JP6821155B2 - Method for producing L-lysine α-oxidase - Google Patents
Method for producing L-lysine α-oxidase Download PDFInfo
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- JP6821155B2 JP6821155B2 JP2016231499A JP2016231499A JP6821155B2 JP 6821155 B2 JP6821155 B2 JP 6821155B2 JP 2016231499 A JP2016231499 A JP 2016231499A JP 2016231499 A JP2016231499 A JP 2016231499A JP 6821155 B2 JP6821155 B2 JP 6821155B2
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- lysox
- lysine
- oxidase
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Landscapes
- Enzymes And Modification Thereof (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は、L-リシンα-オキシダーゼをコードする核酸、該核酸を含むベクター、該核酸が導入された形質転換体、及び該形質転換体を用いたL-リシンα-オキシダーゼの製造方法に関する。さらに、本発明は、抗腫瘍剤に関する。 The present invention relates to a nucleic acid encoding L-lysine α-oxidase, a vector containing the nucleic acid, a transformant into which the nucleic acid has been introduced, and a method for producing L-lysine α-oxidase using the transformant. Furthermore, the present invention relates to antitumor agents.
L-リシンα-オキシダーゼ(LysOX)は、L-リシンの酸化的脱アミノ化反応を触媒するフラビン酵素であり、L-リシンを基質としてα-ケト酸であるα-ケト-ε-アミノカプロン酸、過酸化水素及びアンモニアを生成する(非特許文献１)。LysOXは1980年糸状菌トリコデルマ・ビリデ(Trichoderma viride) Y244-2株から抗腫瘍性及び抗菌性を持つ酵素として発見され(非特許文献１-３)、その後いくつかの海洋生物からも見出されている。しかし、それらの酵素化学的性質と安定性は大きく異なり、トリコデルマ・ビリデY244-2由来のLysOXだけがL-リシンに対して高い基質特異性を有し幅広いpH及び温度で安定であるので、バイオセンサー好適酵素として実用化され、L-リシンの醗酵生産と品質の管理に使用されている。 L-lysine α-oxidase (LysOX) is a flavine enzyme that catalyzes the oxidative deaminolation reaction of L-lysine, and α-keto-ε-aminocaproic acid, which is an α-keto acid using L-lysine as a substrate, Produces hydrogen peroxide and ammonia (Non-Patent Document 1). LysOX was discovered in 1980 as an enzyme with antitumor and antibacterial properties from the Trichoderma viride Y244-2 strain (Non-Patent Documents 1-3), and was subsequently found in several marine organisms. ing. However, their enzymatic chemistries and stability are very different, and only LysOX from Trichoderma viride Y244-2 has high substrate specificity for L-lysine and is stable at a wide range of pH and temperature. It has been put to practical use as an enzyme suitable for sensors, and is used for fermentative production and quality control of L-lysine.
LysOXの他の応用例としては、血中L-リシン濃度を測定することで高リシン血症を始めとする様々な病気の予兆の検出、LysOXは抗腫瘍性を有するが正常細胞には細胞毒性が比較的低いので悪性腫瘍に対する新規医薬品、LysOXのL-リシン酸化反応で生じるα-ケト-ε-アミノカプロン酸の農薬、医薬品及び化粧品の合成中間体であるピペコリン酸合成への応用などが考えられる。 Other applications of LysOX include detecting signs of various diseases such as hyperlysine by measuring blood L-lysine concentration, and LysOX is antitumor but cytotoxic to normal cells. Is relatively low, so it can be applied to new drugs for malignant tumors, pesticides of α-keto-ε-aminocaproic acid generated by the L-lysine oxidation reaction of LysOX, and pipecoline acid synthesis, which is a synthetic intermediate for drugs and cosmetics. ..
このようにバイオセンサーに実用的なトリコデルマ・ビリデY244-2由来のLysOXは、従来、小麦フスマを培地とした固体培養物の水抽出液から精製されており(非特許文献４)、その大量製造には特殊な固体培養設備及び抽出設備が必要である。 In this way, LysOX derived from Trichoderma viride Y244-2, which is practical for biosensors, has conventionally been purified from a water extract of a solid culture using wheat bran as a medium (Non-Patent Document 4), and its mass production Requires special solid culture equipment and extraction equipment.
トリコデルマ・ビリデY244-2の固体培養の水抽出液には、多量の色素と粘度の高い多糖類が含まれている。それらを除去するために、通常の酵素精製に使われているイオン交換樹脂カラム及びゲル濾過カラムの他にも、限外濾過膜処理、アルコール添加による沈殿化工程又は脱色樹脂カラムによる脱色工程が必要となり、精製工程が複雑となり、バイオセンサー用の高純度LysOXを収率良く大量に得ることは非常に困難である。すなわち、糸状菌の固体培養による高純度LysOXの安価大量製造は不可能と言っても良い状況にある。 The water extract of the solid culture of Trichoderma viride Y244-2 contains a large amount of pigment and highly viscous polysaccharides. In order to remove them, in addition to the ion exchange resin column and gel filtration column used for normal enzyme purification, ultrafiltration membrane treatment, precipitation step by adding alcohol, or decolorization step by decolorizing resin column is required. Therefore, the purification process becomes complicated, and it is very difficult to obtain a large amount of high-purity LysOX for a biosensor in good yield. That is, it can be said that low-cost mass production of high-purity LysOX by solid culture of filamentous fungi is impossible.
さらに、糸状菌の固体培養では、コンタミネーションの防止措置が難しく、発酵タンクによる液体培養に比べて、酵素生産の培養管理が難しい。世界的にみても、糸状菌(カビ)の固体培養による酵素製造は、非常に稀な製法になっている。 Furthermore, in solid culture of filamentous fungi, it is difficult to take measures to prevent contamination, and it is more difficult to control the culture of enzyme production than in liquid culture in a fermentation tank. Even in the world, enzyme production by solid culture of filamentous fungi (mold) is a very rare production method.
本発明者らは、最近、トリコデルマ・ビリデY244-2由来のLysOX遺伝子を取得し、放線菌宿主(ストレプトマイセス・リビダンス(Streptomyces lividans) TK24)での発現と酵素生産とに成功している(非特許文献５)。しかしながら、培養期間が18日と長期であり、簡便には酵素を調製することはできない。 The present inventors have recently acquired the LysOX gene derived from Trichoderma viride Y244-2 and succeeded in expression and enzyme production in an actinomycete host (Streptomyces lividans TK24) (Streptomyces lividans TK24). Non-Patent Document 5). However, the culture period is as long as 18 days, and the enzyme cannot be easily prepared.
本発明は、L-リシンα-オキシダーゼの効率的且つ大量生産が可能となる核酸、該核酸を含むベクター、該核酸が導入された形質転換体、及び該形質転換体を用いたL-リシンα-オキシダーゼの製造方法を提供することを目的とする。さらに、本発明は、L-リシンα-オキシダーゼを利用した新たな抗腫瘍剤を提供することを目的とする。 The present invention relates to a nucleic acid capable of efficient and mass production of L-lysine α-oxidase, a vector containing the nucleic acid, a transformant into which the nucleic acid has been introduced, and L-lysine α using the transformant. -It is an object of the present invention to provide a method for producing oxidase. Furthermore, an object of the present invention is to provide a new antitumor agent utilizing L-lysine α-oxidase.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、トリコデルマ・ビリデY244-2由来のLysOXのプレ配列を含む完全長cDNAを大腸菌発現用にコドンを最適化した後に大腸菌に組み入れて発現させ、得られたLysOX前駆体をタンパク質分解酵素によってプレ配列に相当するペプチドを除去したところ、酵素活性が約600倍に増強されるとともに、熱に対する安定性も飛躍的に上昇し、酵素化学的性質も元の酵素と同等となるという知見を得た。 As a result of intensive studies to achieve the above object, the present inventors incorporated a full-length cDNA containing a pre-sequence of LysOX derived from Trichoderma viride Y244-2 into Escherichia coli after optimizing the codon for E. coli expression. When the peptide corresponding to the pre-sequence was removed from the obtained LysOX precursor by proteolytic enzyme, the enzyme activity was enhanced about 600 times and the stability against heat was dramatically increased. It was found that the chemical properties are also equivalent to those of the original enzyme.
本発明は、これら知見に基づき、更に検討を重ねて完成されたものであり、次の核酸、ベクター、形質転換体、L-リシンα-オキシダーゼの製造方法、及び抗腫瘍剤を提供するものである。 The present invention has been completed by further studying based on these findings, and provides the following nucleic acids, vectors, transformants, a method for producing L-lysine α-oxidase, and an antitumor agent. is there.
項１．以下の(a)又は(b)の核酸：
(a) 配列番号１に記載される塩基配列からなる核酸
(b) 配列番号１に記載される塩基配列と80%以上の同一性を有し、且つL-リシンα-オキシダーゼ活性を有するタンパク質をコードする核酸。
項２．項１に記載の核酸を含む発現ベクター。
項３．項１に記載の核酸が導入された形質転換体。
項４．宿主が大腸菌である、項３に記載の形質転換体。
項５．以下の工程を含む、L-リシンα-オキシダーゼの製造方法：
(1) 項３又は４に記載の形質転換体を液体培地で培養し、培養物からL-リシンα-オキシダーゼを回収する工程。
項６．以下の工程を含む、L-リシンα-オキシダーゼの製造方法：
(1) 配列番号２に記載される塩基配列をコドン最適化したコドン最適化核酸が導入された形質転換体を液体培地で培養し、培養物からL-リシンα-オキシダーゼを回収する工程であって、該形質転換体はL-リシンα-オキシダーゼのプレ配列を切断するペプチダーゼを有しない、工程。
項７．更に以下の工程を含む、項５又は６に記載の製造方法：
(2) 工程(1)で得られたL-リシンα-オキシダーゼをペプチダーゼ処理する工程。
項８．以下の(c)又は(d)のタンパク質を含む抗腫瘍剤：
(c) 配列番号３に記載されるアミノ酸配列からなるタンパク質
(d) 配列番号３に記載されるアミノ酸配列において、1〜50個のアミノ酸が欠失、置換、挿入及び／又は付加されたアミノ酸配列からなり、且つL-リシンα-オキシダーゼ活性を有するタンパク質。
Item 1. The following nucleic acids (a) or (b):
(a) Nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1.
