JP2009278907A - Method for modifying sugar chain - Google Patents
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本発明は、酵素学的手法による糖鎖改変方法において、反応に用いたグリコシダーゼの混入がない生成物を得るための方法に関する。 The present invention relates to a method for obtaining a product free from the contamination of glycosidase used in a reaction in a method for modifying a sugar chain by an enzymatic method.
糖鎖は、核酸、タンパク質に次ぐ第三の“鎖”であり、近年、その役割や機能の実態が次第に明らかになるとともに、糖鎖の機能の向上や改変のための糖鎖工学の研究も盛んに行われていることは当業者に周知の通りである。このような状況において、本発明者のグループは、これまでに、グリコシダーゼの１つであるヒアルロニダーゼを用いて糖鎖を２糖単位で加水分解したり転移させたりすることによる糖鎖改変方法を開発している（例えば非特許文献１および非特許文献２を参照のこと）。この方法は、計画的なデザインのもとで糖鎖を２糖またはその倍数のオリゴ糖の単位で改変できることから、糖鎖工学上において非常に利用価値が高いものである。 Sugar chains are the third “chain” after nucleic acids and proteins. In recent years, the roles and functions of these chains have become increasingly clear, and research on sugar chain engineering for improving and modifying sugar chain functions has also been conducted. It is well known to those skilled in the art that this is actively done. In such a situation, the group of the present inventors has so far developed a method for modifying a sugar chain by hydrolyzing or transferring a sugar chain by a disaccharide unit using hyaluronidase which is one of glycosidases. (For example, see Non-Patent Document 1 and Non-Patent Document 2). Since this method can modify a sugar chain with a disaccharide or a multiple of an oligosaccharide unit under a planned design, it is very useful in terms of glycoengineering.
しかしながら、糖鎖の改変対象となるグリコサミノグリカンやプロテオグリカンを含む水溶液にグリコシダーゼを添加して行う反応系（糖転移反応を行う場合にはさらに供与体となるグリコサミノグリカンやプロテオグリカンを添加）では、反応後に生成物を所定の精製手段（ゲルろ過クロマトグラフィーなど）によって精製しても、反応に用いたグリコシダーゼを生成物から除去することが必ずしも容易ではなく、そのため、生成物の生物学的活性を評価したり利用したりする際や生成物を出発原料として新たに糖鎖改変を行う際、生成物に混入したグリコシダーゼが悪影響を及ぼすことが否めないという問題があった。
そこで本発明は、酵素学的手法による糖鎖改変方法において、反応に用いたグリコシダーゼの混入がない生成物を得るための方法を提供することを目的とする。 Accordingly, an object of the present invention is to provide a method for obtaining a product free from the contamination of glycosidase used in a reaction in a method for modifying a sugar chain by an enzymatic method.
上記の点に鑑みてなされた本発明の方法は、請求項１記載の通り、グリコサミノグリカンおよび／またはプロテオグリカンを含む水溶液をグリコシダーゼ固定化カラムに通液し、グリコシダーゼの作用に基づく糖鎖の加水分解反応および／または糖転移反応をカラム内で行うことにより糖鎖を改変することを特徴とする。
また、請求項２記載の方法は、請求項１記載の方法において、グリコシダーゼがヒアルロニダーゼであることを特徴とする。
In the method of the present invention made in view of the above points, as described in claim 1, an aqueous solution containing glycosaminoglycan and / or proteoglycan is passed through a glycosidase-immobilized column, and the sugar chain based on the action of glycosidase is added. The sugar chain is modified by performing a hydrolysis reaction and / or a sugar transfer reaction in a column.
The method according to claim 2 is characterized in that, in the method according to claim 1, the glycosidase is hyaluronidase.
本発明によれば、反応に用いるグリコシダーゼがカラム内に固定化されているので、反応後にグリコシダーゼを除去するための精製工程が不要となる。従って、グリコシダーゼの混入がない高い純度の糖鎖改変生成物の収量の向上を図ることができる。 According to the present invention, since the glycosidase used for the reaction is immobilized in the column, a purification step for removing the glycosidase after the reaction becomes unnecessary. Therefore, it is possible to improve the yield of a high-purity sugar chain modified product free from glycosidase contamination.
