JP4192239B2 - Crystal of budding yeast apyrase and method for producing the crystal - Google Patents
Crystal of budding yeast apyrase and method for producing the crystal Download PDFInfo
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- JP4192239B2 JP4192239B2 JP2003397710A JP2003397710A JP4192239B2 JP 4192239 B2 JP4192239 B2 JP 4192239B2 JP 2003397710 A JP2003397710 A JP 2003397710A JP 2003397710 A JP2003397710 A JP 2003397710A JP 4192239 B2 JP4192239 B2 JP 4192239B2
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- sugar
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Description
本発明は、糖タンパク質の糖鎖付加機構において副産物であるヌクレオチド二リン酸を代謝する、ヌクレオチド二リン酸フォスファターゼの結晶、および該結晶の製造方法に関する。 The present invention relates to a crystal of nucleotide diphosphate phosphatase that metabolizes nucleotide diphosphate which is a by-product in the glycosylation mechanism of glycoprotein, and a method for producing the crystal.
真核生物由来のタンパク質では、アミノ酸のみから成る単純タンパク質ではなく、糖鎖によって修飾された糖タンパク質が頻繁に見い出される。これら糖鎖は、タンパク質の安定性や立体構造の維持に関与したり、細胞間接着などにおける分子間の認識で重要な役割を果たすことが知られている。糖タンパク質の主要な糖鎖としては、アスパラギンに結合するN-linked型とセリン（Ser）あるいはスレオニン（Thr）に結合するO-linked型が挙げられる（非特許文献１）。N-linked型では全てN-アセチルグルコサミン（GlcNAc）がアスパラギンのアミド基に結合している。 In proteins derived from eukaryotes, glycoproteins modified with sugar chains are frequently found, not simple proteins consisting only of amino acids. These sugar chains are known to play an important role in the recognition of molecules between molecules such as intercellular adhesion in the stability of proteins and maintenance of three-dimensional structures. The main sugar chains of glycoprotein include N- linked type that binds to asparagine and O- linked type that binds to serine (Ser) or threonine (Thr) (Non-patent Document 1). In all N- linked types, N -acetylglucosamine (GlcNAc) is linked to the amide group of asparagine.
Asn結合型糖鎖の生合成は、N-アセチルグルコサミン、マンノース、およびグルコースからなる前駆体が脂質キャリアー中間体の上に合成され、まず小胞体（ER）で糖タンパク質の特定の配列（Asn-X-SerまたはThr）に転移される。次にプロセシング（グルコース残基と特定のマンノース残基の切断）を受け、マンノース８残基とN-アセチルグルコサミン２残基からなるM8ハイマンノース型糖鎖（Man8GlcNAc2）が合成される。このハイマンノース型糖鎖を含有するタンパク質はゴルジ体に輸送されて、種々の修飾を受けるが、このゴルジ体での修飾は酵母と哺乳類で大きく異なっている（非特許文献２）。 The biosynthesis of Asn-linked glycans involves the synthesis of a precursor consisting of N-acetylglucosamine, mannose, and glucose on a lipid carrier intermediate, followed by a specific sequence of glycoprotein (Asn- X-Ser or Thr). Next, processing (cleavage of a glucose residue and a specific mannose residue) is performed to synthesize an M8 high mannose type sugar chain (Man 8 GlcNAc 2 ) composed of 8 mannose residues and 2 N -acetylglucosamine residues. The protein containing this high mannose-type sugar chain is transported to the Golgi apparatus and undergoes various modifications. The modification in this Golgi apparatus is greatly different between yeast and mammals (Non-patent Document 2).
哺乳類細胞では、糖鎖修飾を受ける蛋白質の種類によって異なる以下の３種の経路をたどる。1)上記のコア糖鎖が何等変化を受けない場合、2)UDP-N-アセチルグルコサミン（UDP-GlcNAc）のN-アセチルグルコサミン-１-リン酸部分（GlcNAc-1-P）がコア糖鎖のManの６位に付加してMan-6-P-1-GlcNAcとなったのち、このGlcNAc部分だけが除去されて、酸性糖鎖をもつ糖蛋白質に変換される場合、3)コア糖鎖から５分子のManが順次除去されてMan3GlcNAc2となり、これと相前後してGlcNAc、ガラクトース（Gal）、N-アセチルノイラミン酸、別名シアル酸（NeuNAc）などが順次付加して、多様な混成型および複合型糖鎖が混合物として生成する場合の３通りである（非特許文献３）。哺乳類型糖鎖と一口に言っても構造は多岐にわたりそれらの構造が糖タンパク質の機能に大きく関わっていることがわかっている。 Mammalian cells follow the following three pathways that vary depending on the type of protein undergoing glycosylation. 1) When the above core sugar chain is not changed at all, 2) N-acetylglucosamine-1-phosphate part (GlcNAc-1-P) of UDP- N -acetylglucosamine (UDP-GlcNAc) is the core sugar chain When it is added to the 6-position of Man to form Man-6-P-1-GlcNAc, and only this GlcNAc part is removed and converted to a glycoprotein with an acidic sugar chain, 3) Core sugar chain From this, 5 molecules of Man are sequentially removed to become Man3GlcNAc2, and GlcNAc, galactose (Gal), N -acetylneuraminic acid, aka sialic acid (NeuNAc), etc. are added in succession to form a variety of hybrids. There are three cases where complex sugar chains are produced as a mixture (Non-patent Document 3). It is known that mammalian sugar chains have a wide variety of structures, and these structures are greatly related to the functions of glycoproteins.
一方、酵母では、上記のコア糖鎖(Man8GlcNAc2)にマンノースが数残基から100残基以上付加した、マンナン型糖鎖、いわゆる糖外鎖(outer chain)を生成する。多くの場合に、糖外鎖は不均質なタンパク質産物を生成し、タンパク質の精製を困難にしたり、比活性を低下させたりする（非特許文献４）。さらに糖鎖の構造が大きく異なるため、酵母で生産された糖タンパク質は、哺乳類由来のものと同一の生物活性が検出されなかったり、哺乳類動物などに対して強い免疫原性を有することが示されている。 On the other hand, yeast produces a mannan-type sugar chain, a so-called outer chain, in which mannose is added to several to 100 residues of the core sugar chain (Man 8 GlcNAc 2 ). In many cases, the sugar outer chain generates a heterogeneous protein product, which makes it difficult to purify the protein or lowers the specific activity (Non-patent Document 4). Furthermore, because the structure of the sugar chain is greatly different, glycoproteins produced in yeast are not detected to have the same biological activity as those derived from mammals, and are shown to have strong immunogenicity to mammals and the like. ing.
O-linked型ではSerあるいはThrの水酸基に結合する糖が異なる数種類が存在する。その中で、マンノース（Man）が結合した一連の糖鎖はO-linked Man 型として分類される。この他、N-アセチルガラクトサミン（GalNAc）が結合するムチン型、N-アセチルグルコサミン（GlcNAc）が結合するO-linked GlcNAc 型も広く知られている。これらのO-linked型糖鎖はアミノ酸に結合している糖が異なるだけでなく、糖鎖構造全体も大きく違っている（非特許文献５）。O-linked Man 型糖鎖は酵母やカビなどの糖タンパク質でしばしば認められる。酵母の場合、構成糖のほとんどはマンノースであるが、リン酸やガラクトース（Gal）が少数結合している場合が知られている。糖鎖構造は不均一で、酵母の種類によっても異なるが、タンパク質にマンノースが直鎖状に１〜３残基結合し、さらにマンノース、ガラクトース、あるいはマンノースリン酸が転移した７糖以下の構造が知られている（非特許文献６）。 In the O- linked type, there are several types of sugars that bind to the hydroxyl group of Ser or Thr. Among them, a series of sugar chains to which mannose (Man) is bonded is classified as an O- linked Man type. In addition, N - acetylgalactosamine (GalNAc) mucin type of binding, N - acetylglucosamine (GlcNAc) have been widely known O -linked GlcNAc type of binding. These O- linked sugar chains differ not only in the sugars linked to amino acids but also in the overall sugar chain structure (Non-Patent Document 5). O- linked Man-type sugar chains are often found in glycoproteins such as yeast and mold. In the case of yeast, most of the constituent sugars are mannose, but it is known that a small number of phosphates and galactose (Gal) are bound. Although the sugar chain structure is heterogeneous and varies depending on the type of yeast, the structure has 7 or fewer sugars in which mannose is linearly bonded to 1 to 3 residues and mannose, galactose, or mannose phosphate is transferred. It is known (Non-Patent Document 6).