(b) A nucleic acid encoding a protein having 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and having L-lysine α-oxidase activity.
Item 2. An expression vector containing the nucleic acid according to Item 1.
Item 3. A transformant into which the nucleic acid according to Item 1 has been introduced.
Item 4. Item 3. The transformant according to Item 3, wherein the host is Escherichia coli.
Item 5. Method for producing L-lysine α-oxidase including the following steps:
(1) A step of culturing the transformant according to Item 3 or 4 in a liquid medium and recovering L-lysine α-oxidase from the culture.
Item 6. Method for producing L-lysine α-oxidase including the following steps:
(1) A step of culturing a transformant into which a codon-optimized nucleic acid obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO: 2 has been introduced in a liquid medium and recovering L-lysine α-oxidase from the culture. The transformant does not have a peptidase that cleaves the pre-sequence of L-lysine α-oxidase.
Item 7. Item 5. The production method according to Item 5 or 6, further comprising the following steps:
(2) A step of peptidase treatment of the L-lysine α-oxidase obtained in step (1).
Item 8. Antitumor agents containing the following proteins (c) or (d):
(c) Protein consisting of the amino acid sequence set forth in SEQ ID NO: 3
(d) A protein having an amino acid sequence in which 1 to 50 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 3 and having L-lysine α-oxidase activity.
本発明によれば、トリコデルマ・ビリデY244-2由来LysOXを効率良く大量生産することが可能となる。本発明の製造方法で酵素を製造することで、従来はLysOXが高価であったためコスト的に実現できなかったL-リシン比色測定キット、抗腫瘍剤、L-リシンセンサーなどの製品化が可能となることが期待される。 According to the present invention, LysOX derived from Trichoderma viride Y244-2 can be efficiently mass-produced. By producing the enzyme by the production method of the present invention, it is possible to commercialize an L-lysine colorimetric measurement kit, an antitumor agent, an L-lysine sensor, etc., which could not be realized in terms of cost because LysOX was expensive in the past. Is expected to be.
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
なお、本明細書において「含む(comprise)」とは、「本質的にからなる(essentially consist of)」という意味と、「からなる(consist of)」という意味をも包含する。 In addition, in the present specification, "comprise" also includes the meaning of "essentially consist of" and the meaning of "consist of".
本発明において「遺伝子」とは、特に言及しない限り、2本鎖DNA、1本鎖DNA(センス鎖又はアンチセンス鎖)、及びそれらの断片が含まれる。また、本発明において「遺伝子」とは、特に言及しない限り、調節領域、コード領域、エクソン、及びイントロンを区別することなく示すものとする。 Unless otherwise specified, the term "gene" in the present invention includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof. Further, in the present invention, the term "gene" shall be used without distinguishing between a regulatory region, a coding region, an exon, and an intron unless otherwise specified.
また、本発明において、「核酸」、「ヌクレオチド」及び「ポリヌクレオチド」は同義であって、これらはDNA及びRNAの両方を含み、2本鎖であっても1本鎖であってもよい。 Further, in the present invention, "nucleic acid", "nucleotide" and "polynucleotide" are synonymous, and they include both DNA and RNA, and may be double-stranded or single-stranded.
本明細書において、「L-リシンα-オキシダーゼ」は特に明示が無い限りタンパク質を意味するものとするが、遺伝子として解釈することが適切な場合は遺伝子を意味するものとする。また、本明細書において、「L-リシンα-オキシダーゼ」は、特に明示が無い限り、プレ配列を有する前駆体、及びプレ配列が削除された状態の成熟体のいずれも包含する意味である。 In the present specification, "L-lysine α-oxidase" shall mean a protein unless otherwise specified, but when it is appropriate to interpret it as a gene, it shall mean a gene. Further, in the present specification, "L-lysine α-oxidase" is meant to include both a precursor having a pre-sequence and a mature product in which the pre-sequence is deleted, unless otherwise specified.
本発明の核酸は、以下の(a)又は(b)であることを特徴とする。
(a) 配列番号１に記載される塩基配列からなる核酸
(b) 配列番号１に記載される塩基配列と80%以上の同一性を有し、且つL-リシンα-オキシダーゼ活性を有するタンパク質をコードする核酸。
The nucleic acid of the present invention is characterized by the following (a) or (b).
(a) Nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1.
(b) A nucleic acid encoding a protein having 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and having L-lysine α-oxidase activity.
配列番号１に記載される塩基配列は、L-リシンα-オキシダーゼ(L-lysine α-oxidase)をコードする。配列番号１に記載される塩基配列にコードされるL-リシンα-オキシダーゼは、N末端にプレ配列を有する前駆体であり、該プレ配列が切断されることで成熟体となる。配列番号１に記載される塩基配列のオープンリーディングフレームにはアミノ酸617残基がコードされ、その内N末端の77残基がプレ配列と推測される。配列番号１に記載される塩基配列にコードされるアミノ酸配列は、配列番号３に記載されるものである。 The nucleotide sequence shown in SEQ ID NO: 1 encodes L-lysine α-oxidase. The L-lysine α-oxidase encoded by the nucleotide sequence set forth in SEQ ID NO: 1 is a precursor having a pre-sequence at the N-terminal, and becomes a mature product when the pre-sequence is cleaved. The open reading frame of the base sequence shown in SEQ ID NO: 1 encodes 617 amino acid residues, of which 77 residues at the N-terminal are presumed to be presequences. The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 is that set forth in SEQ ID NO: 3.
L-リシンαオキシダーゼ (EC 1.4.3.14)はL-アミノ酸オキシダーゼの一種であり、L-リシンの酸化的脱アミノ化反応により、α-ケト-ε-アミノカプロン酸、アミノ酸及び過酸化水素を生成する(下記式)。
L-リシン + O2 + H2O→α-ケト-ε-アミノカプロン酸+ NH3 + H2O2
L-lysine α-oxidase (EC 1.4.3.14) is a type of L-amino acid oxidase that produces α-keto-ε-aminocaproic acid, amino acids and hydrogen peroxide by the oxidative deamination reaction of L-lysine. (The following formula).
L-lysine + O 2 + H 2 O → α-keto-ε-aminocaproic acid + NH 3 + H 2 O 2
配列番号１に記載される塩基配列がコードするL-リシンα-オキシダーゼは、トリコデルマ・ビリデY244-2株由来のものであり、塩基配列は大腸菌用にコドン最適化されている。 The L-lysine α-oxidase encoded by the nucleotide sequence shown in SEQ ID NO: 1 is derived from the Trichoderma viride Y244-2 strain, and the nucleotide sequence is codon-optimized for Escherichia coli.
上記(b)における配列番号１に記載される塩基配列との同一性としては、好ましくは85%以上、より好ましくは90%以上、更に好ましくは95%以上、更により好ましくは97%以上、特に好ましくは99%以上である。塩基配列の同一性(%)は、当該分野で慣用のプログラム(例えば、BLAST、FASTA等)を初期設定で用いて決定することができる。 The identity with the nucleotide sequence set forth in SEQ ID NO: 1 in (b) above is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 97% or more, particularly. It is preferably 99% or more. Nucleotide sequence identity (%) can be determined using a program commonly used in the art (eg, BLAST, FASTA, etc.) by default.
本発明の核酸は、cDNAライブラリーを鋳型としたPCR法、化学合成、生化学的切断／再結合などの常法で作製することができる。また、改変された核酸の作製も常法に従って行うことができる。 The nucleic acid of the present invention can be prepared by a conventional method such as PCR method using a cDNA library as a template, chemical synthesis, or biochemical cleavage / recombination. In addition, the modified nucleic acid can be prepared according to a conventional method.
本発明の発現ベクターは、上記核酸を含むことを特徴とする。当該発現ベクターとしては、特に制限されず、公知の発現ベクターを広く使用することができる。上記核酸を導入する宿主の種類等を考慮し、適切な発現ベクターを適宜選択すればよい。発現ベクターは、上記核酸以外にも、プロモーター、エンハンサー、ターミネーター、ポリアデニル化シグナル、選択マーカー、複製起点などを含有し得る。発現ベクターは、自立的に複製するベクター、及び宿主細胞に導入された際に宿主細胞のゲノムに組み込まれ、組み込まれた染色体と共に複製されるもののいずれも使用することができる。 The expression vector of the present invention is characterized by containing the above nucleic acid. The expression vector is not particularly limited, and a known expression vector can be widely used. An appropriate expression vector may be appropriately selected in consideration of the type of host into which the nucleic acid is introduced. In addition to the above nucleic acids, the expression vector may contain a promoter, enhancer, terminator, polyadenylation signal, selectable marker, origin of replication and the like. As the expression vector, either a vector that replicates autonomously or a vector that is integrated into the genome of the host cell when introduced into the host cell and replicates together with the integrated chromosome can be used.
発現ベクターの構築、及び当該発現ベクターの細胞への導入法は周知であり、例えば、Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001)等の記載を参考にして実施することができる。 The construction of an expression vector and the method of introducing the expression vector into cells are well known. For example, referring to the description of Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001), etc. Can be carried out.