本発明の方法は、グリコサミノグリカンおよび／またはプロテオグリカンを含む水溶液をグリコシダーゼ固定化カラムに通液し、グリコシダーゼの作用に基づく糖鎖の加水分解反応および／または糖転移反応をカラム内で行うことにより糖鎖を改変することを特徴とするものである。本発明において「糖鎖の改変」とは、グリコシダーゼの作用に基づく糖鎖の加水分解反応によってその構成糖の一部または全部を欠損させること、糖転移反応によって欠損させた構成糖を新たな構成糖に置換して糖鎖を組み換えることや糖鎖に新たな構成糖を付加して糖鎖を伸長することなど、糖鎖の構成糖を人為的に欠損、置換、付加する操作を意味するものとする。 In the method of the present invention, an aqueous solution containing glycosaminoglycan and / or proteoglycan is passed through a glycosidase-immobilized column, and a sugar chain hydrolysis reaction and / or glycosyltransferase reaction based on the action of glycosidase is performed in the column. It is characterized by modifying the sugar chain. In the present invention, “glycan modification” means that a part or all of the constituent sugars are lost by a hydrolysis reaction of a sugar chain based on the action of glycosidase, or a constituent sugar that has been deleted by a transglycosylation reaction is newly constructed. Means an operation to artificially delete, replace, or add a constituent sugar of a sugar chain, such as recombination of a sugar chain by substitution with a sugar or addition of a new constituent sugar to a sugar chain to extend the sugar chain. Shall.
本発明において糖鎖を改変するために用いるグリコシダーゼは、糖鎖の加水分解活性および／または糖転移活性を有するものであればどのようなものであってもよく、例えば、グリコサミノグリカンとして、ヒアルロン酸の他、コンドロイチン、コンドロイチン硫酸Ａ，Ｃ，Ｅ、脱硫酸化コンドロイチン硫酸Ｂなどに作用する精巣性ヒアルロニダーゼ（ＥＣ３．２．１３５：非特許文献１）や、プロテオグリカンのコアタンパク質とグリコサミノグリカン糖鎖の結合部位に存在する共通の橋渡し領域（−ＧｌｃＡ−Ｇａｌ−Ｇａｌ−Ｘｙｌ−セリン）に特異的に作用するエンド−β−キシロシダーゼ、エンド−β−ガラクトシダーゼ、エンド−β−グルクロニダーゼや、エンド−β−キシロシダーゼと同様の酵素活性を有するセルラーゼやキシラナーゼなどのエンド型グリコシダーゼが挙げられる。 The glycosidase used for modifying the sugar chain in the present invention may be any glycosidase having sugar chain hydrolysis activity and / or transglycosylation activity, for example, as glycosaminoglycan, In addition to hyaluronic acid, testicular hyaluronidase (EC 3.2.135: Non-Patent Document 1) acting on chondroitin, chondroitin sulfate A, C, E, desulfated chondroitin sulfate B, and the like, proteoglycan core protein and glycosaminoglycan Endo-β-xylosidase, endo-β-galactosidase, endo-β-glucuronidase, which specifically acts on a common bridging region (-GlcA-Gal-Gal-Xyl-serine) present in the binding site of the sugar chain, -Cellulase and xylose having the same enzyme activity as β-xylosidase Endo-type glycosidase, such as transglutaminase, and the like.