また近年では哺乳類の脳や神経特異的にO-linked Man 型を持つタンパク質の存在が示されており（非特許文献７、８）、O-linked Man 型糖鎖の合成に関わる糖転移酵素の変異が先天性の筋ジストロフィーに眼奇形、神経細胞移動障害を伴う常染色体劣勢遺伝病の一種であるmuscle-eye-brain病の原因であることが明らかとなった（非特許文献９）。 Recently, the existence of proteins with O- linked Man type specific to the mammalian brain and nerves has been shown (Non-patent Documents 7 and 8), and glycosyltransferases involved in the synthesis of O- linked Man type sugar chains have been shown. It has been clarified that the mutation is a cause of muscle-eye-brain disease, which is a kind of autosomal recessive genetic disease accompanied by congenital muscular dystrophy with eye malformation and nerve cell migration disorder (Non-patent Document 9).
糖鎖の伸長はアクセプターとなる糖鎖の非還元末端に単糖が付加されて起こる。この時、付加反応に利用されるのは、単糖そのものではなく、ヌクレオチドの結合により活性化された糖ヌクレオチドと呼ばれるドナーである。糖の付加反応の多くは細胞内の小胞体内腔やゴルジ体で行なわれるのに対し、多くの糖ヌクレオチドは細胞質で（CMP-シアル酸のみ核で）合成される。しかし合成された糖ヌクレオチドは膜を透過できないため、この糖供与体を糖鎖合成の場である細胞内小器官の内腔に運ぶ必要がある。この輸送に関与するのが糖ヌクレオチド輸送体である。糖ヌクレオチドには種々の分子種が存在するが、それぞれの糖ヌクレオチドに対し特異的な糖ヌクレオチド輸送体が存在すると考えられている（非特許文献１０、１１）。 The elongation of the sugar chain occurs when a monosaccharide is added to the non-reducing end of the sugar chain that serves as an acceptor. At this time, what is used for the addition reaction is not a monosaccharide itself but a donor called a sugar nucleotide activated by the binding of nucleotides. Many sugar addition reactions take place in the endoplasmic reticulum lumen and Golgi apparatus, whereas many sugar nucleotides are synthesized in the cytoplasm (CMP-sialic acid only in the nucleus). However, since the synthesized sugar nucleotides cannot penetrate the membrane, it is necessary to transport this sugar donor to the lumen of the organelle, which is the place for sugar chain synthesis. The sugar nucleotide transporter is involved in this transport. There are various molecular species of sugar nucleotides, and it is considered that there is a sugar nucleotide transporter specific for each sugar nucleotide (Non-patent Documents 10 and 11).
糖ヌクレオチド輸送体は、細胞質内にプールされた糖ヌクレオチドを、ヌクレオチド一リン酸（UDP糖はUMP、GDP糖はGMPなど）との対向輸送によって小胞体やゴルジ体内腔に取り入れていると考えられている（非特許文献１２）。対向輸送で細胞質側へ輸送されるモノヌクレオチドは、図1のように生成されると考えられている。まず小胞体やゴルジ体内腔に輸送された糖ヌクレオチドは糖転移反応の糖供与体基質として利用され、ヌクレオチド二リン酸が生成する。このヌクレオチド二リン酸は内腔側に存在するヌクレオチド二リン酸ホスファターゼによって脱リン酸化がおき、ヌクレオチド一リン酸が生成される。このヌクレオチド一リン酸は対向輸送により細胞質側に吐き出され、同時に新たな糖供与体が供給される。このように糖鎖の安定な合成には、糖転移酵素だけでなく、糖受容体、糖供与体となる糖ヌクレオチドの供給に関わる糖ヌクレオチド輸送体、さらに副産物のヌクレオチド二リン酸を代謝するためのヌクレオチド二リン酸ホスファターゼが不可欠であり、これらが一体となって糖鎖合成と糖ヌクレオチド代謝回路を形成している。これらのいずれかの欠損は生体内での糖鎖合成に影響を与え、さらには様々な病態を示すことが示されている。例えば、GDP-フコース輸送体をコードする遺伝子の変異によりおこる、先天性グリコシル化異常症・II型白血球接着不全症（LADII/GDCIIc）が知られており、その解析の結果GDP-フコース輸送体の細胞性免疫に対する関与が示唆されている（非特許文献１３）。 The sugar nucleotide transporter is thought to incorporate sugar nucleotides pooled in the cytoplasm into the endoplasmic reticulum or Golgi body cavity by counter transport with nucleotide monophosphates (such as UMP for UDP sugars and GMP for GDP sugars). (Non-Patent Document 12). Mononucleotides that are transported to the cytoplasmic side by counter transport are thought to be generated as shown in FIG. First, sugar nucleotides transported to the endoplasmic reticulum or Golgi lumen are used as a sugar donor substrate for transglycosylation reaction, and nucleotide diphosphate is generated. This nucleotide diphosphate is dephosphorylated by nucleotide diphosphate phosphatase present on the lumen side, and nucleotide monophosphate is generated. This nucleotide monophosphate is exhaled to the cytoplasm by counter transport, and at the same time, a new sugar donor is supplied. In this way, stable synthesis of sugar chains involves metabolizing not only glycosyltransferases, but also sugar acceptors, sugar nucleotide transporters involved in the supply of sugar nucleotides as sugar donors, and by-product nucleotide diphosphates. Nucleotide diphosphate phosphatases are essential, and they together form a sugar chain synthesis and sugar nucleotide metabolism circuit. It has been shown that any of these defects affects in vivo sugar chain synthesis and further exhibits various pathological conditions. For example, congenital glycosylation / type II leukocyte adhesion deficiency (LADII / GDCIIc) caused by mutations in the gene encoding the GDP-fucose transporter is known. Involvement for cellular immunity has been suggested (Non-patent Document 13).
ヌクレオチド二リン酸ホスファターゼはゴルジ体内腔に触媒部位が配向する膜タンパク質であり、不要となったヌクレオチド二リン酸を脱リン酸化し、ヌクレオチド一リン酸に変換する酵素である。出芽酵母ではこの酵素をコードする遺伝子が２つ見つかっている。１つはGDA1であり、もう一つはYND1である。Gda1タンパク質は518アミノ酸からなり、グアノシン二リン酸（GDP）に比較的高い基質特異性を示すことが示されている（非特許文献１４）。またこの遺伝子の欠損株では、ゴルジ体内腔にGDPが蓄積し、また細胞質側からのGDP-マンノースの取り込みが下がることでマンナン合成量が下がることが分かっている（非特許文献１５）。一方Ynd1タンパク質は630アミノ酸から成るタンパク質で、アミノ末端側500アミノ酸がゴルジ体内腔側、カルボキシル末端側113アミノ酸が細胞質側に配向する膜タンパク質である。Gda1タンパク質と異なり、GDPのみならず他のヌクレオチド二リン酸も分解できる。さらにアデノシン三リン酸などのヌクレオチド三リン酸も分解することができるATPase活性を持つため、別名アピラーゼとも呼ばれる。この遺伝子の欠損株もgda1欠損株と同様、糖鎖合成に影響を与えるが、その作用は若干異なっていると考えられている。例えばポリアクリルアミノゲル電気泳動におけるキチナーゼの移動度を比較すると、ynd1破壊株のキチナーゼの移動度は野生型酵母由来キチナーゼより早く、gda1破壊株由来のキチナーゼより遅いことから、Ynd1タンパク質は糖鎖の延長に重要な機能を果たしているかもしれない。さらに、これらの遺伝子の二重破壊株は、酵母の出芽や細胞壁の強さに大きく影響することが示されている。（非特許文献１４） Nucleotide diphosphate phosphatase is a membrane protein whose catalytic site is oriented in the Golgi body cavity, and is an enzyme that dephosphorylates nucleotide diphosphate that is no longer needed and converts it into nucleotide monophosphate. Two genes encoding this enzyme have been found in Saccharomyces cerevisiae. One is GDA1 and the other is YND1 . Gda1 protein consists of 518 amino acids and has been shown to exhibit relatively high substrate specificity for guanosine diphosphate (GDP) (Non-patent Document 14). In addition, it has been found that in this gene-deficient strain, the amount of mannan synthesis decreases due to the accumulation of GDP in the Golgi body cavity and the decrease in the uptake of GDP-mannose from the cytoplasm side (Non-patent Document 15). On the other hand, the Ynd1 protein is a protein composed of 630 amino acids, and is a membrane protein in which 500 amino acids on the amino terminal side are oriented on the Golgi lumen and 113 amino acids on the carboxyl terminal side are oriented on the cytoplasm side. Unlike Gda1 protein, it can degrade not only GDP but also other nucleotide diphosphates. In addition, it has an ATPase activity that can degrade nucleotide triphosphates such as adenosine triphosphate, so it is also called apyrase. The strain lacking this gene, like the gda1-deficient strain, affects sugar chain synthesis, but its action is considered to be slightly different. For example, comparing the mobility of chitinase in polyacrylaminogel electrophoresis, the mobility of the chindase of the ynd1 disruption strain is faster than that of the chitinase derived from the wild-type yeast and slower than that of the chitinase derived from the gda1 disruption strain. It may play an important function in extension. Furthermore, double disruption strains of these genes have been shown to greatly affect yeast budding and cell wall strength. (Non-Patent Document 14)
これらの相同遺伝子は他の酵母のみならず、カビから哺乳類まで幅広く保存されている。ヒトではＣＤ39（ENTPD1）として知られるectoapyrase familyやlysosomal apyrase-like 1 (LYSAL1) familyなどが知られており、少なくとも８つの相同遺伝子が見つかっている。 These homologous genes are conserved not only from other yeasts but also from fungi to mammals. In humans, the ectoapyrase family known as CD39 (ENTPD1) and the lysosomal apyrase-like 1 (LYSAL1) family are known, and at least 8 homologous genes have been found.