本発明の形質転換体は、上記核酸が導入されていることを特徴とする。核酸の導入は上記のように当該核酸を含む発現ベクターを利用すること等の公知の方法により行うことができる。宿主細胞への発現ベクターの導入は、コンピテントセル法、プロトプラスト法、エレクトロポーレーション法、リン酸カルシウム法、リポフェクション法、リポソーム法、マイクロインジェクション法、酢酸リチウム法等の周知の方法により行うことができる。上記核酸を導入する宿主としては、例えば、細菌(例えば、大腸菌(Escherichia coli)、ストレプトマイセス属、ロドコッカス(Rhodococcus)属、ストレプトコッカス(Streptococcus)属、スタフィロコッカス(Staphylococcus)属)、真菌(例えば、酵母(サッカロマイセス(Saccharomyces)属、シゾサッカロマイセス(Schizosaccharomyces)属など)、アスペルギルス(Aspergillus)属)、昆虫細胞(例えば、ドロソフィラ(Drosophila)S2、スポドプテラ(Spodoptera)SF)などが挙げられる。宿主としては、L-リシンα-オキシダーゼのプレ配列を切断するペプチダーゼを有しないものが望ましい。宿主としては、大腸菌が好ましい。 The transformant of the present invention is characterized in that the above nucleic acid is introduced. The nucleic acid can be introduced by a known method such as using an expression vector containing the nucleic acid as described above. The expression vector can be introduced into a host cell by a well-known method such as competent cell method, protoplast method, electroporation method, calcium phosphate method, lipofection method, liposome method, microinjection method, lithium acetate method and the like. Examples of the host into which the above nucleic acid is introduced include bacteria (for example, Escherichia coli, Streptomyces, Rhodococcus, Streptococcus, Staphylococcus), and fungi (for example). , Yeast (genus Saccharomyces, genus Schizosaccharomyces, etc.), genus Aspergillus), insect cells (eg, Drosophila S2, Spodoptera SF) and the like. It is desirable that the host does not have a peptidase that cleaves the pre-sequence of L-lysine α-oxidase. Escherichia coli is preferred as the host.
上記核酸以外であっても、L-リシンα-オキシダーゼをコードするトリコデルマ・ビリデY244-2株由来の配列番号２に記載される塩基配列をコドン最適化したコドン最適化核酸を、宿主に導入する核酸として使用することもできる。この場合、宿主としてはL-リシンα-オキシダーゼのプレ配列を切断するペプチダーゼを有しないものが使用される。ここでのコドン最適化とは、元の塩基配列におけるコドンを宿主細胞で使用頻度の高いコドンに変更することをいう。 In addition to the above nucleic acids, a codon-optimized nucleic acid obtained by codon-optimizing the nucleotide sequence set forth in SEQ ID NO: 2 derived from Trichoderma viride Y244-2 strain encoding L-lysine α-oxidase is introduced into the host. It can also be used as a nucleic acid. In this case, a host that does not have a peptidase that cleaves the pre-sequence of L-lysine α-oxidase is used. The codon optimization here means changing the codon in the original base sequence to a codon that is frequently used in the host cell.
本発明のL-リシンα-オキシダーゼの製造方法は、以下の工程を含むことを特徴とする。
(1) 上記形質転換体を液体培地で培養し、培養物からL-リシンα-オキシダーゼを回収する工程。
The method for producing L-lysine α-oxidase of the present invention is characterized by including the following steps.
(1) A step of culturing the transformant in a liquid medium and recovering L-lysine α-oxidase from the culture.
本発明の製造方法に使用する液体培地としては、上記形質転換体が生育できL-リシンα-オキシダーゼを産生できる培地であれば特に制限されず、炭素源、窒素源、無機塩などの栄養源を含有する合成培地又は天然培地を使用することができる。例えば、培地の炭素源としては、グルコース、スクロース、フラクトース、マルトース、グリセリン、デキストリン、デンプン、オリゴ糖、糖蜜、麦芽エキス、有機酸等が挙げられる。また、窒素源としては、各種ペプトン、酵母エキス、コーンスティープリカー、大豆粉、フスマエキス、肉エキス、カゼイン、アミノ酸、尿素等の有機窒素源、硝酸塩、アンモニウム塩等の無機窒素源等が挙げられる。無機塩類としては、ナトリウム塩、カリウム塩、マグネシウム塩、鉄塩、その他の金属塩等が挙げられる。さらに、その他の栄養源としては、ビタミン類、アミノ酸類、核酸類等が挙げられる。 The liquid medium used in the production method of the present invention is not particularly limited as long as it is a medium capable of growing the above transformant and producing L-lysine α-oxidase, and is a nutrient source such as a carbon source, a nitrogen source, and an inorganic salt. A synthetic medium or a natural medium containing the above can be used. For example, examples of the carbon source of the medium include glucose, sucrose, fructose, maltose, glycerin, dextrin, starch, oligosaccharide, molasses, malt extract, organic acid and the like. Examples of the nitrogen source include organic nitrogen sources such as various peptones, yeast extracts, corn steep liquors, soybean flour, bran extract, meat extracts, casein, amino acids and urea, and inorganic nitrogen sources such as nitrates and ammonium salts. Examples of the inorganic salts include sodium salts, potassium salts, magnesium salts, iron salts, and other metal salts. Furthermore, examples of other nutrient sources include vitamins, amino acids, nucleic acids and the like.
培養は、液体培養であって、一般的な微生物の培養方法等に従って行うことができる。培養は、通気攪拌培養、振盪培養等によって行うことができる。また、培養方式については、回分培養、流加培養及び連続培養の何れの方式によっても行うことができる。培養条件(温度、pH、培養時間等)は、培養する形質転換体の生育特性に応じて適宜設定することができ、培養温度としては、通常10〜40℃、好ましくは30〜37℃であり、pHとしては、通常pH4〜8、好ましくはpH5〜7である。培養時間としては、通常10〜30時間、好ましくは16〜20時間である。 The culture is a liquid culture and can be carried out according to a general method for culturing microorganisms. The culture can be carried out by aeration stirring culture, shaking culture and the like. Further, as the culture method, any of batch culture, fed-batch culture and continuous culture can be used. The culture conditions (temperature, pH, culture time, etc.) can be appropriately set according to the growth characteristics of the transformant to be cultured, and the culture temperature is usually 10 to 40 ° C, preferably 30 to 37 ° C. The pH is usually pH 4 to 8, preferably pH 5 to 7. The culturing time is usually 10 to 30 hours, preferably 16 to 20 hours.
培養により得られた培養物(例えば、菌体、培養上清など)には、L-リシンα-オキシダーゼが蓄積されている。菌体内に蓄積したL-リシンα-オキシダーゼの菌体からの溶出は、常法に従って行うことができる。具体的には、菌体を超音波、フレンチプレス、高圧ホモジナイザー等の機械的破砕処理、シクロヘキサン、トルエン、酢酸エチル等による処理、リゾチームによる溶菌処理などの方法が挙げられる。このようにして得られたL-リシンα-オキシダーゼ溶出液及びL-リシンα-オキシダーゼ含有培養上清は必要に応じて更に精製工程に供することもできる。精製方法としては、例えば、溶媒抽出、クロマトグラフィー(例えば、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、ハイドロキシアパタイトカラムクロマトグラフィー、疎水性相互作用クロマトグラフィー、ゲルろ過クロマトグラフィー等)、膜処理(例えば、メンブレンフィルター、限外濾過、精密濾過、逆浸透等)、活性炭処理、超臨界流体抽出処理、蒸留処理、晶析などを挙げることができ、これらを単独で又は２種以上を任意の順序で適宜組み合わせて行うことができる。 L-lysine α-oxidase is accumulated in the culture obtained by culturing (for example, bacterial cells, culture supernatant, etc.). The elution of L-lysine α-oxidase accumulated in the cells from the cells can be carried out according to a conventional method. Specific examples thereof include methods such as mechanical crushing treatment of cells with ultrasonic waves, French press, high-pressure homogenizer, treatment with cyclohexane, toluene, ethyl acetate, etc., and lysis treatment with lysozyme. The L-lysine α-oxidase eluate and the L-lysine α-oxidase-containing culture supernatant thus obtained can be further subjected to a purification step, if necessary. Purification methods include, for example, solvent extraction, chromatography (eg, affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, hydrophobic interaction chromatography, gel filtration chromatography, etc.), membrane treatment (eg, membrane). Filter, ultrafiltration, precision filtration, back-penetration, etc.), activated carbon treatment, supercritical fluid extraction treatment, distillation treatment, crystallization, etc., which may be used alone or in combination of two or more in any order. Can be done.
工程(1)で得られたL-リシンα-オキシダーゼは、プレ配列を有する前駆体であることが望ましい。このようにプレ配列を有する前駆体の状態で産生させることで、L-リシンα-オキシダーゼの生産量を増加させることができる。 The L-lysine α-oxidase obtained in step (1) is preferably a precursor having a pre-sequence. By producing in the state of a precursor having a pre-sequence in this way, the production amount of L-lysine α-oxidase can be increased.
本発明のL-リシンα-オキシダーゼの製造方法は、更に以下の工程を含み得る。
(2) 工程(1)で得られたL-リシンα-オキシダーゼをペプチダーゼ処理する工程。
The method for producing L-lysine α-oxidase of the present invention may further include the following steps.
(2) A step of peptidase treatment of the L-lysine α-oxidase obtained in step (1).
工程(1)で得られたL-リシンα-オキシダーゼは、プレ配列を有するものである場合は、ペプチダーゼ処理を行うことにより、天然の酵素と同様の活性、基質特異性及び熱安定性を有するL-リシンα-オキシダーゼ成熟体を得ることができる。 When the L-lysine α-oxidase obtained in step (1) has a pre-sequence, it has the same activity, substrate specificity and thermal stability as a natural enzyme by treating with peptidase. A mature L-lysine α-oxidase can be obtained.
