WO2002004602A1 - Transformed yeast presenting protein a on the cell surface layer - Google Patents

Transformed yeast presenting protein a on the cell surface layer Download PDF

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Publication number
WO2002004602A1
WO2002004602A1 PCT/JP2001/005788 JP0105788W WO0204602A1 WO 2002004602 A1 WO2002004602 A1 WO 2002004602A1 JP 0105788 W JP0105788 W JP 0105788W WO 0204602 A1 WO0204602 A1 WO 0204602A1
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yeast
protein
fragment
igg
transformed yeast
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PCT/JP2001/005788
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French (fr)
Japanese (ja)
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Atsuo Tanaka
Mitsuyoshi Ueda
Yutaka Teranishi
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Mitsubishi Chemical Corporation
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Publication of WO2002004602A1 publication Critical patent/WO2002004602A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a transformed yeast which displays protein A or a fragment thereof on the cell surface, and a method for analyzing the Fc portion of IgG using the same.
  • the present invention relates to a method of expressing, on a cell surface, protein A or a fragment thereof obtained by introducing a yeast expression vector containing protein A or a fragment thereof having an affinity for the Fc portion of IgG into yeast. And a method for analyzing the Fc portion of IgG using the same.
  • heterologous proteins using genetic recombination technology is used in various industries, and Escherichia coli, yeast, insect cells, animal cells, and the like are mainly used as hosts.
  • yeast which is a eukaryotic microorganism
  • Yeast mass culture methods have been established, and expression systems using various yeasts as hosts have been developed.
  • post-translational modifications such as acetylation, phosphorylation and addition of sugar chains of the produced protein are considered to be similar to those of animal cells.
  • E. coli prokaryotic methods
  • yeast prokaryotic methods
  • E. coli are not always effective for all proteins, and reproduce complex post-translational modifications of eukaryotic proteins or the same conformational structure as the natural form. It is not always easy.
  • Escherichia coli also has a specific endotoxin, which may be a contaminant in the final product.
  • methods using animal cells or insect cells are more difficult to handle than microorganisms, require more cultivation, and have lower production efficiency. Therefore, we will establish a protein display method using yeast as a host. If it is possible, it will be very useful in screening for a protein having a desired function or a new function.
  • Protein A of Gram-positive bacterium Staphylococcus aureus has a selective and specific affinity for the Fc portion of IgG produced in humoral immunity. Therefore, if yeast capable of expressing protein A on the cell surface can be prepared, specific adsorption of combinatorial antibody libraries and antibody components with Fc, and stable supply of catalytic antibodies with interesting activities will be possible. This is useful for screening new functional molecules. Disclosure of the invention
  • An object of the present invention is to provide a yeast capable of expressing and displaying protein A or a fragment thereof on the cell surface.
  • Another problem to be solved by the present invention is to provide a method for detecting or isolating the Fc portion of IgG using yeast capable of expressing and displaying protein A or a fragment thereof on the cell surface.
  • the present inventors have conducted intensive studies and found that by transforming the gene encoding the ZZ domain of protein A into yeast using a certain yeast expression vector, the ZZ domain of protein A was transferred to the yeast cell surface.
  • the present inventors have found that they can be expressed and presented in the present invention, and have completed the present invention.
  • a transformed yeast displaying protein A or a fragment thereof on the cell surface (1) A transformed yeast displaying protein A or a fragment thereof on the cell surface.
  • yeast expression vector is a multicopy plasmid or a chromosome-integrated plasmid.
  • a yeast expression vector comprising a promoter, a secretory signal sequence, a DNA encoding protein A or a fragment thereof, a yeast-derived aglutinin gene or a fragment thereof in this order in the 5 'to 3' direction.
  • FIG. 1 shows the structure of plasmid pEZZ18.
  • FIG. 2 shows the sequence of the gene encoding the ZZ domain.
  • FIG. 3 is a diagram showing the construction of the cell surface expression multicopy plasmid pCZZ and the chromosome integration plasmid pICZZ of the domain.