YND1とGDA1とはアミノ酸レベルで27％の相同性があるが、その基質特異性は大きく異なっており、触媒領域の高次構造が異なっていることが予想される。また、ヌクレオチド二リン酸のみならずヌクレオチド三リン酸をも分解するメカニズムの解明には高次構造の情報が重要である。したがってYnd1タンパク質の高次構造を解明することで、これらの酵素の基質特異性を説明できるようになると考えられる。 Although YND1 and GDA1 have 27% homology at the amino acid level, their substrate specificities are greatly different, and it is expected that the higher-order structure of the catalytic region is different. In addition, higher-order structure information is important for elucidating the mechanism of degrading not only nucleotide diphosphates but also nucleotide triphosphates. Therefore, elucidating the higher-order structure of the Ynd1 protein may explain the substrate specificity of these enzymes.
X線解析によりタンパク質の高次構造を解明するためには、Ynd1タンパク質を結晶化する必要がある。しかしながら、これまでYnd1タンパク質が結晶化されたという報告例はない。
本発明は、このような状況に鑑みてなされたものであり、その目的は糖タンパク質の糖鎖付加機構において副産物であるヌクレオチド二リン酸を代謝する、Ynd1タンパク質の結晶の提供にある。また、本発明は、該結晶の製造方法の提供をも目的とする。 The present invention has been made in view of such circumstances, and an object thereof is to provide a crystal of Ynd1 protein that metabolizes nucleotide diphosphate which is a byproduct in the glycosylation mechanism of glycoprotein. Another object of the present invention is to provide a method for producing the crystal.
本発明者は、上記の課題を解決すべく、Ynd1タンパク質の生産および結晶化を試みた。その結果、タンパク質生産における適切な宿主ベクター系の選択および膨大な数の結晶化条件の試行錯誤により、遂に、Ynd1タンパク質の結晶化に成功するに至った。 The present inventor tried to produce and crystallize the Ynd1 protein in order to solve the above problems. As a result, Ynd1 protein was finally successfully crystallized by selecting an appropriate host vector system for protein production and trial and error of a large number of crystallization conditions.
即ち、本発明は、Ynd1タンパク質の結晶およびその製造方法に関し、より具体的には、
〔１〕 Ynd1タンパク質の結晶であって、斜方晶に属し、空間群212121である結晶
〔２〕 Ynd1タンパク質の結晶であって、格子定数がa=93.0,b=103.6,c=145.6(Å)である結晶
〔３〕 Ynd1タンパク質の結晶であって、0.1mm角以上の大きさを有する結晶
〔４〕 基質との複合体である、〔１〕から〔３〕に記載の結晶
〔５〕 基質がヌクレオチド-1リン酸である、〔４〕に記載の結晶
〔６〕 Ynd1タンパク質の結晶の製造方法であって、ハンギングドロップ法において、7-12% PEG、10-20 %エチレングリコールを用いる方法
〔７〕 結晶がYnd1タンパク質とその基質との複合体である、〔６〕に記載の方法
〔８〕 基質がヌクレオチド-1リン酸である、〔７〕に記載の方法
を提供するものである。
That is, the present invention relates to a crystal of Ynd1 protein and a production method thereof, more specifically,
[1] Ynd1 protein crystal belonging to orthorhombic crystal and space group 2 1 2 1 2 1 [2] Ynd1 protein crystal having lattice constants of a = 93.0, b = 103.6, Crystals of c = 145.6 (タ ン パ ク 質) [3] Ynd1 protein crystals having a size of 0.1 mm square or more [4] A complex with a substrate, [1] to [3] [5] The method for producing a crystal of [6] Ynd1 protein according to [4], wherein the substrate is nucleotide-1 phosphate, wherein 7-12% PEG, 10-20 [7] The method according to [6], wherein the crystal is a complex of Ynd1 protein and its substrate [8] The method according to [7], wherein the substrate is nucleotide-1 phosphate Is to provide.
本発明のYnd1タンパク質の結晶を用い、高次構造を解明することで、該タンパク質の触媒領域の構造が明らかとなり、該タンパク質基質特異性を説明できるようになる。また、これまで哺乳類から原核生物を含めてATPaseの高次構造は解明されておらず、結晶を利用することで、ヌクレオチド二リン酸のみならずヌクレオチド三リン酸をも分解するメカニズムを明らかにすることができる。 By elucidating the higher-order structure using the Ynd1 protein crystal of the present invention, the structure of the catalytic region of the protein is clarified, and the protein substrate specificity can be explained. In addition, the higher-order structure of ATPase from mammals to prokaryotes has not been elucidated so far, and the mechanism of degrading not only nucleotide diphosphate but also nucleotide triphosphate is clarified by using crystals. be able to.
また、酵素の欠損が糖鎖合成や、ひいては細胞の増殖に影響を与えるため、Ynd1タンパク質に特異的な阻害剤が見つかれば、抗真菌剤としての応用も考えられる。酵素を大量に生産し、その阻害剤をスクリーニングすることも可能であるが、高次構造の情報からバーチャルスクリーニングにより化合物を同定することも可能となる。 In addition, since enzyme deficiency affects sugar chain synthesis and thus cell growth, if an inhibitor specific to the Ynd1 protein is found, its application as an antifungal agent is also conceivable. Enzymes can be produced in large quantities and their inhibitors can be screened, but it is also possible to identify compounds by virtual screening from higher order structure information.
さらに、Ynd1タンパク質は、ヌクレオチド二リン酸やヌクレオチド三リン酸を分解してリン酸を生成するため、リン酸量を指標とした試料中の核酸の定量においても有用である。また、Ynd1タンパク質の結晶化は、Ynd1タンパク質の安定性や保存性を高める上でも有用である。 Furthermore, since the Ynd1 protein decomposes nucleotide diphosphates and nucleotide triphosphates to generate phosphate, it is also useful for quantification of nucleic acids in samples using the amount of phosphate as an indicator. In addition, crystallization of Ynd1 protein is also useful for improving the stability and storage stability of Ynd1 protein.
本発明は、Ynd1タンパク質の結晶を提供する。本発明における「Ynd1タンパク質」は、完全なタンパク質でもよく、また、ヌクレオチド二リン酸ホスファターゼ活性（あるいはATPアーゼ活性）を有する部分ペプチドであってもよい。また、該活性を有する限り、1若しくは複数（例えば、30,20,10,5,または3アミノ酸以内）のアミノ酸が、付加、欠失、置換などされた変異体であってもよい。典型的なYnd1タンパク質のアミノ酸配列を配列番号：２に、該タンパク質をコードするDNAの塩基配列を配列番号：１に示す。 The present invention provides a crystal of Ynd1 protein. The “Ynd1 protein” in the present invention may be a complete protein or a partial peptide having nucleotide diphosphate phosphatase activity (or ATPase activity). Moreover, as long as it has this activity, it may be a mutant in which one or a plurality of amino acids (for example, within 30, 20, 10, 5, or 3 amino acids) are added, deleted or substituted. The amino acid sequence of a typical Ynd1 protein is shown in SEQ ID NO: 2, and the base sequence of the DNA encoding the protein is shown in SEQ ID NO: 1.