ペプチダーゼとしては、L-リシンα-オキシダーゼのプレ配列を切断できるものであれば特に制限なく使用できる。ペプチダーゼとしては、エンドペプチダーゼ及びエキソペプチダーゼが挙げられ、前者としてはアミノペプチダーゼ、ジペプチダーゼ、ジペプチジルペプチダーゼ、トリペプチジルペプチダーゼ及びカルボキシペプチダーゼが挙げられ、後者としてはセリンプロテアーゼ、システインプロテアーゼ、アスパラギン酸プロテアーゼ及びメタロプロテアーゼが挙げられる。これらは単独又は２種以上を組み合わせて使用することができる。 The peptidase can be used without particular limitation as long as it can cleave the pre-sequence of L-lysine α-oxidase. Examples of the peptidase include endopeptidase and exopeptidase, the former includes aminopeptidase, dipeptidase, dipeptidase peptidase, tripeptidyl peptidase and carboxypeptidase, and the latter include serine protease, cysteine protease, aspartic protease and metaro. Proteases can be mentioned. These can be used alone or in combination of two or more.
具体的なペプチダーゼとしては、ペプシン、トリプシン、キモトリプシン、ズブチリシン、サーモライシン、パパイン、キモパパイン、ブロメライン等が挙げられ、これらは単独又は２種以上を組み合わせて使用することができる。 Specific examples of pepsinase include pepsin, trypsin, chymotrypsin, subtilisin, thermolysin, papain, chymopapain, bromelain and the like, and these can be used alone or in combination of two or more.
ペプチダーゼの使用量は、処理するL-リシンα-オキシダーゼの量、反応温度及び反応時間に応じて、適切な使用量を適宜設定することができる。ペプチダーゼ処理の時間は、L-リシンα-オキシダーゼのプレ配列の切断が完了するまでの時間であれば特に制限されず、例えば、1〜48時間の範囲から選択される。 The amount of peptidase used can be appropriately set according to the amount of L-lysine α-oxidase to be treated, the reaction temperature and the reaction time. The time of the peptidase treatment is not particularly limited as long as it is the time until the cleavage of the pre-sequence of L-lysine α-oxidase is completed, and is selected from the range of, for example, 1 to 48 hours.
ペプチダーゼ処理のpHは、使用酵素の至適pHに対応して適宜設定することができ、例えば、pH2〜12、好ましくはpH6〜10、より好ましくはpH7〜9の範囲である。pHは、酸、アルカリ剤、又は緩衝剤の添加により調整することができる。ペプチダーゼ処理の温度は、使用酵素の至適温度に対応して適宜設定することができ、例えば、30〜70℃の範囲から選択される。 The pH of the peptidase treatment can be appropriately set according to the optimum pH of the enzyme used, and is, for example, in the range of pH 2 to 12, preferably pH 6 to 10, and more preferably pH 7 to 9. The pH can be adjusted by adding an acid, alkaline agent, or buffer. The temperature of the peptidase treatment can be appropriately set according to the optimum temperature of the enzyme used, and is selected from, for example, the range of 30 to 70 ° C.
ペプチダーゼ処理の停止は、ペプチダーゼを失活又は除去することにより行う。失活操作は加熱処理、フェニルメチルスルフォニルフルオライド(PMSF)、エチレンジアミン4酢酸(EDTA)等の添加などにより行うことができる。 The peptidase treatment is stopped by inactivating or removing the peptidase. The deactivation operation can be performed by heat treatment, addition of phenylmethylsulfonylfluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), or the like.
バイオセンサーにおいて、L-リシンα-オキシダーゼを使用する場合の固定化方法としては、物理吸着法、イオン結合法、包括法、共有結合法などタンパク質の固定化方法として公知の方法を利用できる。 As an immobilization method when L-lysine α-oxidase is used in a biosensor, a method known as a protein immobilization method such as a physical adsorption method, an ionic bonding method, a comprehensive method, or a covalent bonding method can be used.
バイオセンサーでは、L-リシンの検出のために、L-リシンα-オキシダーゼ固定化体の近傍に該酵素の触媒する反応により減少する酸素、増加する過酸化水素及びアンモニウムイオンのいずれか1つを検知する電気化学的検出手段が備えられていることが望ましい。中でも、生成する過酸化水素を検知することが望ましく、過酸化水素の高感度計測には、アンペロメトリー等の電気化学的な手法を使用することができる。 In the biosensor, for the detection of L-lysine, one of oxygen decreased, hydrogen peroxide and ammonium ion increased by the enzyme-catalyzed reaction is placed in the vicinity of the L-lysine α-oxidase immobilized substance. It is desirable that an electrochemical detection means for detection is provided. Above all, it is desirable to detect the generated hydrogen peroxide, and an electrochemical method such as amperometry can be used for high-sensitivity measurement of hydrogen peroxide.
固定化された酵素に試料を接触させて反応させる方式としては、バッチ方式、フロー方式等が挙げられる。 Examples of the method of bringing the sample into contact with the immobilized enzyme to cause a reaction include a batch method and a flow method.
本発明の抗腫瘍剤は、以下の(c)又は(d)のタンパク質を含むことを特徴とする。
(c) 配列番号３に記載されるアミノ酸配列からなるタンパク質
(d) 配列番号３に記載されるアミノ酸配列において、1〜50個のアミノ酸が欠失、置換、挿入及び／又は付加されたアミノ酸配列からなり、且つL-リシンα-オキシダーゼ活性を有するタンパク質。
The antitumor agent of the present invention is characterized by containing the following proteins (c) or (d).
(c) Protein consisting of the amino acid sequence set forth in SEQ ID NO: 3
(d) A protein having an amino acid sequence in which 1 to 50 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 3 and having L-lysine α-oxidase activity.
配列番号３に記載されるアミノ酸配列は、トリコデルマ・ビリデY244-2株由来のL-リシンα-オキシダーゼのプレ配列を有する前駆体のアミノ酸配列である。このようなL-リシンα-オキシダーゼのプレ配列を有する前駆体が抗腫瘍活性を有することは今まで知られていなかった。 The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of a precursor having a pre-sequence of L-lysine α-oxidase derived from Trichoderma viride Y244-2 strain. It has not been known until now that a precursor having such a pre-sequence of L-lysine α-oxidase has antitumor activity.
上記(d)のペプチドにおいて、欠失、置換、挿入及び／又は付加されるアミノ酸の個数は、好ましくは1〜30個、より好ましくは1〜15個、更に好ましくは1〜10個、特に好ましくは1〜5個である。特定のアミノ酸配列において、1個若しくは2個以上のアミノ酸を欠失、置換、挿入及び／又は付加させる技術は公知である。 In the peptide of (d) above, the number of amino acids deleted, substituted, inserted and / or added is preferably 1 to 30, more preferably 1 to 15, still more preferably 1 to 10, and particularly preferably. Is 1 to 5. Techniques for deleting, substituting, inserting and / or adding one or more amino acids in a particular amino acid sequence are known.
上記の(c)又は(d)のタンパク質には、その塩や誘導体も含まれる。 The above-mentioned proteins (c) or (d) also include salts and derivatives thereof.
本発明における「抗腫瘍剤」は、抗癌剤、抗腫瘍薬剤、抗腫瘍医薬組成物等と表現される場合もある。 The "antitumor agent" in the present invention may be expressed as an anticancer agent, an antitumor agent, an antitumor pharmaceutical composition and the like.
抗腫瘍剤として調製する場合、上記のタンパク質をそのまま使用するか、又は医薬品において許容される無毒性の担体、希釈剤若しくは賦形剤とともに、タブレット(素錠、糖衣錠、発泡錠、フィルムコート錠、チュアブル錠、トローチ剤などを含む)、カプセル剤、丸剤、粉末剤(散剤)、細粒剤、顆粒剤、液剤、懸濁液、乳濁液、シロップ、ペースト、注射剤(使用時に、蒸留水又はアミノ酸輸液や電解質輸液等の輸液に配合して液剤として調製する場合を含む)などの形態に調製して、医薬用の製剤にすることが可能である。 When prepared as an antitumor agent, the above proteins can be used as is, or with non-toxic carriers, diluents or excipients allowed in pharmaceuticals, tablets (plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, Chewable tablets, troches, etc.), capsules, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, excipients (distilled when used) It is possible to prepare a pharmaceutical preparation by preparing it in a form such as (including the case where it is mixed with an infusion solution such as water or an amino acid infusion solution or an electrolyte infusion solution and prepared as a liquid preparation).
本発明の抗腫瘍剤における上記のタンパク質の含量は、抗腫瘍剤全量中0.0001〜100質量%、好ましくは0.001〜99.9質量%、より好ましくは0.01〜99質量%の範囲から適宜選択することが可能である。 The content of the above protein in the antitumor agent of the present invention can be appropriately selected from the range of 0.0001 to 100% by mass, preferably 0.001 to 99.9% by mass, and more preferably 0.01 to 99% by mass in the total amount of the antitumor agent. Is.
本発明の抗腫瘍剤の投与方法は特に限定されず、例えば、動脈内投与、静脈内投与、口腔内投与、直腸投与、経腸投与、経皮投与、経口投与などにより行うことができる。 The method of administering the antitumor agent of the present invention is not particularly limited, and for example, it can be administered by intraarterial administration, intravenous administration, oral administration, rectal administration, enteral administration, transdermal administration, oral administration, or the like.
本発明の抗腫瘍剤は、ヒトを含む哺乳動物に対して投与される。 The antitumor agent of the present invention is administered to mammals including humans.