グリコシダーゼ固定化カラムの作製は、酵素固定化カラムの一般的な作製方法に従って行うことができる。その一例としては、ＣＮＢｒ（臭化シアン）活性化セファロースなどのＣＮＢｒによって活性化させたビーズ状のアガロースゲルにグリコシダーゼをカップリングさせてガラス製のカラムに充填する方法が挙げられる（グリコシダーゼを固定する担体はビニルポリマーなどの合成樹脂からなるビーズなどであってもよい）。グリコシダーゼをカラム内に固定化することで、その酵素活性の安定化が図られるため、カラムを繰り返し利用できる。また、作製したカラムは３〜５℃で保存することにより、少なくとも３ヶ月はグリコシダーゼの酵素活性を維持しておくことができる（６ヶ月経過後においても約７０％の酵素活性の維持が可能）。 The glycosidase-immobilized column can be produced according to a general production method for an enzyme-immobilized column. One example is a method in which a glycosidase is coupled to a beaded agarose gel activated with CNBr, such as CNBr (cyanogen bromide) activated sepharose, and packed into a glass column (glycosidase is immobilized). The carrier may be beads made of a synthetic resin such as vinyl polymer). Since the enzyme activity is stabilized by immobilizing glycosidase in the column, the column can be used repeatedly. In addition, by storing the prepared column at 3 to 5 ° C., the enzyme activity of glycosidase can be maintained for at least 3 months (approximately 70% of enzyme activity can be maintained even after 6 months). .
グリコシダーゼ固定化カラム内での糖鎖の加水分解反応や糖転移反応は、糖鎖改変対象とするグリコサミノグリカンやプロテオグリカンを含む水溶液を、予め至適な緩衝溶液で緩衝化したカラムに通液し（糖転移反応を行う場合にはさらに供与体となるグリコサミノグリカンやプロテオグリカンを通液）、カラム内を所定の反応条件に保つことで行うことができる。例えば、グリコシダーゼとして精巣性ヒアルロニダーゼを用いる場合、好適には３６〜３８℃、ｐＨ３．０〜６．５の条件下において、糖鎖改変対象とするグリコサミノグリカンやプロテオグリカンの糖鎖を、その非還元末端側から構成糖が２糖ずつ遊離するように加水分解することができる。また、好適には２〜５０℃、ｐＨ５．０〜８．５の条件下において、反応系内に共存させた供与体となるグリコサミノグリカンやプロテオグリカンの糖鎖を加水分解し、その非還元末端側から構成糖を２糖ずつ遊離させるとともに、遊離した構成糖を２糖ずつ糖鎖改変対象とするグリコサミノグリカンやプロテオグリカンの糖鎖の非還元末端側に次々に転移させて糖鎖を伸長することができる。糖鎖をより長く伸長させるためには反応温度は低い方が望ましく、例えば２〜２６℃の反応温度では１０個以上の２糖単位を、１６℃以下の反応温度では２０個以上の２糖単位を効率的に転移させることができる。なお、言うまでもないことであるがグリコシダーゼとしてヒアルロニダーゼを用いる場合には糖鎖改変対象や供与体となるグリコサミノグリカンやプロテオグリカンはヒアルロニダーゼの作用を受けるものでなければならない。所定の反応時間（例えば１０分間〜３日間）が経過した後、カラムに洗浄液として例えば水や塩化ナトリウム水溶液や酢酸アンモニウム水溶液などを通液して反応液を流出させ、必要な精製手段を適用することで、グリコシダーゼの混入がない糖鎖改変生成物を得ることができる。なお、精製手段は、反応液に含まれる目的生成物や未反応出発原料やこれらの分解物などの物理化学的性状を考慮し、ゲルろ過クロマトグラフィー、陽イオン交換クロマトグラフィー、陰イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、透析、限外ろ過などから適宜採用すればよい。 In the glycosidase-immobilized column, sugar chain hydrolysis and transglycosylation are conducted through a column in which an aqueous solution containing glycosaminoglycan or proteoglycan to be modified is previously buffered with an optimal buffer solution. (In the case of carrying out a sugar transfer reaction, the glycosaminoglycan or proteoglycan serving as a donor is further passed through), and the column interior can be maintained under predetermined reaction conditions. For example, when testicular hyaluronidase is used as a glycosidase, the sugar chain of glycosaminoglycan or proteoglycan to be subjected to sugar chain modification is preferably removed under conditions of 36 to 38 ° C. and pH 3.0 to 6.5. Hydrolysis can be performed so that the constituent sugars are released from the reducing end side by two sugars. In addition, the glycosaminoglycan or proteoglycan sugar chain serving as a donor coexisting in the reaction system is preferably