本発明の結晶の一つの好ましい態様は、斜方晶に属し、空間群212121の結晶である。「空間群」とは、結晶の対象要素の配置をいう。 One preferred embodiment of the crystal of the present invention is a crystal belonging to orthorhombic crystal and having space group 2 1 2 1 2 1 . “Space group” refers to the arrangement of target elements of a crystal.
他の好ましい態様は、格子定数がa=93.0,b=103.6,c=145.6(Å)の結晶である。「格子定数」とは、単位格子の形と大きさを表現している数字の組み合わせをいい、「単位格子」とは、基本的な平行六面体のブロックをいう。 Another preferred embodiment is a crystal having lattice constants of a = 93.0, b = 103.6, and c = 145.6 (Å). “Lattice constant” refers to a combination of numbers expressing the shape and size of a unit cell, and “unit cell” refers to a basic parallelepiped block.
他の好ましい態様は、0.1mm角以上（好ましくは、0.2,0.3,0.4または0.5mm角以上）の大きさを有する結晶である。結晶は、例えば、四方両錘形でありうる。 Another preferred embodiment is a crystal having a size of 0.1 mm square or more (preferably 0.2, 0.3, 0.4 or 0.5 mm square or more). The crystal can be, for example, a quadrilateral bipyramidal.
本発明の結晶は、基質との複合体として調製することができる。本発明において「基質」とは、タンパク質（酵素）の基質結合部位に結合しうる化合物を指し、天然由来の化合物であっても、人工的な化合物であってもよい。また、種々の薬剤候補化合物であってもよい。好ましい基質は、ヌクレオチド-1リン酸、ヌクレオチド二リン酸、あるいはヌクレオチド三リン酸である。 The crystal of the present invention can be prepared as a complex with a substrate. In the present invention, the “substrate” refers to a compound that can bind to a substrate binding site of a protein (enzyme), and may be a naturally occurring compound or an artificial compound. Various drug candidate compounds may also be used. Preferred substrates are nucleotide-1 phosphate, nucleotide diphosphate, or nucleotide triphosphate.
本発明の結晶を得るためには、まず、精製されたYnd1タンパク質を取得する必要がある。精製されたYnd1タンパク質は、Ynd1タンパク質をコードするポリヌクレオチドを含むベクターを宿主に導入し、宿主において発現したYnd1タンパク質を回収することにより調製することができる。 In order to obtain the crystal of the present invention, it is first necessary to obtain a purified Ynd1 protein. The purified Ynd1 protein can be prepared by introducing a vector containing a polynucleotide encoding the Ynd1 protein into a host and recovering the Ynd1 protein expressed in the host.
標的遺伝子のDNA遺伝子断片の単離は、Saccharomyces cerevisiaeのゲノムプロジェクトにより、その染色体上の配座が既知である（Goffeau et al., Nature, 387 (suppl.), 1-105 (1997)） ことから、米国ATCC（American Type Culture Collection）など公的な機関から標的遺伝子の近傍を含む遺伝子断片の分与を受けることが可能である（ATCC Recombinant DNA materials, 3rd edition, 1993）。また、一般的手法にしたがってS. cerevisiaeからゲノムDNAを抽出し、目的遺伝子を選別することにより、取得することが可能である。S.cerevisiaeからゲノムDNAの抽出は、例えば、Cryer らの方法（Methods in Cell Biology, 12, 39-44 (1975)）およびP. Philippsenらの方法（Methods Enzymol., 194, 169-182 (1991) ）に従って行なうことができる。 Isolation of the DNA gene fragment of the target gene is known by Saccharomyces cerevisiae genome project (Goffeau et al., Nature, 387 (suppl.), 1-105 (1997)) Can receive gene fragments including the vicinity of the target gene from public institutions such as the American Type Culture Collection (ATCC) (ATCC Recombinant DNA materials, 3rd edition, 1993). It can also be obtained by extracting genomic DNA from S. cerevisiae and selecting the target gene according to a general method. Extraction of genomic DNA from S. cerevisiae can be carried out, for example, by the method of Cryer et al. (Methods in Cell Biology, 12, 39-44 (1975)) and the method of P. Philippsen et al. (Methods Enzymol., 194, 169-182 (1991). )).
標的遺伝子は、PCR法により増幅することができる。PCR法は、インビトロ（in vitro）でDNAの特定断片をその領域の両端のセンス・アンチセンスプライマー、耐熱性DNAポリメラーゼ、DNA増幅システム等の組み合わせを用いて約２-３時間で数十万倍以上に増幅できる技術であるが、標的遺伝子の増幅には、プライマーとして25-30merの合成1本鎖DNAを、鋳型としてゲノムDNAを用いる。 The target gene can be amplified by PCR. The PCR method is several hundred thousand times in a few hours using a combination of a specific fragment of DNA in vitro (sense / antisense primer, heat-resistant DNA polymerase, DNA amplification system) at both ends of the region. This technique can be amplified as described above. For amplification of a target gene, 25-30mer synthetic single-stranded DNA is used as a primer, and genomic DNA is used as a template.
上記操作における、DNAの細胞への導入およびこれによる形質転換の方法としては、一般的な方法、例えば、ベクターとしてファージを用いる場合は、大腸菌宿主にこれを感染させる方法等により、効率よく宿主にDNAを取り込ませることができる。またプラスミドを用いて酵母を形質転換する方法としては、リチウム塩で処理して自然にDNAを取込みやすい状態にしてプラスミドを取り込ませる方法や、あるいは電気的にDNAを細胞内に導入する方法を採用できる（Becker and Guarente, Methods Enzymol., 194, 182-187 (1991)）。 As a method for introducing DNA into a cell and transforming it in the above operation, a general method, for example, when a phage is used as a vector, a method of infecting an E. coli host with this, etc., can be efficiently introduced into the host. DNA can be incorporated. In addition, as a method of transforming yeast using a plasmid, a method in which the plasmid is incorporated by treating it with a lithium salt to make it easy to incorporate DNA, or a method of electrically introducing DNA into cells is adopted. (Becker and Guarente, Methods Enzymol., 194, 182-187 (1991)).
また、上記操作における、DNAの単離・精製等は何れも常法、例えば大腸菌の場合、アルカリ/SDS法とエタノール沈殿によるDNA抽出、さらにRNase処理、PEG沈殿法などによりDNAを精製できる。また、遺伝子のDNA配列の決定等も通常の方法、例えばジデオキシ法（Sanger et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)）等により行なうことができる。さらに、上記DNA塩基配列の決定は、市販のシークエンスキット等を用いることによっても容易に行ない得る。 In the above operation, DNA can be purified by conventional methods, for example, in the case of Escherichia coli, DNA extraction by alkali / SDS method and ethanol precipitation, RNase treatment, PEG precipitation method, etc. The determination of the DNA sequence of a gene and the like can also be performed by a conventional method such as the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)). Furthermore, the determination of the DNA base sequence can be easily performed by using a commercially available sequence kit or the like.
また、Ynd1タンパク質は本来膜貫通タンパク質であるので、C末端側の膜貫通領域を欠失させ、N末端側に分泌シグナルを付加したタンパク質を発現させることによって、細胞外に可溶化型酵素として製造させることが可能である。組換え体ポリヌクレオチドを導入する宿主細胞としては、原核細胞、真核細胞（動物細胞、酵母細胞、カビ細胞、昆虫細胞など）のように、遺伝子組換え技術で用いられる細胞を用いることができる。例えば、原核細胞としては大腸菌、真核細胞のうち酵母細胞としては、出芽酵母のほか、異種タンパク質発現に適しているメタノール資化性酵母があげられる。メタノール資化性酵母はPichia pastoris, Hansenula polymorpha, Yarrowia lipolitica, Candida boidinii, Ogatae minuta等が知られており、いずれも利用可能である。Pichia pastorisについては、発現用ベクターが市販品として入手可能であり、公知の方法によって形質転換を行なうことができる。 Since Ynd1 protein is originally a transmembrane protein, it is produced as a solubilized enzyme outside the cell by expressing a protein with a secretory signal added to the N-terminal side by deleting the transmembrane region on the C-terminal side. It is possible to make it. As a host cell into which a recombinant polynucleotide is introduced, cells used in gene recombination techniques such as prokaryotic cells and eukaryotic cells (animal cells, yeast cells, mold cells, insect cells, etc.) can be used. . For example, Escherichia coli as the prokaryotic cell, and yeast cells among the eukaryotic cells include budding yeast and methanol-utilizing yeast suitable for heterologous protein expression. Methanol-assimilating yeasts are known, such as Pichia pastoris , Hansenula polymorpha , Yarrowia lipolitica , Candida boidinii , Ogatae minuta, and the like. Regarding Pichia pastoris , an expression vector is commercially available, and can be transformed by a known method.