本発明の抗腫瘍剤の投与量は、患者の体重、年齢、性別、症状などの種々の条件に応じて適宜決定することができる。 The dose of the antitumor agent of the present invention can be appropriately determined according to various conditions such as body weight, age, sex, and symptoms of the patient.
本発明の抗腫瘍剤により治療できる癌の種類は、特に制限されず、例えば、胃癌、直腸癌、結腸癌、小腸癌、食道癌、肝臓癌、膵臓癌、肺癌、咽頭癌、腎癌、胆のう及び胆管癌、頭頸部癌、前立腺癌、膀胱癌、乳癌、子宮癌、卵巣癌、皮膚癌、悪性黒色腫、脳腫瘍、白血病、悪性リンパ腫等が挙げられる。 The type of cancer that can be treated by the antitumor agent of the present invention is not particularly limited, and for example, gastric cancer, rectal cancer, colon cancer, small intestinal cancer, esophageal cancer, liver cancer, pancreatic cancer, lung cancer, pharyngeal cancer, renal cancer, and biliary cyst. And bile duct cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, skin cancer, malignant melanoma, brain tumor, leukemia, malignant lymphoma and the like.
本発明の製造方法により、トリコデルマ・ビリデY244-2由来LysOXを効率良く大量生産することができる。さらに、本発明の製造方法により生産されたLysOX前駆体をペプチダーゼ処理することで得られるLysOXは、元の酵素と同等の酵素化学的性質(活性、基質特異性及び熱安定性)を有している。 According to the production method of the present invention, LysOX derived from Trichoderma viride Y244-2 can be efficiently mass-produced. Furthermore, LysOX obtained by peptidase treatment of the LysOX precursor produced by the production method of the present invention has the same enzymatic chemical properties (activity, substrate specificity and thermal stability) as the original enzyme. There is.
LysOXは、L-リシンを検出するためのバイオセンサー、農薬、医薬品及び化粧品の合成中間体であるピペコリン酸の合成、悪性腫瘍に対する新規医薬品の有効成分などに産業上応用される可能性がある。以前はLysOXが高価であったため実現できなかったが、本発明の製造方法によりLysOXのコストが低下することでL-リシン比色測定キット、抗腫瘍剤、L-リシンセンサーなどの製品化が可能となることが期待される。 LysOX has the potential to be industrially applied to biosensors for detecting L-lysine, synthesis of pipecolic acid, which is a synthetic intermediate for pesticides, pharmaceuticals and cosmetics, and active ingredients for new pharmaceuticals for malignant tumors. Previously, LysOX was expensive and could not be realized, but the manufacturing method of the present invention reduces the cost of LysOX, making it possible to commercialize L-lysine colorimetric measurement kits, antitumor agents, L-lysine sensors, etc. Is expected to be.
以下、本発明を更に詳しく説明するため実施例を挙げる。しかし、本発明はこれら実施例等になんら限定されるものではない。 Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.
試験例１：大腸菌を用いた発現系構築
[使用菌株及びベクター]
プラスミド構築などの形質転換体の宿主菌には大腸菌Escherichia coli TOP10及びEscherichia coli BL21(DE3)を使用した。発現用ベクターにはpCold IVを用いた。
Test Example 1: Construction of expression system using Escherichia coli
[Strains and vectors used]
Escherichia coli TOP10 and Escherichia coli BL21 (DE3) were used as host bacteria for transformants such as plasmid construction. PCold IV was used as the expression vector.
[使用培地]
組換え大腸菌の培養には、以下の培地を使用した。
・2×YT培地[1.6%ポリペプトン、1%酵母エキス、0.5% NaCl (1.5%寒天粉末)]
・SOC培地[2%ペプトン、0.5%酵母エキス、0.05%塩化ナトリウム/100 mlに対し、オートクレーブ済み1 Mグルコースを2 ml添加し、使用直前に1 M塩化マグネシウム1 ml、1 M硫酸マグネシウム1 mlを加えた]
組換えプラスミドを保有するE. coliを培養する際は、50 mg/mlアンピシリン(Amp)を終濃度50μg/mlとなるように添加した。E. coli BL21を培養するときは50 mg/ml カナマイシン(Kan)を終濃度50μg/mlとなるように添加した。
[Medium used]
The following media were used for culturing recombinant Escherichia coli.
・ 2 × YT medium [1.6% polypeptone, 1% yeast extract, 0.5% NaCl (1.5% agar powder)]
・ Add 2 ml of autoclaved 1 M glucose to SOC medium [2% peptone, 0.5% yeast extract, 0.05% sodium chloride / 100 ml, 1 ml magnesium chloride 1 ml, 1 M magnesium sulfate 1 ml immediately before use. Added]
When culturing E. coli carrying the recombinant plasmid, 50 mg / ml ampicillin (Amp) was added to a final concentration of 50 μg / ml. When culturing E. coli BL21, 50 mg / ml kanamycin (Kan) was added to a final concentration of 50 μg / ml.
[実験操作]
・核酸の定量法
スペクトラムの測定にはUV-1200を使用し、2本鎖DNAの濃度はOD260＝1.0のとき50μg/mlとして求めた。RNAの濃度はOD260＝1.0のとき40μg/mlとして求めた。また、純度はOD260/OD280＝1.7から2.0が好ましいとした。
[Experimental operation]
-Nucleic acid quantification method UV-1200 was used for spectrum measurement, and the concentration of double-stranded DNA was determined as 50 μg / ml when OD 260 = 1.0. The RNA concentration was determined as 40 μg / ml when OD 260 = 1.0. The purity is preferably OD 260 / OD 280 = 1.7 to 2.0.
(1) PCR反応
LysOXの配列の増幅には大腸菌用にコドンの最適化を行った配列においてはKOD-Plus-(東洋紡社)を使用した。
(1) PCR reaction
For the amplification of the LysOX sequence, KOD-Plus- (Toyobo Co., Ltd.) was used for the codon-optimized sequence for Escherichia coli.
(2) プラスミドの構築
プラスミド構築はGeneArt(登録商標) Seamless Cloning and Assembly (サーモフィッシャー サイエンティフィック社)を使用し、相同組換えによるインサート導入を行った。
(2) Construction of plasmid For plasmid construction, GeneArt (registered trademark) Seamless Cloning and Assembly (Thermo Fisher Scientific Co., Ltd.) was used, and inserts were introduced by homologous recombination.
(3) 大腸菌の形質転換
コンピテントセルを氷上で解凍し、コンピテントセルの液量の10分の1容量までのライゲーション反応液を添加し、軽く懸濁した後、氷上で30分静置した。これに、SOC培地を500μl添加し、37℃で1時間インキュベートした後、遠心(14,000 rpm，1分間)した。100μl程度残るように上清を除去し、菌体をピペッティングにより懸濁をし、LBプレート(抗生物質含有)にスプレッドして、37℃で一晩培養した。
(3) Escherichia coli transformation competent cells were thawed on ice, a ligation reaction solution up to 1/10 of the volume of the competent cells was added, lightly suspended, and then allowed to stand on ice for 30 minutes. .. To this, 500 μl of SOC medium was added, and the mixture was incubated at 37 ° C. for 1 hour and then centrifuged (14,000 rpm, 1 minute). The supernatant was removed so that about 100 μl remained, the cells were suspended by pipetting, spread on an LB plate (containing antibiotics), and cultured at 37 ° C. overnight.
(4) コロニーダイレクトPCR
組換え体のインサートの確認にはコロニーからのプラスミドDNA精製が不要で、形質転換後、生えてきたコロニーから直接PCRを行うコロニーダイレクトPCR法を用いた。
(4) Colony direct PCR
Purification of plasmid DNA from colonies was not required to confirm the insert of the recombinant, and the colony direct PCR method was used in which PCR was performed directly from the colonies that had grown after transformation.
(5) プラスミド抽出
プラスミド抽出はバイオラッド社のQuantum Prep Plasmid Mini Prep Kitを使用した。培養液を集菌後、上清を廃棄した。200μlのResuspension Solutionに再懸濁し、250μlのLysis Solutionを加えてゆるやかに懸濁後、250μlのNeutralization Solutionを添加し、ゆるやかに懸濁後、12,000×gで5分間遠心を行った。上清をスピンフィルターへと回収し、200μlのQuantum Matrixを加えて懸濁し、12,000×gで30秒間遠心を行い、流出液を廃棄した。洗浄として、500μlのWater Bufferを加え、12,000×gで30秒間遠心し流出液を廃棄、これを2回繰り返した。スピンフィルターを1.5 mlチューブへと移し、100μlの滅菌水を添加し、12,000×gで1分遠心後、溶出液を精製プラスミド溶液とした。
(5) plasmid extraction For plasmid extraction, Bio-Rad's Quantum Prep plasmid Mini Prep Kit was used. After collecting the culture solution, the supernatant was discarded. It was resuspended in 200 μl of Resuspension Solution, 250 μl of Lysis Solution was added to suspend it gently, 250 μl of Neutralization Solution was added, and the suspension was gently suspended, and then centrifuged at 12,000 × g for 5 minutes. The supernatant was collected on a spin filter, suspended by adding 200 μl of Quantum Matrix, centrifuged at 12,000 × g for 30 seconds, and the effluent was discarded. For washing, 500 μl of Water Buffer was added, and the mixture was centrifuged at 12,000 × g for 30 seconds to discard the effluent, and this was repeated twice. The spin filter was transferred to a 1.5 ml tube, 100 μl of sterile water was added, and the mixture was centrifuged at 12,000 × g for 1 minute, and the eluate was used as a purified plasmid solution.