hydrolyzed under the conditions of 2 to 50 ° C. and pH 5.0 to 8.5, and the non-reduction thereof is performed. The constituent sugars are released from the terminal side by two sugars, and the released constituent sugars are transferred one after another to the non-reducing terminal side of the glycosaminoglycan or proteoglycan sugar chain to be modified. Can stretch. In order to extend the sugar chain longer, it is desirable that the reaction temperature is lower, for example, 10 or more disaccharide units at a reaction temperature of 2 to 26 ° C, and 20 or more disaccharide units at a reaction temperature of 16 ° C or less. Can be efficiently transferred. Needless to say, when hyaluronidase is used as a glycosidase, glycosaminoglycan or proteoglycan serving as a sugar chain modification target or donor must be subject to the action of hyaluronidase. After a predetermined reaction time (for example, 10 minutes to 3 days) has passed, for example, water, an aqueous solution of sodium chloride, an aqueous solution of ammonium acetate, or the like is passed through the column as a washing solution, and the reaction solution is discharged, and necessary purification means are applied. Thus, a sugar chain modification product free from glycosidase contamination can be obtained. The purification means is based on gel filtration chromatography, cation exchange chromatography, anion exchange chromatography in consideration of the physicochemical properties of the target product, unreacted starting materials and their decomposition products contained in the reaction solution. , Affinity chromatography, dialysis, ultrafiltration, etc. may be employed as appropriate.
なお、糖鎖改変対象がプロテオグリカンの場合、予めプロテオグリカンにプロテアーゼを作用させてコアタンパク質をペプチドに断片化してからその水溶液をグリコシダーゼ固定化カラムに通液するようにしてもよい。本発明の「プロテオグリカン」には、コアタンパク質をペプチドに断片化したグリカンも含まれるものとする。 In addition, when the sugar chain modification target is a proteoglycan, a protease may act on the proteoglycan in advance to fragment the core protein into a peptide, and the aqueous solution may be passed through a glycosidase-immobilized column. The “proteoglycan” of the present invention includes glycans obtained by fragmenting the core protein into peptides.
また、本発明の方法によれば、例えば、グリコシダーゼを用いてプロテオグリカンのグリコサミノグリカン糖鎖の加水分解反応を行ってその構成糖の一部または全部を欠損させた後、糖転移反応を行って欠損させた構成糖を新たな構成糖に置換して糖鎖を組み換えたり、さらに新たな構成糖を付加して糖鎖を伸長したりする連続工程を、グリコシダーゼ固定化カラムを組み込んだ図１に例示する糖鎖改変装置を用いて繰り返し行うことができる。 In addition, according to the method of the present invention, for example, glycosidase is used to hydrolyze the glycosaminoglycan sugar chain of proteoglycan so that a part or all of the constituent sugars are lost, and then a transglycosylation reaction is performed. The glycosidase-immobilized column is incorporated into the continuous process of replacing sugar chains that have been deleted in this way with new constituent sugars to recombine sugar chains or adding new constituent sugars to extend sugar chains. It can be repeated using the sugar chain modifying apparatus exemplified in the above.
