-17 | 2011-03-31 | Tottori Institute Of Industrial Technology | Spheroid-forming promoter |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2004505615A (en) | 2000-06-23 | 2004-02-26 | ワーナー−ランバート・カンパニー、リミテッド、ライアビリティ、カンパニー | Methods for isolation and purification of proteins, resulting proteins |
| WO2003042236A1 (en) | 2001-11-13 | 2003-05-22 | The Nisshin Oillio, Ltd. | Process for producing concentrated/purified protein using clay mineral composition |
| JP2008069118A (en) | 2006-09-15 | 2008-03-27 | Hiroshima Univ | Sweat antigen adsorbent |
| JP2010538299A (en) | 2007-09-06 | 2010-12-09 | ウオーターズ・テクノロジーズ・コーポレイシヨン | Isolation method of functional polymer |
| JP2011062129A (en) | 2009-09-17 | 2011-03-31 | Tottori Institute Of Industrial Technology | Spheroid-forming promoter |
Non-Patent Citations (1)
| Title |
|---|
| 日本分子生物学会年会プログラム，2016年，３９巻，１Ｐ－０３１０ |
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