(6) タンパク質定量法
タンパク質定量はブラッドフォードの方法に基づくバイオラッド社のプロテインアッセイキットを使用した。1.6 mlの酵素溶液に対して0.4 mlのアッセイ試薬を添加し、10〜15分室温で反応後、595 nmの吸収をUV 1200 (島津製作所社)で測定した。BSAを用いて作製した検量線よりタンパク質濃度を算出した。
(6) Protein quantification method For protein quantification, Bio-Rad's protein assay kit based on Bradford's method was used. 0.4 ml of the assay reagent was added to 1.6 ml of the enzyme solution, and after reacting at room temperature for 10 to 15 minutes, absorption at 595 nm was measured with UV 1200 (Shimadzu Corporation). The protein concentration was calculated from the calibration curve prepared using BSA.
(7) LysOX活性測定(MBTH法(α-ケト酸の定量))
酵素反応は70 mMカリウムリン酸バッファー(pH7.4)中、50 mM L-リシン及び300 U/mlカタラーゼの存在下で行った。酵素溶液(0.01〜0.3 U/ml) 0.1 ml，0.1 Mカリウムリン酸バッファー(pH7.4) 0.7 ml及び300 U/mlカタラーゼ0.1 mlを混合し30℃で5分間プレインキュベートした。酵素反応開始は0.5 M L-リシンを0.1 ml添加することで行い、30℃で10分間反応を行った。酵素反応停止は、25%TCAを0.1 ml添加することで行い、反応停止後に1 M酢酸バッファー(pH5.0)を2 ml及び0.1%MBTHを0.8 ml添加し、50℃で30分間インキュベートした。反応液を冷却後316 nmの吸光度を測定した。
(7) LysOX activity measurement (MBTH method (quantification of α-keto acid))
The enzymatic reaction was carried out in 70 mM potassium phosphate buffer (pH 7.4) in the presence of 50 mM L-lysine and 300 U / ml catalase. Enzyme solution (0.01-0.3 U / ml) 0.1 ml, 0.1 M potassium phosphate buffer (pH 7.4) 0.7 ml and 300 U / ml catalase 0.1 ml were mixed and pre-incubated at 30 ° C. for 5 minutes. The enzyme reaction was started by adding 0.1 ml of 0.5 mL-lysine, and the reaction was carried out at 30 ° C. for 10 minutes. The enzyme reaction was stopped by adding 0.1 ml of 25% TCA. After the reaction was stopped, 2 ml of 1 M acetate buffer (pH 5.0) and 0.8 ml of 0.1% MBTH were added, and the mixture was incubated at 50 ° C. for 30 minutes. After cooling the reaction solution, the absorbance at 316 nm was measured.
1 Uは、カタラーゼの存在下、30℃で1分間に1μmolのα-ケトアミノカプロン酸を生成する酵素量とした。 1 U was defined as the amount of enzyme that produces 1 μmol of α-ketoaminocaproic acid per minute at 30 ° C. in the presence of catalase.
(8) LysOXの濃縮(硫酸アンモニウム濃縮)
本培養を終えた培地を遠心分離し、菌体を回収し、3倍量の破砕緩衝液(pH7.4)に懸濁し、破砕機によって菌体破砕を行った。これを20-65%飽和になるように硫酸アンモニウムを添加し、氷中で30分間撹拌後、沈殿を回収した。沈殿をできるだけ少量の20 mMリン酸カリウム緩衝液(pH 7.4)に懸濁し、透析した。
(8) Concentration of LysOX (Ammonium sulfate concentration)
The medium after the main culture was centrifuged, the cells were collected, suspended in 3 times the amount of crushing buffer (pH 7.4), and the cells were crushed by a crusher. Ammonium sulfate was added to this to make it 20-65% saturated, and the mixture was stirred in ice for 30 minutes, and the precipitate was recovered. The precipitate was suspended in as little 20 mM potassium phosphate buffer (pH 7.4) as possible and dialyzed.
[結果]
(1) コドンの最適化
LysOXの塩基配列は大腸菌では見られないレアコドンを多く含むため、発現量が抑えられていることが推測された。このため、LysOXのコドンの最適化を図った。大腸菌用にコドンの最適化を行ったDNA配列を配列番号１に示す。また、前駆体配列と成熟体配列との2種類を調製した。
[result]
(1) Codon optimization
Since the base sequence of LysOX contains many rare codons not found in Escherichia coli, it was speculated that the expression level was suppressed. Therefore, the codon of LysOX was optimized. The codon-optimized DNA sequence for E. coli is shown in SEQ ID NO: 1. In addition, two types, a precursor sequence and a mature sequence, were prepared.
(2) 発現用プラスミドの作製
pCold IVプラスミドベクターのNdeI-EcoRIサイトへの挿入を行うため、前もってTAクローニングされたpET24a-LysOX(full3)を鋳型にしてインサート[LysOX(full3)，(mature3)]の増幅を行った。使用したプライマーを以下の表１に示す。
(2) Preparation of expression plasmid
In order to insert the pCold IV plasmid vector into the NdeI-EcoRI site, the insert [LysOX (full3), (mature3)] was amplified using the previously TA-cloned pET24a-LysOX (full3) as a template. The primers used are shown in Table 1 below.
pCold IVプラスミドベクターの制限酵素処理(NdeI、EcoRI)を行い、DNA cleanup kitを使用し、精製した。これをGene-Art(登録商標) Seamless Cloning and Assembly (サーモフィッシャー サイエンティフィック社)の相同組換え法によりインサート導入を行い、反応液をE. coli BL21(DE3)に導入し、形質転換させた。 The pCold IV plasmid vector was treated with restriction enzymes (NdeI, EcoRI) and purified using a DNA cleanup kit. This was inserted by the homologous recombination method of Gene-Art (registered trademark) Seamless Cloning and Assembly (Thermo Fisher Scientific), and the reaction solution was introduced into E. coli BL21 (DE3) and transformed. ..
生えてきたコロニーを培養し、プラスミド抽出後、制限酵素処理を行い、アガロースゲル電気泳動法及びDNAシーケンスでインサートが導入されているかを確認した。
LysOX (full3)：大腸菌用にコドンを最適化したプレ配列を含む前駆体の塩基配列
LysOX (mature3)：大腸菌用にコドンを最適化したプレ配列が除かれた成熟体の塩基配列
The growing colonies were cultured, and after plasmid extraction, restriction enzyme treatment was performed, and it was confirmed by agarose gel electrophoresis and DNA sequence whether the insert was introduced.
LysOX (full3): Precursor base sequence containing codon-optimized presequences for E. coli
LysOX (mature3): Codon-optimized pre-sequenced base sequence for E. coli
(3) LysOXの発現と活性の確認
pCold IV-LysOX(full3，mature3)を用いてE. coli BL21(DE3)を形質転換させた物を5 mlの2YT(Amp)培地で37℃，16時間，230 rpmで前培養した後、5 mlの2×YT培地(Amp)で本培養を2時間，180 rpmで行った。15℃，30分間静置(コールドショック)し、終濃度1 mMの イソプロピルチオガラクトシド(IPTG)を添加し、15℃で24時間，180 rpmで誘導を行った。培養終了後、菌体を集菌し、超音波菌体破砕を行い、粗酵素溶液とし遠心分離により可溶性画分に分けた。可溶性画分について活性測定を行った。活性測定の結果を表２に示す。
(3) Confirmation of LysOX expression and activity
E. coli BL21 (DE3) transformed with pCold IV-LysOX (full3, mature3) was precultured in 5 ml of 2YT (Amp) medium at 37 ° C. for 16 hours at 230 rpm, and then 5 Main culture was performed in ml of 2 × YT medium (Amp) for 2 hours at 180 rpm. The mixture was allowed to stand at 15 ° C. for 30 minutes (cold shock), isopropylthiogalactoside (IPTG) at a final concentration of 1 mM was added, and induction was performed at 15 ° C. for 24 hours at 180 rpm. After completion of the culture, the cells were collected, ultrasonically disrupted, prepared as a crude enzyme solution, and separated into soluble fractions by centrifugation. The activity of the soluble fraction was measured. The results of the activity measurement are shown in Table 2.
表２の結果から、プレ配列を含むLysOX前駆体の方が高い活性を有しており、大腸菌においてLysOX成熟体と比べて発現量が高いことが分かった。 From the results in Table 2, it was found that the LysOX precursor containing the pre-sequence has higher activity and the expression level in Escherichia coli is higher than that in the mature LysOX.
試験例２：LysOX前駆体からLysOX成熟体の調製
[使用菌株及び使用培地]
・使用菌株：E. coli BL21/pCold IV-LysOX(full3)
・使用培地：2×YT培地(Amp final 50μg/ml)[1.6%ポリペプトン、1%酵母エキス、0.5% NaCl]
Test Example 2: Preparation of LysOX mature product from LysOX precursor
[Strain used and medium used]
・ Strain used: E. coli BL21 / pCold IV-LysOX (full3)
-Medium used: 2 x YT medium (Amp final 50 μg / ml) [1.6% polypeptone, 1% yeast extract, 0.5% NaCl]
[使用試薬]
・ペプチダーゼである、ストレプトマイセス・グリセウス(Streptomyces griseus) 由来ペプチダーゼ(SGP)、トリプシン及びキモトリプシンはシグマ-アルドリッチ社の製品を使用した。
・DEAE-トヨパール 650 M、Butyl-トヨパール 650 Mの各担体は東洋紡社から購入した。
[Reagents used]
-Peptidase (SGP), trypsin and chymotrypsin derived from Streptomyces griseus, which are peptidases, were manufactured by Sigma-Aldrich.
-DEAE-Toyopearl 650 M and Butyl-Toyopearl 650 M carriers were purchased from Toyobo.