図１に例示する糖鎖改変装置は、糖鎖の加水分解反応を行うための第１のグリコシダーゼ固定化カラム（第１ＣＭ）と糖転移反応を行うための第２のグリコシダーゼ固定化カラム（第２ＣＭ）を直列に連結したものであり、第１ＣＭと第２ＣＭの間には第１ＣＭ内で行う糖鎖の加水分解反応によって得られる生成物の精製手段としての限外ろ過膜ユニットが配置され、第２ＣＭの下流には送液手段としてのペリスタポンプが配置されている。
以下、グリコシダーゼ固定化カラムとしてヒアルロニダーゼ固定化カラムを組み込んだ糖鎖改変装置の使用方法の一例について順を追って説明する。
まず、（１）から出発原料（糖鎖改変対象）であるプロテオグリカンを含む水溶液を第１ＣＭにアプライした後、第１ＣＭの出口のコックを閉じ、第１ＣＭ内で糖鎖の加水分解反応を所定の条件下（例えば０．１Ｍ酢酸ナトリウム緩衝液−０．１５Ｍ ＮａＣｌ，ｐＨ４．０〜５．５，３７℃）で数時間〜一晩行う。この過程により、プロテオグリカンのグリコサミノグリカン糖鎖は、橋渡し領域の４糖を含む６糖を残して消化され、糖鎖欠損プロテオグリカンと主として４糖からなるグリコサミノグリカン糖鎖の分解断片が第１ＣＭ内に生成する。
次に、第１ＣＭの出口のコックを開き、（１）から水を加え、ペリスタポンプを作動させて装置内の流路に反応液が流れるようにすることで、第１ＣＭから流出した反応液に含まれる糖鎖欠損プロテオグリカンと糖鎖分解断片を、限外ろ過膜ユニット（例えばＰｅｌｌｉｃｏｎ（商品名） ＸＬデバイス，バイオマックス−５，日本ミリポア社）を循環する過程で分離し（反応液の循環速度は例えば３〜４ｍＬ／ｍｉｎが望ましい）、糖鎖分解断片を流路の外に排出するとともに、糖鎖欠損プロテオグリカンを第２ＣＭに導入する。
次に、（２）から糖転移反応を行うための供与体（ドナー）となる長鎖のグリコサミノグリカン（例えばヒアルロン酸であれば１５０〜２５０糖、コンドロイチンやコンドロイチン硫酸Ａであれば１００〜１５０糖、コンドロイチン硫酸Ｃであれば５０〜１００糖が好適である）を第２ＣＭの入口のコックを開いて導入し、糖転移反応を所定の条件下（例えば０．１Ｍ Ｔｒｉｓ−ＨＣｌ緩衝液，ｐＨ７．０，３７℃）で数時間〜一晩行う。第２ＣＭ内を糖転移反応の至適条件とするために、供与体となるグリコサミノグリカンは、例えば０．２〜１Ｍ Ｔｒｉｓ−ＨＣｌ緩衝液に溶解して第２ＣＭに導入することが望ましい。この過程により、供与体としたグリコサミノグリカンの糖鎖が加水分解されてその非還元末端側から構成糖が２糖ずつ遊離するとともに、遊離した構成糖が２糖ずつ糖鎖欠損プロテオグリカンが有する６糖からなる糖鎖の非還元末端側に次々に転移して糖鎖が伸長する。所定の反応時間が経過した後、（１）から水を加え、ペリスタポンプを作動させて生成物（糖鎖改変プロテオグリカン）を回収する。
なお、第１ＣＭ内での副反応である糖転移反応と第２ＣＭ内での副反応である糖鎖の加水分解反応を極力抑制し、第１ＣＭ内での糖鎖の加水分解反応と第２ＣＭ内での糖転移反応を効率的に行うためには、第２ＣＭの酵素活性は第１ＣＭの酵素活性よりも低いことが望ましい。従って、第１ＣＭとしたカラムを繰り返し利用することで酵素活性が低下したら、新しいカラムに交換するとともに、酵素活性が低下したカラムは第２ＣＭとして使用することで、カラムの有効利用を図ることができる。
The sugar chain modifying apparatus illustrated in FIG. 1 includes a first glycosidase-immobilized column (first CM) for performing a sugar chain hydrolysis reaction and a second glycosidase-immobilized column (second CM) for performing a sugar transfer reaction. ) Are connected in series, and between the first CM and the second CM, an ultrafiltration membrane unit is disposed as a means for purifying a product obtained by hydrolysis reaction of sugar chains performed in the first CM. A peristaltic pump as a liquid feeding means is arranged downstream of 2CM.
Hereinafter, an example of a method for using a sugar chain altering apparatus incorporating a hyaluronidase-immobilized column as a glycosidase-immobilized column will be described step by step.