発現用ベクター中、目的遺伝子の発現に利用するプロモーターとしては、恒常的に発現が誘導される解糖系の遺伝子、例えばグリセルアルデヒド-3-リン酸デヒドロゲナーゼのプロモーターが利用できる。さらにはメタノール資化性酵母の場合、メタノールによって誘導されるメタノール代謝系遺伝子、例えばアルコールオキシダーゼ（AOX）などのプロモーターが利用可能である。 In the expression vector, as a promoter used for expression of the target gene, a glycolytic gene whose expression is constantly induced, for example, a promoter of glyceraldehyde-3-phosphate dehydrogenase can be used. Further, in the case of methanol-assimilating yeast, a methanol metabolic gene induced by methanol, for example, a promoter such as alcohol oxidase (AOX) can be used.
効率的にYnd1タンパク質を生産するためには、図２に示すようなプラスミドを構築し、適した制限酵素で直線化した後、ゲノム上に導入することでPichia pastorisを形質転換することが好ましい。 In order to efficiently produce the Ynd1 protein, it is preferable to transform a Pichia pastoris by constructing a plasmid as shown in FIG. 2, linearizing it with a suitable restriction enzyme, and introducing it into the genome.
上記の形質転換は、それぞれの宿主について一般的に行われている方法で行う。例えば、宿主が大腸菌であれば、塩化カルシウム法その他の方法により作製したコンピテント細胞に組換えポリヌクレオチドを含むベクタ−を温度ショック法あるいはエレクトロポレ−ション法により導入することが可能である(Sambrook, J, Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), 1.74-1.85)。宿主が酵母細胞であれば、塩化リチウム法その他の方法により作製したコンピテント細胞に組換えポリヌクレオチドを含むベクタ−を温度ショック法あるいはエレクトロポレ−ション法により導入することが可能である(Becker, D.M. and Guarente, L., Methods Enzymol., (1991), 194, 182-187)。宿主が動物細胞であれば、増殖期等の細胞に組換え体ポリヌクレオチドを含むベクタ−をリン酸カルシウム法、リポフェクション法またはエレクトロポレ−ション法により導入することが可能である(Sambrook, J, Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), 16.30-16.55)。 The transformation is performed by a method generally performed for each host. For example, if the host is Escherichia coli, a vector containing the recombinant polynucleotide can be introduced into competent cells prepared by the calcium chloride method or other methods by the temperature shock method or the electroporation method (Sambrook). , J, Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), 1.74-1.85). If the host is a yeast cell, a vector containing the recombinant polynucleotide can be introduced into competent cells produced by the lithium chloride method or other methods by the temperature shock method or electroporation method (Becker, DM and Guarente, L., Methods Enzymol., (1991), 194, 182-187). If the host is an animal cell, a vector containing the recombinant polynucleotide can be introduced into cells in the growth phase by the calcium phosphate method, lipofection method or electroporation method (Sambrook, J, Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), 16.30-16.55).
このようにして得られた形質転換体を培地に培養することにより、Ynd1p タンパク質を産生させることが可能である。形質転換体を培養する場合、培養に使用される培地としては、それぞれの宿主が生育可能な培地ならば良い。例えば、宿主が大腸菌であればLB培地などを用い、宿主が酵母であればYPD培地などを用いる。宿主がメタノール資化性酵母であればDifco社から供給される各種の培地成分とグリセロールを炭素源とした培地で菌体を増殖した後、メタノールを培地中に添加することで目的タンパク質を生産できる。動物細胞であれば、Dulbecco's MEM に動物血清を加えたものなどを用いる。培養は、それぞれの宿主について一般的に用いられている条件で行う。例えば、宿主が大腸菌ならば約30〜37℃で、約3〜24時間行い、必要により通気や撹拌を加えることができる。宿主が酵母であれば約25〜37℃で、約12時間〜2週間行い、必要により通気や撹拌を加えることができる。宿主が動物細胞であれば約32〜37℃で、5%CO2、100% 湿度の条件で約24時間〜2週間行い、必要により気相の条件を変えたり撹拌を加えることができる。 Ynd1p protein can be produced by culturing the transformant thus obtained in a medium. When culturing the transformant, the medium used for the culture may be a medium in which each host can grow. For example, if the host is Escherichia coli, LB medium is used, and if the host is yeast, YPD medium is used. If the host is a methanol-assimilating yeast, the target protein can be produced by growing the cells in various medium components supplied from Difco and medium using glycerol as a carbon source, and then adding methanol to the medium. . For animal cells, use Dulbecco's MEM supplemented with animal serum. Culturing is performed under conditions generally used for each host. For example, if the host is Escherichia coli, it is carried out at about 30 to 37 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added. If the host is yeast, it is carried out at about 25 to 37 ° C. for about 12 hours to 2 weeks, and if necessary, aeration or agitation can be added. If the host is an animal cell, it is carried out at about 32 to 37 ° C. under conditions of 5% CO 2 and 100% humidity for about 24 hours to 2 weeks. If necessary, the gas phase conditions can be changed or agitation can be added.
上記の培養物（培養液、培養菌体）から糖タンパク質を単離精製するためには、通常のタンパク質の単離、精製法を用いればよい。例えば、菌体内に存在する糖タンパク質については培養終了後、細胞を遠心分離により回収し水系緩衝液に懸濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、ダイノミル等により細胞を破砕し、無細胞抽出液を得、これを遠心分離することにより上清を得る。一方培養上清に存在する糖タンパク質については培養終了後、細胞を遠心分離により分離し、限外ろ過、塩析法等により濃縮する。以降の操作は通常のタンパク質の単離精製法、即ち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（DEAE）セファロース等を用いた陰イオン交換クロマトグラフィー法、スルホプロピル（SP）セファロース（ファルマシア社製）等を用いた陽イオン交換クロマトグラフィー法、フェニルセファロース等を用いた疎水性クロマトグラフィー法、セファクリル等を用いた分子篩を用いたゲルろ過法、群特異性担体、レクチンリガンド、金属キレートなどを用いたアフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の手法を単独あるいは組み合わせて用い、精製標品を得ることができる。 In order to isolate and purify glycoproteins from the above culture (culture medium, cultured cells), ordinary protein isolation and purification methods may be used. For example, for the glycoprotein present in the cells, after culturing, the cells are collected by centrifugation and suspended in an aqueous buffer, and then disrupted by an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. A cell-free extract is obtained and centrifuged to obtain a supernatant. On the other hand, the glycoprotein present in the culture supernatant is separated by centrifugation after completion of the culture, and concentrated by ultrafiltration, salting-out, or the like. Subsequent operations include isolation and purification of ordinary proteins, ie, solvent extraction, salting out using ammonium sulfate, desalting, precipitation using organic solvents, anion exchange chromatography using diethylaminoethyl (DEAE) sepharose, etc. Chromatography method, cation exchange chromatography method using sulfopropyl (SP) Sepharose (Pharmacia), hydrophobic chromatography method using phenyl sepharose, gel filtration method using molecular sieve using Sephacryl, etc. A purified sample can be obtained by using methods such as affinity chromatography using group-specific carriers, lectin ligands, metal chelates, etc., electrophoresis methods such as chromatofocusing methods and isoelectric focusing alone or in combination. it can.