  • FIG. 4 shows the sequence of a fragment gene encoding Fc of human IgG1.
  • FIG. 5 is a diagram showing the structure of the multicopy plasmid pCGFP-Fc. BEST MODE FOR CARRYING OUT THE INVENTION
  • Protein A of Gram-positive bacterium Staphylococcus aureus has a selective and specific affinity for the Fc portion of IgG produced in humoral immunity.
  • the yeast surface expression vector developed by the present inventors we succeeded for the first time to display the ZZ domain, which encodes the part of protein A that reacts with the Fc part of IgG, on the yeast cell surface. did.
  • the domain ZZ domain of protein A By displaying the domain ZZ domain of protein A on the surface of yeast cells, it is possible to perform specific adsorption of combinatorial antibody libraries and antibody components having Fc, and stable supply of catalytic antibodies having interesting activities. This is useful for screening new functional molecules.
  • the technology of the present invention can be applied to the establishment of a new separation and purification method using a secreted protein having Fc.
  • the yeast of the present invention is a transformed yeast obtained by transforming a yeast expression vector having DNA encoding protein A or a fragment thereof into yeast.
  • the yeast of the present invention can display protein A or a fragment thereof on the cell surface.
  • the expression vector used in the present invention contains a yeast-derived aglutinin gene or a fragment thereof.
  • yeast for obtaining the aglutinin gene include a group of microorganisms described as yeast in "Classification and Identification of Microorganisms", edited by Takeharu Hasegawa (Society Press, Sen-ichi, 1975).
  • preferred yeasts are those belonging to the genus Saccharomyces, and include, for example, Saccharomyces cerevisiae MT8-1. Detailed properties of yeast are described in Tajima, M. et al. 3 Yeast 1, 67-77 (1985).
  • the agglutinin may be any of a-agglutinin and hyaglutinin.
  • the molecular structure of hyaglutinin is, in order from the N-terminal, a secretory signal sequence, an agglutinin active region, a cell surface internal region, and a GPI anchor (glycosylation). (Phosphatidylinositol) It has a configuration of an adhesion signal region.
  • the fragment of the aglutinin gene region can express a heterologous protein on the yeast cell surface.
  • a particularly preferred example is a gene sequence encoding 320 amino acids from the C-terminal of Saccharomyces spp. And a DNA fragment containing the region.
  • This DNA fragment contains the GPI anchor attachment signal region.
  • This DNA fragment can also be obtained by cutting out a plasmid pGA11 containing the fragment described in Murai, T. et al. Applied and Environmental Microbiology, 63, 1366-1366 (1997) with a restriction enzyme. .
  • the expression vector of the present invention containing the agglutinin gene or a fragment thereof and the DNA encoding protein A or a fragment thereof can be obtained by introducing the gene into an expression vector usually used in yeast.
  • Preferred examples of the expression vector include, for example, pICAS 1 (Murai et al., Appl. Environ. Microbiol., 64, 4857-4861 (1998)), pCAS1 (Murai et al., Appl. Environ. Microbiol. ., 64, 4857-4861 (1998)).
  • the expression vector used in the present invention usually contains a promoter and a secretory signal sequence (for example, a fungal-derived secretory signal sequence).
  • the promoter is not particularly limited as long as it is a promoter generally used for expression in yeast, but a preferred column is glyceroaldehyde triphosphate dehydrogenase of Saccharomyces cerevisiae. Promote of Ichinose (GAPDH) (Sawani-Hatanaka, H., T. et al., Biosci. Biotechnol.
  • Examples of the secretory signal sequence include a signal region usually used for expression and secretion in yeast, for example, glucoa derived from Rhizopus oryzae.
  • the secretory signal region of the Mirase gene can be used.
  • the above-mentioned promoter and secretory signal sequence can be obtained by a known method, for example, by using a primer prepared based on the known sequence and performing a PCR method using the genomic DNA of the microorganism as type III. From where you can get.
  • DNA encoding protein A or a fragment thereof is used as DNA encoding a protein to be expressed and displayed on the cell surface.