First, after applying an aqueous solution containing the proteoglycan as a starting material (subject to sugar chain modification) from (1) to the first CM, the outlet cock of the first CM is closed, and the hydrolysis reaction of the sugar chain is performed in the first CM. The reaction is performed for several hours to overnight under the conditions (for example, 0.1 M sodium acetate buffer-0.15 M NaCl, pH 4.0 to 5.5, 37 ° C.). Through this process, the glycosaminoglycan sugar chain of proteoglycan is digested leaving 6 sugars including the 4 sugars in the bridging region, and a glycosaminoglycan sugar chain fragment composed mainly of sugar chain-deficient proteoglycans and tetrasaccharides Generate in 1CM.
Next, the outlet cock of the first CM is opened, water is added from (1), and the peristaltic pump is operated to allow the reaction liquid to flow through the flow path in the apparatus, so that it is included in the reaction liquid flowing out from the first CM. The sugar chain-deficient proteoglycan and the glycosylated fragment are separated in the process of circulating through an ultrafiltration membrane unit (for example, Pellicon (trade name) XL device, Biomax-5, Nihon Millipore). For example, 3-4 mL / min is desirable), and the sugar chain degrading fragment is discharged out of the flow path, and the sugar chain deficient proteoglycan is introduced into the second CM.
Next, from (2), a long-chain glycosaminoglycan serving as a donor (donor) for carrying out the transglycosylation reaction (for example, 150 to 250 sugars for hyaluronic acid, 100 to 250 for chondroitin or chondroitin sulfate A) 150 sugar, 50 to 100 sugars are suitable for chondroitin sulfate C), and the cock at the entrance of the second CM is opened, and the transglycosylation reaction is carried out under predetermined conditions (for example, 0.1 M Tris-HCl buffer, pH 7.0, 37 ° C.) for several hours to overnight. In order to make the second CM the optimum condition for the transglycosylation reaction, it is desirable that the glycosaminoglycan serving as a donor is dissolved in, for example, 0.2 to 1 M Tris-HCl buffer and introduced into the second CM. Through this process, the sugar chain of the glycosaminoglycan used as a donor is hydrolyzed to release two constituent sugars from the non-reducing end side, and the released constituent sugars are contained in sugar chain-deficient proteoglycans by two sugars. The sugar chain is extended by successively transferring to the non-reducing end side of the sugar chain consisting of hexasaccharides. After a predetermined reaction time has elapsed, water is added from (1), and the peristaltic pump is operated to recover the product (sugar chain-modified proteoglycan).
The sugar transfer reaction, which is a side reaction in the first CM, and the hydrolysis reaction of the sugar chain, which is a side reaction in the second CM, are suppressed as much as possible, and the hydrolysis reaction of the sugar chain in the first CM and the second CM In order to efficiently carry out the transglycosylation reaction, the enzyme activity of the second CM is preferably lower than the enzyme activity of the first CM. Therefore, when the enzyme activity is reduced by repeatedly using the first CM column, the column is replaced with a new column, and the column having the reduced enzyme activity is used as the second CM, so that the column can be effectively used. .
以下、本発明を実施例によって詳細に説明するが、本発明は以下の記載に限定して解釈されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted.
参考例１：ヒアルロニダーゼ固定化カラムの作製
１．２ｇのウシ精巣性ヒアルロニダーゼ（Ｔｙｐｅ１−Ｓ，シグマ社）と、膨潤させる前の乾燥重量で３０ｇ（膨潤させた後におけるタンパク質濃度が１０ｍｇ／ｍＬ）のＧＥヘルスケア社のＣＮＢｒ活性化セファロース４ＦＦを、０．１Ｍリン酸緩衝液（ｐＨ７．０）中、室温で２時間または４℃で１８時間かけてカップリングさせ、直径が１５ｍｍで長さが１０ｃｍのガラス製のカラムに充填することで作製した（充填した担体のベッドボリュームは３．６ｍＬで高さは２ｃｍ）。カップリング効率は１１．８６％で、担体１ｍＬ（ベッドボリューム）あたりのヒアルロニダーゼのタンパク質量と力価はそれぞれ１．２ｍｇと５３４Ｕ程度であった。このようにして作製したカラムは使用時まで４℃で保存した。
Reference example 1: Preparation of hyaluronidase-immobilized column 1.2 g of bovine testicular hyaluronidase (Type1-S, Sigma) and 30 g of dry weight before swelling (protein concentration after swelling is 10 mg / mL) GE Healthcare CNBr activated Sepharose 4FF was coupled in 0.1 M phosphate buffer (pH 7.0) for 2 hours at room temperature or 18 hours at 4 ° C., 15 mm in diameter and 10 cm in length. (The bed volume of the packed carrier is 3.6 mL and the height is 2 cm). The coupling efficiency was 11.86%, and the protein amount and titer of hyaluronidase per 1 mL (bed volume) of the carrier were about 1.2 mg and 534 U, respectively. The column thus prepared was stored at 4 ° C. until use.