[操作]
前培養：2×YT培地(Amp, final 50 μg/mL) 5 mL, 37℃, 18時間, 230 rpm
本培養：2×YT培地(Amp, final 50 μg/mL) 120 mL + 前培養液 1.2 mL, 220 rpm, 30℃, O.D.600＝0.4以上になるまで培養。
コールドショック：振盪培養停止後、30分間，15℃で冷却。その後、IPTG(終濃度1 mM)を添加。
低温誘導：15℃, 48時間, 180 rpm
集菌：遠心分離(4℃, 6000 rpm, 25分間)
洗浄：集菌菌体を、20 mM KPB (pH 7.4)で懸濁し、その後遠心(7000 rpm, 20分間)。
菌体破砕：洗浄後、洗浄バッファーを捨て、新たに破砕バッファーを添加して懸濁し、ソニケーターを用いて懸濁液を破砕(40分間)。破砕後、7000 rpm, 30分, 4℃で遠心し、上清を回収。
硫安分画：菌体破砕液上清に硫安を65%飽和になるように添加。30分間静置後、7800 rpm, 20分, 4℃で遠心。
透析：沈殿を20 mM KPB (pH 7.4)で溶かし、外液に20%飽和硫安・20 mM KPB (pH 7.4)を用いて透析。
精製：透析後のサンプルをButyl-トヨパール 650 M、DEAE-トヨパール 650 M及びゲル濾過セファデックス S300を用いて精製
[operation]
Preculture: 2 × YT medium (Amp, final 50 μg / mL) 5 mL, 37 ° C, 18 hours, 230 rpm
Main culture: 2 × YT medium (Amp, final 50 μg / mL) 120 mL + preculture solution 1.2 mL, 220 rpm, 30 ° C, OD600 = 0.4 or more.
Cold shock: After stopping shaking culture, cool at 15 ° C for 30 minutes. Then, IPTG (final concentration 1 mM) was added.
Cold induction: 15 ° C, 48 hours, 180 rpm
Collection: Centrifuge (4 ° C, 6000 rpm, 25 minutes)
Washing: Collected cells are suspended at 20 mM KPB (pH 7.4) and then centrifuged (7000 rpm, 20 minutes).
Crushing cells: After washing, discard the washing buffer, add a new crushing buffer to suspend, and crush the suspension using a sonicator (40 minutes). After crushing, centrifuge at 7000 rpm, 30 minutes, 4 ° C, and collect the supernatant.
Ammonium sulfate fraction: Ammonium sulfate is added to the supernatant of the cell disruption solution so that it is 65% saturated. After standing for 30 minutes, centrifuge at 7800 rpm, 20 minutes, 4 ° C.
Dialysis: Dissolve the precipitate with 20 mM KPB (pH 7.4) and dialyze with 20% saturated ammonium sulfate and 20 mM KPB (pH 7.4) in the external solution.
Purification: Purify post-dialysis samples using Butyl-Toyopearl 650 M, DEAE-Toyopearl 650 M and Gel Filtration Sephadex S300
[酵素分解]
精製酵素を処理する各種ペプチダアーゼ(1.トリプシン、2.キモトリプシン、3.SGP)の1 mg/ml溶液を作製した。精製酵素(LysOX前駆体)をタンパク質濃度が2.0 mg/mlになるよう希釈し、希釈した精製酵素に1/50量の各種ペプチダアーゼを添加した。また、コントロールとしてペプチダーゼを添加しない物も作製した。30℃, 4時間インキュベートした後、終濃度1 mMになるようにEDTA及びPMSF(ジメチルスルホキシドで溶解させた物)を添加し、酵素反応を停止させた。
[Enzymatic decomposition]
A 1 mg / ml solution of various peptidaases (1. trypsin, 2. chymotrypsin, 3.SGP) to be treated with the purified enzyme was prepared. The purified enzyme (LysOX precursor) was diluted to a protein concentration of 2.0 mg / ml, and 1/50 amount of various peptidaase was added to the diluted purified enzyme. In addition, as a control, a product to which peptidase was not added was also produced. After incubating at 30 ° C. for 4 hours, EDTA and PMSF (dissolved in dimethyl sulfoxide) were added to a final concentration of 1 mM to stop the enzymatic reaction.
[タンパク質定量法、LysOX活性測定]
試験例１と同様に行った。
[Protein quantification method, LysOX activity measurement]
The procedure was the same as in Test Example 1.
[結果]
酵素分解を行ったサンプルについて、タンパク質濃度測定、LysOX活性測定(MBTH法)、及びSDS-PAGEを行った。その結果を表３及び図１に示す。
[result]
Protein concentration measurement, LysOX activity measurement (MBTH method), and SDS-PAGE were performed on the enzymatically degraded sample. The results are shown in Table 3 and FIG.
表３の結果から、精製されたLysOX前駆体を各種ペプチダーゼで処理することでその比活性は大幅に上昇した。未処理の時と比べてその比活性は最大で60〜70倍近く上昇することが確認された。また、図１のSDS-PAGEによるバンド位置の確認から、LysOX前駆体とはバンドの位置が変化し、約56 kDaのところにバンドが現れることが分かった。LysOX成熟体の分子量は56 kDaであり、これに合致していた。 From the results in Table 3, the specific activity of the purified LysOX precursor was significantly increased by treating it with various peptidases. It was confirmed that the specific activity increased up to 60 to 70 times as much as that in the untreated state. Further, from the confirmation of the band position by SDS-PAGE in FIG. 1, it was found that the band position changed from that of the LysOX precursor and the band appeared at about 56 kDa. The molecular weight of the mature LysOX was 56 kDa, which was consistent with this.
また、ペプチダーゼ処理後の精製酵素サンプルとペプチダーゼ未処理の精製酵素サンプルのN末端アミノ酸配列を解析すると次のようになった。
1.ペプチダーゼ未処理 MDNVD
2.トリプシン処理 AE
3.キモトリプシン処理 AE
4.SGP処理 AEE
In addition, the N-terminal amino acid sequences of the purified enzyme sample after peptidase treatment and the purified enzyme sample not treated with peptidase were analyzed as follows.
1. Peptidase untreated MDNVD
2. Trypsin treatment AE
3. Chymotrypsin treatment AE
4. SGP processing AEE
ペプチダーゼ処理サンプルの方からはLysOX前駆体からプレ配列部分を除いたLysOX成熟体のN末端アミノ酸配列と同じアミノ酸残基が検出されており、ペプチダーゼ処理によってLysOX前駆体のプレ配列が除去されることが示唆される結果となった。一方で、ペプチダーゼ未処理サンプルのN末端アミノ酸配列は、LysOX前駆体のプレ配列のN末端アミノ酸配列と同じ配列が検出されており、ペプチダーゼ未処理の酵素はLysOX成熟体にプレ配列がついたままの物が多いことがわかった。 In the peptidase-treated sample, the same amino acid residue as the N-terminal amino acid sequence of the LysOX mature product excluding the pre-sequence part from the LysOX precursor was detected, and the pre-sequence of the LysOX precursor was removed by the peptidase treatment. Was suggested. On the other hand, in the N-terminal amino acid sequence of the peptidase-untreated sample, the same sequence as the N-terminal amino acid sequence of the pre-sequence of the LysOX precursor was detected, and the peptidase-untreated enzyme remained pre-sequenced to the LysOX mature product. It turned out that there are many things.
試験例３：L-リシンに対する活性及び基質特異性の検討
次のサンプル(1)〜(3)を以下の試験で使用した。
(1) LysOX前駆体原液(上記で調製及び精製した物)
(2) SGP処理LysOX(30℃,3時間)
(3) T.virideフスマ培養LysOX
Test Example 3: Examination of activity and substrate specificity for L-lysine The following samples (1) to (3) were used in the following tests.
(1) LysOX precursor stock solution (prepared and purified above)
(2) SGP treatment LysOX (30 ℃, 3 hours)
(3) T.viride Fusuma culture LysOX
＜サンプル(2)及び(3)の調製＞
(2) SGP処理したLysOX成熟体
LysOX前駆体原液を1.5倍希釈し、以下の処理を行った。処理液は更に2倍希釈されるので、最終的に前駆体タンパク質濃度は3 mg/ml程度に調整したことになった。
100μL LysOX前駆体原液の1.5倍希釈液
100μL 0.02M KPB, pH7.4
10μL 10 mg/mL SGP
<Preparation of samples (2) and (3)>
(2) SGP-treated LysOX mature body
The LysOX precursor stock solution was diluted 1.5-fold and the following treatment was performed. Since the treatment solution was further diluted 2-fold, the precursor protein concentration was finally adjusted to about 3 mg / ml.
1.5-fold dilution of 100 μL LysOX precursor stock solution
100 μL 0.02M KPB, pH 7.4
10 μL 10 mg / mL SGP
上記の混合液を30℃、3時間インキュベーションした後、7倍希釈し、アミノ酸23種類について活性測定した(サンプルは原液の22.05倍希釈)。 The above mixed solution was incubated at 30 ° C. for 3 hours, then diluted 7-fold, and the activity of 23 amino acids was measured (sample was diluted 22.05-fold from the stock solution).
(3) 元菌から培養・精製したLysOX
トリコデルマ・ビリデY244-2株をフスマ培養した培養物から精製したLysOX 100 U/mlを4倍希釈し、アミノ酸23種類について活性を測定した。
(3) LysOX cultured and purified from the original bacteria
LysOX 100 U / ml purified from a culture in which Trichoderma viride Y244-2 strain was cultured in bran was diluted 4-fold, and the activity of 23 amino acids was measured.