本発明の結晶の調製においては、このようにして得た精製タンパク質標品につき、結晶化条件のスクリーニングを行なう。スクリーニングには、透析法、蒸気拡散法、バッチ法、自由界面拡散法、濃縮法、温度勾配法などが利用できる。蒸気拡散法においてはハンギングドロップ法、シッティングドロップ法、サンドイッチドロップ法などが使用可能である。使われる沈殿剤は一般的な試薬を購入し、適当に濃度調節・混合して調製できる。また市販の沈殿剤のセットを購入することも可能である。しかしながら、X線回折実験に適した単結晶を調製するためには、無限に考えられる結晶化条件の中から好適な条件を選択しなければならない。ハンギングドロップ法において、タンパク質濃度7-15 mg/mlの場合には、沈殿剤として7-12% PEGを用い、エチレングリコール濃度を10-20 %とするのが好適な条件である（本発明者は、タンパク質濃度10mg/ml の場合、PEG濃度7-12 %、エチレングリコール濃度10-20 %の条件を、また、PEG 濃度8%の場合、タンパク質濃度7-15 mg/ml、エチレングリコール濃度10-20%の条件を本発明の結晶を製造するために用いることができることを確認している）。基質との共結晶を得る場合には、基質濃度としては、10mMの条件を適用することができる。本発明の結晶を製造するために最も好ましいのは、実施例に記載の条件である。本発明は、このように本発明の結晶の製造方法をも提供するものである。 In the preparation of the crystal of the present invention, the purified protein preparation thus obtained is screened for crystallization conditions. Dialysis, vapor diffusion, batch, free interface diffusion, concentration, temperature gradient, etc. can be used for screening. In the vapor diffusion method, a hanging drop method, a sitting drop method, a sandwich drop method, or the like can be used. The precipitating agent to be used can be prepared by purchasing a general reagent and adjusting and mixing the concentration appropriately. It is also possible to purchase a set of commercially available precipitants. However, in order to prepare a single crystal suitable for an X-ray diffraction experiment, a suitable condition must be selected from infinitely considered crystallization conditions. In the hanging drop method, when the protein concentration is 7-15 mg / ml, 7-12% PEG is used as the precipitating agent and the ethylene glycol concentration is 10-20% (the present inventors are preferred conditions). When the protein concentration is 10 mg / ml, the PEG concentration is 7-12% and the ethylene glycol concentration is 10-20% .When the PEG concentration is 8%, the protein concentration is 7-15 mg / ml and the ethylene glycol concentration is 10%. -20% condition can be used to produce the crystals of the present invention). When obtaining a co-crystal with a substrate, a substrate concentration of 10 mM can be applied. Most preferred for producing the crystals of the present invention are the conditions described in the examples. Thus, the present invention also provides a method for producing the crystal of the present invention.
得られた結晶の構造解析は、例えば、重原子同型置換法や多波長異常散乱（分散）法により行なうことができる。構造解析によりYnd1タンパク質の三次元構造が明らかとなれば、形状相補性や相互作用エネルギーなどの評価により、Ynd1タンパク質に結合する化合物をコンピュータ上でスクリーニングすることも可能となる。 The structural analysis of the obtained crystal can be performed by, for example, the heavy atom isomorphous substitution method or the multiwavelength anomalous scattering (dispersion) method. If the three-dimensional structure of the Ynd1 protein is clarified by structural analysis, a compound that binds to the Ynd1 protein can be screened on a computer by evaluating shape complementarity and interaction energy.
以下、本発明を実施例により、さらに具体的に説明するが本発明はこれら実施例に制限されるものではない。
(1) 参考例および実施例に用いた試薬類
特に指定の無い試薬類は、シグマ製、和光純薬製あるいはナカライテスク製の最高グレードのものを使用した。また、制限酵素はTAKARA製のものを用いた。結晶のスクリーニングにはハンプトン製の「クリスタルスクリーニングキット」およびJENA BIOSCIENCE 社製のJBScreenキットを用いた。
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
(1) Reagents used in Reference Examples and Examples Reagents not specified were those of the highest grade made by Sigma, Wako Pure Chemical or Nacalai Tesque. Moreover, the restriction enzyme made from TAKARA was used. For crystal screening, a “crystal screening kit” manufactured by Hampton and a JBScreen kit manufactured by JENA BIOSCIENCE were used.
(2) 実施例に用いた機器類
目的のDNA断片を増幅する際には、TAKARA製Tharmal Cycler 650を、また、遺伝子配列決定には、ABI PLISM 310 DNA
Sequencer（Perkin-Elmer社製）を使用した。大量培養には、ジャーファーメンターシステム（東京理化器械社製）を用いた。
(2) Equipment used in Examples When amplifying a target DNA fragment, TAKARA's Thermal Cycler 650 is used. For gene sequencing, ABI PLISM 310 DNA is used.
Sequencer (Perkin-Elmer) was used. A jar fermenter system (manufactured by Tokyo Rika Kikai Co., Ltd.) was used for mass culture.
〔実施例１〕 可溶型Ynd1タンパク質の発現系の構築
出芽酵母YND1 遺伝子配列は、 GenBankデータベースにAF203695で登録されている（Gao, X.-D., Kaigorodov, V. and Jigami, Y. (1999) J. Biol. Chem. 274, 21450-21456）。 酵母W303-1Aより常法に従いゲノムDNAを抽出し、プライマーA(CCCTCGAGAAAAGAATGCTCATAGAAAACACTAATGATC:配列番号：３)とプライマーB（ATTTGCGGCCGCTCAATGATGATGATGATGATGAGACTCCAGTAACTTGCCAGGTATG：配列番号：４）を用いてPCRで増幅を行なった。増幅した遺伝子はYnd1タンパク質の配列番号：５に示す部分アミノ酸配列（Met1-Ser487；）のC末端側にヒスチジンが6残基付加したような融合タンパク質（以下可溶型Ynd1タンパク質と略する）をコードしている。得られたPCR産物をMicrospin S-400カラム（Amersham Bioscience社製）を用いて塩とプライマーを除いた。精製したPCR産物をXhoIとNotIで切断を行ない、同様にXhoIとNotIで切断したpPIC9(Invitrogen社製)にライゲーション反応を行い、PCR産物を挿入した。PCR産物の塩基配列をシークエンサーで確認後、SalIでプラスミドの切断を行ない、直鎖状にした（図２）。これを用いて、Pichia pastoris SMD1168株（Invitrogen社製）の形質転換を行なった。コンピテントセルの調製、キュベット、電気穿孔の条件はInvitrogenのプロトコールに従った。SD-His（2％グルコース、0.67 % Yeast Nitrogen Base w/o amino acids (Difco社製)、核酸塩基、およびヒスチジンを除くアミノ酸混合物（20-400 mg/L））培地のプレートにまいて、30℃で2日間培養し、形質転換体を得た。
[Example 1] Construction of soluble Ynd1 protein expression system The budding yeast YND1 gene sequence is registered in the GenBank database as AF203695 (Gao, X.-D., Kaigorodov, V. and Jigami, Y. ( 1999) J. Biol. Chem. 274, 21450-21456). Genomic DNA was extracted from yeast W303-1A according to a conventional method, and amplified by PCR using primer A (CCCTCGAGAAAAGAATGCTCATAGAAAACACTAATGATC: SEQ ID NO: 3) and primer B (ATTTGCGGCCGCTCAATGATGATGATGATGATGAGACTCCAGTAACTTGCCAGGTATG: SEQ ID NO: 4). The amplified gene is a fusion protein in which 6 residues of histidine are added to the C-terminal side of the partial amino acid sequence (Met1-Ser487;) shown in SEQ ID NO: 5 of Ynd1 protein (hereinafter abbreviated as soluble Ynd1 protein). Code. Salts and primers were removed from the obtained PCR product using a Microspin S-400 column (Amersham Bioscience). The purified PCR product subjected to cleavage with Xho I and Not I, likewise and ligated reaction pPIC9 cut with Xho I and Not I (Invitrogen Co.), PCR products were inserted. After confirming the base sequence of the PCR product with a sequencer, the plasmid was cleaved with Sal I to make it linear (FIG. 2). Using this, transformation of Pichia pastoris SMD1168 strain (manufactured by Invitrogen) was performed. Competent cell preparation, cuvettes, and electroporation conditions followed Invitrogen's protocol. Spread on a plate of SD-His (2% glucose, 0.67% Yeast Nitrogen Base w / o amino acids (Difco), nucleobase and amino acid mixture excluding histidine (20-400 mg / L)) medium, 30 After culturing at 2 ° C. for 2 days, a transformant was obtained.
形質転換体よりゲノムDNAを調製し、PCR法により、増幅した遺伝子がP. pastorisのゲノム上にインテグレーションされていることを確認した。この発現株をAWP-1株と命名した。 Genomic DNA was prepared from the transformant, and it was confirmed by PCR that the amplified gene was integrated on the genome of P. pastoris . This expression strain was named AWP-1 strain.