実施例１：ヒアルロニダーゼ固定化カラムを用いた糖鎖欠損プロテオグリカンの調製（糖鎖の加水分解反応）
平衡化緩衝液（０．１Ｍ酢酸ナトリウム緩衝液−０．１５Ｍ ＮａＣｌ，ｐＨ５．０）で平衡化したヒアルロニダーゼ固定化カラムに、出発原料（糖鎖改変対象）であるプロテオグリカンとしてヒト尿中トリプシンインヒビター（ＵＴＩ：わかもと製薬社）を担体１ｍＬ（ベッドボリューム）あたり約１０ｍｇアプライし（平衡化緩衝液を溶媒として使用）、３７℃で１８時間保持して糖鎖の加水分解反応を行って糖鎖を分解させた後、反応液をカラムから排出させ、限外ろ過膜（アミコン社）を用いて精製・濃縮することで、糖鎖欠損プロテオグリカンを得た。
Example 1: Preparation of a sugar chain-deficient proteoglycan using a hyaluronidase-immobilized column (sugar chain hydrolysis reaction)
A human urinary trypsin inhibitor (proteoglycan as a starting material (sugar chain modification target) is applied to a hyaluronidase-immobilized column equilibrated with an equilibration buffer (0.1 M sodium acetate buffer-0.15 M NaCl, pH 5.0). Apply about 10 mg of UTI (Wakamoto Pharmaceutical Co., Ltd.) per 1 mL (bed volume) of the carrier (using the equilibration buffer as a solvent) and hold at 37 ° C. for 18 hours to hydrolyze the sugar chain to decompose the sugar chain Then, the reaction solution was discharged from the column, and purified and concentrated using an ultrafiltration membrane (Amicon) to obtain a sugar chain-deficient proteoglycan.
実施例２：ヒアルロニダーゼ固定化カラムを用いた糖鎖改変プロテオグリカンの調製（糖転移反応）
平衡化緩衝液（０．１Ｍ Ｔｒｉｓ−ＨＣｌ緩衝液，ｐＨ７．０）で平衡化したヒアルロニダーゼ固定化カラムに、受容体（アクセプター）としての実施例１で調製した糖鎖欠損プロテオグリカンと供与体（ドナー）としての約２００糖からなるヒアルロン酸（紀文フードケミファ社）を同時にアプライした。それぞれのアプライ量は、担体１ｍＬ（ベッドボリューム）あたり約１５ｍｇと約２ｍｇとした（平衡化緩衝液を溶媒として使用）。４℃で１８時間保持して糖転移反応を行って糖鎖を伸長させた後、反応液をカラムから排出させて濃縮することで、糖鎖改変プロテオグリカンを得た。
Example 2: Preparation of a sugar chain-modified proteoglycan using a hyaluronidase-immobilized column (glycosyl transfer reaction)
The sugar chain-deficient proteoglycan prepared in Example 1 as a receptor (acceptor) and a donor (donor) were placed on a hyaluronidase-immobilized column equilibrated with an equilibration buffer (0.1 M Tris-HCl buffer, pH 7.0). ) Hyaluronic acid (Kibun Food Chemifa) consisting of about 200 sugars was simultaneously applied. The amount of each applied was about 15 mg and about 2 mg per 1 mL (bed volume) of the carrier (using an equilibration buffer as a solvent). After maintaining the sugar chain at 4 ° C. for 18 hours to carry out a sugar transfer reaction to extend the sugar chain, the reaction solution was discharged from the column and concentrated to obtain a sugar chain-modified proteoglycan.