＜方法＞
１．電極法による活性測定
ハンザテック社製酸素電極(酸素電極がセルの底に密着)のジャケットに恒温水を循環させて電極チャンバー全体を30℃に保ち、電極セルに1 mlの10 mMアミノ酸基質溶液(0.1 M KPB, pH7.4)を入れて密閉した。酸素濃度を示す電極からの信号が安定した後、チャンバーセルの蓋の細い穴から10μlのLysOX被験酸素液をインジェクションして、密閉セル内の10 mMアミノ酸基質溶液(0.1 M KPB, pH7.4)中に含まれている酸素(30℃で0.23μmol/ml)の減少速度を測定した。
<Method>
1. 1. Activity measurement by the electrode method A constant temperature water is circulated through the jacket of an oxygen electrode manufactured by Hansa Tech (the oxygen electrode is in close contact with the bottom of the cell) to keep the entire electrode chamber at 30 ° C. 0.1 M KPB, pH 7.4) was added and sealed. After the signal from the electrode indicating the oxygen concentration has stabilized, 10 μl of LysOX test oxygen solution is injected through the narrow hole in the lid of the chamber cell, and a 10 mM amino acid substrate solution (0.1 M KPB, pH 7.4) in a closed cell. The rate of decrease of oxygen contained therein (0.23 μmol / ml at 30 ° C) was measured.
２．SDS-PAGE
各サンプルとDTT＋Ez溶液(アトー社)を1:1の割合で混合し(計20μL)、 100℃、5分間加熱、冷却・遠心後、全量(20μL)をアプライした。希釈バッファーは0.02 M KPB, pH7.4を使用した。
2. 2. SDS-PAGE
Each sample and DTT + Ez solution (Ato) were mixed at a ratio of 1: 1 (20 μL in total), heated at 100 ° C for 5 minutes, cooled and centrifuged, and then the total amount (20 μL) was applied. The dilution buffer used was 0.02 M KPB, pH 7.4.
＜結果＞
サンプル(1)〜(3)の活性測定の結果を以下の表に示す。
<Result>
The results of activity measurement of samples (1) to (3) are shown in the table below.
LysOX前駆体で、L-Lysへの活性は0.45 U/mLであるが、これを30℃、3時間SGP処理すると276.0 U/mLとなり、613倍の活性上昇を示した。また、前駆体をSGP処理したサンプルは、T.virideから精製したLysOXとほぼ同様の基質特異性を示した。 The activity of LysOX precursor to L-Lys was 0.45 U / mL, but when it was treated with SGP at 30 ° C for 3 hours, it became 276.0 U / mL, showing a 613-fold increase in activity. In addition, the SGP-treated sample of the precursor showed almost the same substrate specificity as LysOX purified from T. viride.
SDS-PAGEの結果を図２に示す。図２から、LysOX前駆体をSGP処理すると、T.virideから精製したLysOXと同等のサブユニットとなっていた。 The result of SDS-PAGE is shown in FIG. From FIG. 2, when the LysOX precursor was treated with SGP, it became a subunit equivalent to LysOX purified from T. viride.
試験例４：熱安定性試験
次のサンプル(i)〜(ii)を以下の試験で使用した。
(i) LysOX前駆体原液
(ii) SGP添加(30℃, 3時間, SGP処理)
Test Example 4: Thermal stability test The following samples (i) to (ii) were used in the following tests.
(i) LysOX precursor stock solution
(ii) Addition of SGP (30 ℃, 3 hours, SGP treatment)
＜方法＞
SGP添加
700μL LysOX前駆体原液の10倍希釈液
700μL 0.02M KPB, pH7.4
70μL 1 mg/mL SGP
<Method>
SGP addition
700 μL LysOX precursor stock solution 10-fold dilution
700 μL 0.02M KPB, pH 7.4
70 μL 1 mg / mL SGP
上記の混合液を30℃、3時間インキュベーションし、続いて40〜90℃で100μLずつ30分間インキュベーション後に、急冷した。各サンプル10μLを10 mM L-Lys溶液(0.1 M KPB, pH7.4) 1 mLにインジェクションし、活性測定を行った(サンプルは原液の21倍希釈)。 The above mixture was incubated at 30 ° C. for 3 hours, followed by incubation at 40 to 90 ° C. for 100 μL each for 30 minutes, and then rapidly cooled. 10 μL of each sample was injected into 1 mL of a 10 mM L-Lys solution (0.1 M KPB, pH 7.4) and the activity was measured (the sample was diluted 21-fold with the stock solution).
LysOX前駆体原液は、30、50、60℃で30分間インキュベーションした後、20μLを10 mM L-Lys溶液(0.1 M KPB, pH7.4) 1 mLにインジェクションし、活性測定を行った。 The LysOX precursor stock solution was incubated at 30, 50, and 60 ° C. for 30 minutes, and then 20 μL was injected into 1 mL of a 10 mM L-Lys solution (0.1 M KPB, pH 7.4) to measure the activity.
活性の測定は試験例３と同様に行った。 The activity was measured in the same manner as in Test Example 3.
＜結果＞
各サンプルの活性測定の結果を表５及び図３に示す。
<Result>
The results of activity measurement of each sample are shown in Table 5 and FIG.
SGP処理したLysOXの熱安定性を検討した結果、30分間の処理で30℃から60℃まで活性はほぼ維持され、65℃で90.7%、70℃で37.4%となり、75℃で失活した。したがって、SGP処理したLysOXは、60℃，30分間の処理で安定と言える。 As a result of examining the thermal stability of LysOX treated with SGP, the activity was almost maintained from 30 ° C to 60 ° C after 30 minutes of treatment, 90.7% at 65 ° C, 37.4% at 70 ° C, and inactivated at 75 ° C. Therefore, it can be said that LysOX treated with SGP is stable after treatment at 60 ° C for 30 minutes.
LysOX前駆体では、50℃で34.4%、60℃でゼロとなり、加熱に対して不安定であった。 The LysOX precursor was 34.4% at 50 ° C and zero at 60 ° C, which was unstable to heating.
試験例５：LysOXのがん細胞に対する細胞毒性の検討
＜方法＞
・MTSアッセイ法による細胞生存率の測定
大腸菌形質転換体由来LysOX前駆体及びペプチダーゼ処理して作製したLysOX成熟体の抗腫瘍性を調べるため、ヒト腫瘍細胞株としてA431(ヒト扁平上皮がん細胞株)及びMDA-MB-231(ヒト乳線癌細胞株)の2種類を用い、ウォータージャケット型CO2/マルチガスインキュベータ(アステック社)で培養した。
Test Example 5: Examination of Cytotoxicity of LysOX to Cancer Cells <Method>
-Measurement of cell viability by MTS assay
In order to investigate the antitumor properties of LysOX precursors derived from Escherichia coli transformants and LysOX mature products prepared by peptidase treatment, A431 (human squamous cell line) and MDA-MB-231 (human milk) were used as human tumor cell lines. Two types of line cancer cell lines) were used and cultured in a water jacket type CO 2 / multi-gas incubator (Astec).
1×105個/mlの濃度のA431及びMDA-MB-231細胞株を96穴プレートの各ウェルに100μlずつ播種し、37℃で24時間、CO2/マルチガスインキュベータで5%CO2・20%O2条件下で培養した。1, 2.5, 5, 10 mU/mlの濃度になるようにLysOX前駆体或いはLysOX成熟体を各細胞株に添加し、3日間培養した。培養3日後に、MTSアッセイを行った。各ウェルに20μlのMTSアッセイ試薬(CellTiter 96 AQueous One Solution Cell Proliferation Assay，プロメガ社)を添加し、37℃, 1時間インキュベートした後に490 nmの吸光度を測定した。細胞生存率はコントロール(無添加)に対する吸光度の割合として算した。各細胞株に対するアッセイは4連で行った。 100 μl of A431 and MDA-MB-231 cell lines at a concentration of 1 × 10 5 cells / ml were seeded in each well of a 96-well plate at 37 ° C for 24 hours, 5% CO 2 in a CO 2 / multi-gas incubator. The cells were cultured under 20% O 2 conditions. LysOX precursor or LysOX mature product was added to each cell line to a concentration of 1, 2.5, 5, 10 mU / ml and cultured for 3 days. After 3 days of culture, an MTS assay was performed. 20 μl of MTS assay reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega) was added to each well, and the mixture was incubated at 37 ° C. for 1 hour, and then the absorbance at 490 nm was measured. Cell viability was calculated as the ratio of absorbance to control (no additives). The assay for each cell line was performed in 4 series.
＜結果＞
がん細胞A431細胞株及びMDA-MB-231細胞株に対するLysOX前駆体及びLysOX成熟体の細胞毒性を調べた結果を図４に示す。LysOX前駆体及びLysOX成熟体ともに10.0 mU/mlの濃度で添加した際に、両がん細胞に対して顕著な細胞毒性を示した。
<Result>
The results of examining the cytotoxicity of LysOX precursor and LysOX mature product against cancer cell A431 cell line and MDA-MB-231 cell line are shown in FIG. Both LysOX precursor and LysOX mature were significantly cytotoxic to both cancer cells when added at a concentration of 10.0 mU / ml.
Claims (2)
(1) 配列番号２に記載される塩基配列をコドン最適化したコドン最適化核酸が導入された形質転換体を液体培地で培養し、培養物からL-リシンα-オキシダーゼを回収する工程であって、該形質転換体はL-リシンα-オキシダーゼのプレ配列を切断するペプチダーゼを有しない、工程。 Method for producing L-lysine α-oxidase including the following steps:
(1) A step of culturing a transformant into which a codon-optimized nucleic acid obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO: 2 has been introduced in a liquid medium and recovering L-lysine α-oxidase from the culture. The transformant does not have a peptidase that cleaves the pre-sequence of L-lysine α-oxidase.
(2) 工程(1)で得られたL-リシンα-オキシダーゼをペプチダーゼ処理する工程。 The production method according to claim 1 , further comprising the following steps:
(2) A step of peptidase treatment of the L-lysine α-oxidase obtained in step (1).
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