次にAWP-1株を小スケールで培養し、可溶型Ynd1タンパク質の発現を確認した。5 mlのBMGY培地（1％酵母エキス、2％ポリペプトン、100 mM リン酸カリウム緩衝液(pH 6.0)、1.34％ Yeast Nitrogen Base、1％グリセロール）で1日培養後、集菌し、上清を除いた。これに5 mlのBMMY培地（1％酵母エキス、2％ポリペプトン、100mM リン酸カリウム緩衝液 (pH 6.0)、1.34％ Yeast Nitrogen Base、0.5％メタノール）を添加・懸濁し、さらに3日培養を行なった。集菌後、上清15 μlをSDS-ポリアクリルアミドゲル電気泳動（SDS-PAGE）に供し、親株（SMD1168株）の上清との比較を行なった。その結果、AWP-1株の上清を流したレーンのみ、分子量約50 kDa付近に染色されるバンドを確認することができた。 Next, the AWP-1 strain was cultured on a small scale, and the expression of soluble Ynd1 protein was confirmed. Cultivate for 1 day in 5 ml BMGY medium (1% yeast extract, 2% polypeptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% Yeast Nitrogen Base, 1% glycerol) Excluded. 5 ml of BMMY medium (1% yeast extract, 2% polypeptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% Yeast Nitrogen Base, 0.5% methanol) is added and suspended in this, and further cultured for 3 days. It was. After collection, 15 μl of the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and compared with the supernatant of the parent strain (SMD1168 strain). As a result, a band stained with a molecular weight of about 50 kDa could be confirmed only in the lane in which the supernatant of the AWP-1 strain was passed.
〔実施例２〕可溶型Ynd1タンパク質の活性測定
可溶型Ynd1タンパク質の活性測定はGaoらの方法（Gao, X.-D., Kaigorodov, V. and Jigami, Y. (1999) J. Biol. Chem. 274, 21450-21456）を一部改変して行なった。Ynd1タンパク質精製標品を終濃度で0.2 M imidazole buffer, 10 mM MnCl2, 0.1% Triton X-100, 2 mM UDP, pH 7.6になるように調製した酵素反応液に加え、100μlになるように調製した。なお他の基質に対する活性を測定する場合は、UDPの代わりに同濃度でヌクレオチド二リン酸またはヌクレオチド三リン酸を加えて、同様の反応を行った。基質30℃、5分間反応させた後、80μlの60%(w/v)過塩素酸を加え、反応を停止した。遊離したリン酸をEnzChekTMPhosphate Assay Kit（Molecular probes社製）を用いて定量した。なお1 unitは1分間に1μmolのリン酸を遊離する酵素量として定義した。その結果、AWP-1株の培養上清中にYnd1タンパク質活性が確認された。
[Example 2] Activity measurement of soluble Ynd1 protein The activity of soluble Ynd1 protein was measured by the method of Gao et al. (Gao, X.-D., Kaigorodov, V. and Jigami, Y. (1999) J. Biol). Chem. 274, 21450-21456) was partially modified. Ynd1 protein purified preparation is added to the enzyme reaction solution prepared to a final concentration of 0.2 M imidazole buffer, 10 mM MnCl 2 , 0.1% Triton X-100, 2 mM UDP, pH 7.6, and adjusted to 100 μl did. When measuring the activity against other substrates, nucleotide diphosphate or nucleotide triphosphate was added at the same concentration instead of UDP, and the same reaction was performed. After reacting the substrate at 30 ° C. for 5 minutes, 80 μl of 60% (w / v) perchloric acid was added to stop the reaction. The liberated phosphoric acid was quantified using EnzChek ™ Phosphate Assay Kit (Molecular probes). One unit was defined as the amount of enzyme that liberates 1 μmol of phosphate per minute. As a result, Ynd1 protein activity was confirmed in the culture supernatant of the AWP-1 strain.
〔実施例３〕 可溶型Ynd1タンパク質の大量調製法の構築
可溶型Ynd1タンパク質を大量調製するために、培養をスケールアップして行なった。まず100 mlのBMGY培地（1％酵母エキス、2％ポリペプトン、100 mM リン酸カリウム緩衝液 (pH 6.0)、1.34％ Yeast Nitrogen Base、1％グリセロール）で前培養を行なった後、６Ｌのrich BMGY培地（1％酵母エキス、6％ポリペプトン、100 mM リン酸カリウム緩衝液 (pH 6.0)、1.34％ Yeast Nitrogen Base、1％グリセロール）、30℃で培養を行なった。Glycerolの消費の終点は酸素溶存濃度（DO）値を指標とし、DO値の上昇とともにメタノールの添加を開始した。同時に酸素発生装置を用いて、酸素ガスを培地にブローし、DO値10％を維持するようにした。また温度を26℃で培養し、メタノールは連続的に添加した（約10 ml/hr）。48時間後、培養上清を回収した。
[Example 3] Construction of large-scale preparation method of soluble Ynd1 protein In order to prepare a large amount of soluble Ynd1 protein, the culture was scaled up. First, pre-culture in 100 ml of BMGY medium (1% yeast extract, 2% polypeptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% Yeast Nitrogen Base, 1% glycerol), and then 6 L of rich BMGY Culture was performed at 30 ° C. in a medium (1% yeast extract, 6% polypeptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% Yeast Nitrogen Base, 1% glycerol). The end point of Glycerol consumption was determined by the oxygen dissolved concentration (DO) value as an index, and the addition of methanol was started as the DO value increased. At the same time, using an oxygen generator, oxygen gas was blown into the medium to maintain a DO value of 10%. Moreover, it culture | cultivated at 26 degreeC and methanol was added continuously (about 10 ml / hr). After 48 hours, the culture supernatant was collected.
得られた培養上清はマイクローザUFラボモジュールACP-1010（旭化成社製）を用いて濃縮・脱塩し、NaOHでpHを7.0に調整した。次にTALON Metal Affinity Resin (Clontech社製) 25 mlを空カラムに詰めた。バッファーA（20 mM リン酸ナトリウム、0.3 M NaCl、pH 7.0）でカラムを平衡化し、濃縮した培養上清をカラムに添加した。カラムを10倍容のバッファーAで洗浄した後、バッファーB（20 mM リン酸ナトリウム、0.3 M NaCl、0.15 M イミダゾール、pH 7.0）で溶出を行なった。溶出した画分の一部をSDS-PAGEで泳動し、精製度を確認した。次いで予めバッファーC（20 mM Tris-HCl, 150 mM NaCl, pH 7.5）で平衡化したHiLoad16/60 Superdex 200pg（Amersham Bioscience社製）でゲル濾過を行なった。一定量ずつ分取を行ない、各画分を電気泳動し、また実施例2に従い活性を測定して、可溶型Ynd1タンパク質の画分を取得した。これらをまとめて可溶型Ynd1タンパク質標品を得た。 The obtained culture supernatant was concentrated and desalted using Microza UF Lab Module ACP-1010 (manufactured by Asahi Kasei Corporation), and the pH was adjusted to 7.0 with NaOH. Next, 25 ml of TALON Metal Affinity Resin (Clontech) was packed in an empty column. The column was equilibrated with buffer A (20 mM sodium phosphate, 0.3 M NaCl, pH 7.0), and the concentrated culture supernatant was added to the column. The column was washed with 10 volumes of buffer A and then eluted with buffer B (20 mM sodium phosphate, 0.3 M NaCl, 0.15 M imidazole, pH 7.0). A portion of the eluted fraction was electrophoresed on SDS-PAGE to confirm the degree of purification. Subsequently, gel filtration was performed with HiLoad16 / 60 Superdex 200 pg (manufactured by Amersham Bioscience) previously equilibrated with buffer C (20 mM Tris-HCl, 150 mM NaCl, pH 7.5). A fixed amount was fractionated, each fraction was electrophoresed, and the activity was measured according to Example 2 to obtain a fraction of soluble Ynd1 protein. These were combined to obtain a soluble Ynd1 protein preparation.
次に酵素により可溶型Ynd1タンパク質のN-結合型糖鎖を切断した。可溶型Ynd1タンパク質 1 mgに対し、Endoglycosidase H（endoH；New England Biolab社製）を 1000 unit添加し、37℃、3時間処理した。反応後、HiTrapQ HP（Amersham Bioscience社製）でendoHを除去した。HiTrapQ HPカラムをバッファーD（20 mM リン酸ナトリウム緩衝液, 15 mM NaCl, pH 7.0）で平衡化した後、酵素反応液をカラムに供した。10倍容のバッファーDで洗浄の後、NaClの濃度を500 mMまで直線的に増加させることにより、可溶型Ynd1タンパク質とendoHを分離した。可溶型Ynd1タンパク質の脱糖鎖はSDS-PAGE上での移動度の変化で確認した。 Next, the N -linked sugar chain of the soluble Ynd1 protein was cleaved with an enzyme. To 1 mg of soluble Ynd1 protein, 1000 units of Endoglycosidase H (endoH; manufactured by New England Biolab) was added and treated at 37 ° C. for 3 hours. After the reaction, endoH was removed with HiTrapQ HP (Amersham Bioscience). The HiTrapQ HP column was equilibrated with buffer D (20 mM sodium phosphate buffer, 15 mM NaCl, pH 7.0), and then the enzyme reaction solution was applied to the column. After washing with 10 volumes of buffer D, the soluble Ynd1 protein and endoH were separated by linearly increasing the NaCl concentration to 500 mM. The deglycosylated chain of soluble Ynd1 protein was confirmed by the change in mobility on SDS-PAGE.