（生成物の純度の評価）
実施例１で得られた糖鎖欠損プロテオグリカンと実施例２で得られた糖鎖改変プロテオグリカンの純度を、従来法（試験管内で行う反応系）で得られた糖鎖欠損プロテオグリカンと糖鎖改変プロテオグリカンの純度と比較した結果を図２に示す（ＣＢＢ染色ＳＤＳ−ＰＡＧＥ：いずれも反応液の状態のもの）。なお、各レーンは以下の通りである。
レーン１：プロテオグリカン
レーン２：実施例１で得られた糖鎖欠損プロテオグリカン
レーン３：実施例２で得られた糖鎖改変プロテオグリカン
レーン４：従来法で得られた糖鎖欠損プロテオグリカン
レーン５：従来法で得られた糖鎖改変プロテオグリカン
レーン６：ヒアルロニダーゼ
図２から明らかなように、本発明の方法によるレーン２とレーン３では、目的生成物のバンド（矢印１）の他にも出発原料由来のバンド（矢印２で示される低分子ＵＴＩなど）が認められるが、従来法によるレーン４とレーン５で認められるヒアルロニダーゼのバンドは認められず、本発明の方法によれば、従来法に比較して高い純度で目的生成物を調製できることがわかった。なお、種々の大きさやベッドボリュームのヒアルロニダーゼ固定化カラムを作製して同様の実験を行っても、結果は同じであった。
(Evaluation of product purity)
The purity of the sugar chain-deficient proteoglycan obtained in Example 1 and that of the sugar chain-modified proteoglycan obtained in Example 2 were compared with the sugar chain-deficient proteoglycan and sugar chain-modified proteoglycan obtained by the conventional method (reaction system carried out in vitro). FIG. 2 shows the result of comparison with the purity of (CBB-stained SDS-PAGE: both in the reaction solution state). Each lane is as follows.
Lane 1: Proteoglycan
Lane 2: sugar chain-deficient proteoglycan obtained in Example 1
Lane 3: sugar chain-modified proteoglycan obtained in Example 2
Lane 4: Sugar chain-deficient proteoglycan obtained by the conventional method
Lane 5: Sugar chain-modified proteoglycan obtained by the conventional method
Lane 6: Hyaluronidase As is apparent from FIG. 2, in the lane 2 and lane 3 according to the method of the present invention, in addition to the target product band (arrow 1), a band derived from the starting material (low molecular weight indicated by arrow 2). UTI, etc.) is recognized, but the hyaluronidase band observed in lanes 4 and 5 by the conventional method is not recognized, and the target product can be prepared with higher purity than the conventional method according to the method of the present invention. I understood. In addition, even if the hyaluronidase fixed column of various sizes and bed volumes was prepared and the same experiment was performed, the result was the same.
本発明は、酵素学的手法による糖鎖改変方法において、反応に用いたグリコシダーゼの混入がない生成物を得るための方法を提供することができる点において産業上の利用可能性を有する。 INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide a method for obtaining a product free from the contamination of glycosidase used in a reaction in a sugar chain modification method by an enzymatic method.
Claims (2)
The method according to claim 1, wherein the glycosidase is hyaluronidase.
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| JP2011252115A (en) * | 2010-06-03 | 2011-12-15 | Hirosaki Univ | Manufacturing method of hybrid sugar chain |
| JP2016079402A (en) * | 2014-10-16 | 2016-05-16 | 地方独立行政法人青森県産業技術センター | Decomposition method of proteoglycan, and decomposition product |
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| JP2016079402A (en) * | 2014-10-16 | 2016-05-16 | 地方独立行政法人青森県産業技術センター | Decomposition method of proteoglycan, and decomposition product |
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