前述の方法で調整したタンパク質溶液(濃度20mg/mL以上)を充分量のミリQ水に対して16時間以上透析した。透析後、溶液を遠心(15krpm、10分)し、不溶性の残渣を取り除いた。 The protein solution (concentration of 20 mg / mL or more) prepared by the above method was dialyzed against a sufficient amount of milli-Q water for 16 hours or more. After dialysis, the solution was centrifuged (15 krpm, 10 minutes) to remove insoluble residues.
その後BCA法によりタンパク質濃度を決定し、結晶化時の濃度に調製した。この溶液に必要に応じて結晶化添加剤あるいは酵素基質、2価金属イオンなどを加え、結晶化用ストック溶液とした。 Thereafter, the protein concentration was determined by the BCA method and adjusted to the concentration at the time of crystallization. If necessary, crystallization additives, enzyme substrates, divalent metal ions and the like were added to this solution to obtain a crystallization stock solution.
〔実施例４〕可溶型Ynd1タンパク質の結晶化条件の検討
結晶化実験は全てハンギングドロップによる蒸気拡散法で行った。タンパク質ドロップはストックのタンパク質溶液とリザーバー溶液とを1対1で混合し調製した。
[Example 4] Examination of crystallization conditions for soluble Ynd1 protein All crystallization experiments were performed by the vapor diffusion method using hanging drop. The protein drop was prepared by mixing the stock protein solution and the reservoir solution 1: 1.
タンパク質ドロップは表面をシリコンで撥水処理を施した丸形カバーグラス上に調製した。リザーバーの容器は24穴の細胞培養用プレートを用いた。カバーグラスでプレートの穴に蓋をする形で密閉した。機密性を高めるためカバーグラスとプレートの間には真空グリスを塗布した。 The protein drop was prepared on a round cover glass whose surface was water-repellent with silicon. A 24-well cell culture plate was used as the reservoir container. The plate was sealed with a cover glass so as to cover the hole of the plate. Vacuum grease was applied between the cover glass and the plate to enhance confidentiality.
1〜3マイクロリットルのタンパク質ドロップを0.5mLのリザーバー溶液に対して平衡化した。このように作製したプレートを4℃あるいは20℃で1〜7日間放置した。 1-3 microliters of protein drop was equilibrated against 0.5 mL of reservoir solution. The plate thus prepared was left at 4 ° C. or 20 ° C. for 1 to 7 days.
結晶の観察は随時、オリンパス社製の偏光顕微鏡あるいは双眼実体顕微鏡で行った。 The observation of the crystal was performed at any time with a polarizing microscope or a binocular stereomicroscope manufactured by Olympus.
最初の結晶化条件のスクリーニングにはJENA BIOSCIENCE 社製JBScreenキットを用いた。このキットには文献でよく報告されている240の結晶化条件で構成されている。 The JBScreen kit manufactured by JENA BIOSCIENCE was used for screening of the initial crystallization conditions. This kit consists of 240 crystallization conditions well reported in the literature.
本タンパク質の1次スクリーニングはタンパク質濃度 10mg/mLで行い、キットの全ての条件に対し、基質(UDP)を加えたものと無添加のものの2種類を行った。合計480通りの条件を検討した。 The primary screening of this protein was performed at a protein concentration of 10 mg / mL, and for all conditions of the kit, two types were added, one with and without substrate (UDP) added. A total of 480 conditions were examined.
このスクリーニングにより、いくつかの条件で微結晶が得られた。これらの条件をもとにより大きな結晶が得られるよう結晶化条件の最適化するためにタンパク質濃度 12種類と沈殿剤濃度 64種類の全ての組み合わせで結晶化実験を行い、大きな結晶(0.1mm角以上)を得たが、X線回折像から、上述の条件で得られた結晶は全て多結晶であることが判明した。 This screening yielded microcrystals under several conditions. In order to optimize the crystallization conditions so that large crystals can be obtained based on these conditions, crystallization experiments were conducted with all combinations of 12 protein concentrations and 64 precipitating agent concentrations. However, from the X-ray diffraction image, it was found that all the crystals obtained under the above conditions were polycrystalline.
結晶を単結晶化するため、以下のことを行った。
(1)72の結晶化添加剤の検討
(2)精製用タグの検討
(3)別のスクリーニングキットの検討
(4)精製法の検討
In order to make the crystal into a single crystal, the following was performed.
(1) Examination of 72 crystallization additives
(2) Examination of purification tags
(3) Examination of another screening kit
(4) Examination of purification method
約 35,000回にも及ぶ結晶化実験の結果、遂に高分解能の単結晶が得られる条件を見出した。その条件を表１に、得られた結晶を図３に示す。 As a result of about 35,000 crystallization experiments, we finally found the conditions for obtaining high-resolution single crystals. The conditions are shown in Table 1, and the obtained crystals are shown in FIG.
結晶は四方両錘形で大きさは0.2-0.5mm角であった。 The crystal was a tetragonal bipyramidal shape with a size of 0.2-0.5 mm square.
〔実施例５〕X線回折実験
X線回折実験は高エネルギー加速器研究機構の放射光施設(PFーBL6A、BL18B)あるいは(AR-NW)あるいは回転対陰極式X線発生器FRーDで行った。結晶は常法に従いクライオループにマウントし、液体窒素中で急速凍結した。測定は全てクライオ条件(100K)で行った。以下、最もよいデータセットの収集条件です。
[Example 5] X-ray diffraction experiment
X-ray diffraction experiments were performed at the Synchrotron Radiation Facility (PF-BL6A, BL18B) or (AR-NW) of the High Energy Accelerator Research Organization or the rotating anti-cathode X-ray generator FR-D. The crystals were mounted on a cryoloop according to a conventional method and snap frozen in liquid nitrogen. All measurements were performed under cryo conditions (100K). Below are the best data collection conditions.
X線回折データの収集にはR-AXIS III (IP 300mm×300mm)を用いた。線源はFR-D回転対陰極式X線発生器(波長Cuκα線1.5418Å、加速電圧50kV、加速電流60mA)を使用した。カメラ長は180mmで測定した。測定はデータ収集プログラムd*trek (crystal clear)で制御し、プログラムd*trekによりデータ処理を行った。表２にデータ収集の概要を示す。 R-AXIS III (IP 300 mm × 300 mm) was used for collecting X-ray diffraction data. The radiation source used was an FR-D rotating anti-cathode X-ray generator (wavelength Cuκα radiation 1.5418 mm, acceleration voltage 50 kV, acceleration current 60 mA). The camera length was measured at 180 mm. The measurement was controlled by a data collection program d * trek (crystal clear), and data processing was performed by the program d * trek. Table 2 shows an overview of data collection.
結晶は斜方晶に属し、空間群P212121、格子定数はa=93.00 b=103.57 c=145.59 (Å)であった。非対称単位中に3分子のモノマーが存在すると仮定すると、Vm＝1.95 Å3/Daであった。結晶は最大で2.3Å分解能以上まで反射を示した。135枚のX線回折像をd*trekで処理した結果、2.3Å分解能までの回折強度データセットを得た。データの完全性(99.4%)と精度(Rmerge=0.057)が良好であるので、この結晶を用いて、2.3Å分解能での構造解析が可能であると考えられる。 The crystal belonged to orthorhombic crystal, space group P2 1 2 1 2 1 , and lattice constant a = 93.00 b = 103.57 c = 145.59 (Å). Assuming that there are 3 molecules of monomer in the asymmetric unit, Vm = 1.95 / 3 / Da. The crystals reflected up to 2.3 mm resolution or higher. As a result of processing 135 X-ray diffraction images with d * trek, a diffraction intensity data set up to 2.3 mm resolution was obtained. Since the completeness of the data (99.4%) and accuracy (Rmerge = 0.057) are good, it is considered that structural analysis with 2.3Å resolution is possible using this crystal.
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