  • Protein A is a protein having a molecular weight of about 42,000, which accounts for 5% of the cell wall component of the Gram-positive bacterium Staphylococcus aureus (Yellow Staphylococcus aureus). ) Specifically binds to the Fc fragment. By utilizing this property of protein A, it can be bound to an appropriate carrier and used for quantification of antibody-producing cells, purification of IgG, separation of antibody-bound cells, and the like.
  • Fragments of Protein A that can be used in the present invention preferably include at least a portion that has an affinity for the Fc portion of an IgG. More specifically, a region encoding a portion that reacts with the Fc portion of IgG includes a ZZ domain.
  • promoter In a preferred embodiment of the expression vector used in the present invention, promoter, secretory signal sequence, DNA encoding protein A or a fragment thereof, yeast-derived aglutinin gene or a fragment thereof (for example, C-terminal of ⁇ -agglutinin) D ⁇ fragment) that encodes the fragment in this order.
  • the promoter, secretory signal sequence, multicloning site, and agglutinin gene or a fragment thereof are arranged in this order in the plasmid vector.
  • it can be constructed by introducing a protein or a DN thereof encoding a fragment thereof into the multicloning site.
  • Conventional recombinant DNA techniques are used to construct such a recombinant expression vector. Technique is available.
  • the expression vector used in the present invention may contain a selection marker gene such as an antibiotic resistance gene.
  • examples of yeast to be transformed by the above-described recombinant vector include, for example, "Classification and Identification of Microorganisms" (edited by Takeharu Hasegawa).
  • yeast is not particularly limited, and includes, for example, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula and the like.
  • yeast belonging to the genus Saccharomyces and specific examples thereof include Saccharomyces cerevisiae.
  • the detailed properties of yeast are described in Tajima, M. et al., Yeast 1, 67-77 (1985).
  • Transformation of yeast with an expression vector can be performed by a conventional method, and a transformant can be obtained by, for example, a lithium acetate method, an electric pulse method, a protoplast method, or the like. Methods for culturing transformants are also known and described, for example, in "MD Rose et al.,-Methods In Yeast Genetics", Cold Sprmg Harbor Laboratory Press (1990).
  • the yeast of the present invention can display protein A or a fragment thereof on the cell surface, it is possible to screen for any substance that interacts with the displayed protein A or a fragment thereof (for example, the Fc portion of IgG). it can.
  • a transformed yeast capable of displaying the protein A of the present invention or a fragment thereof on the cell surface with a sample containing the Fc portion of IgG or a protein fragment containing the same (for example, a combinatorial antibody library).
  • a sample containing the Fc portion of IgG or a protein fragment containing the same for example, a combinatorial antibody library.
  • the type and labeling method for labeling the protein fragment can be appropriately selected from those known to those skilled in the art.
  • an enzyme for example, horseradish peroxidase, alkaline phosphatase, glucose oxidase,? -Galactosidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme , Maletodehydrogenase, glucose-6-phospho-todehydrogenase and the like can be used as labels.
  • the labeled antibody can be detected by reacting the substrate of the above-mentioned labeled enzyme and measuring the reaction by color development or the like.
  • a substrate such as 2,2'-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is used.
  • ABTS 2,2'-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid
  • a dye for example, the antibody or a fragment thereof can be labeled with a fluorescent dye such as FITC (fluorescein isothiosinate) or TRITC (tetramethylrhodamine B isothiosinate).
  • colloid metal and colored latex can be used as the label.
  • the colloid metal include metal colloid particles which are dispersed particles of gold sol, silver sol, selenium sol, tellurium sol and platinum sol.
  • the size of colloidal metal particles is usually about 3 to 60 dishes in diameter.
  • a synthetic latex such as polystyrene latex colored with respective pigments such as red and blue can be given.
  • Natural latex such as natural rubber latex can be used as the latex.
  • the size of the colored latex can be selected from several tens to several hundreds of nm in diameter. Commercially available products can be used for these coloring markers as they are. Alternatively, it can be produced by a method known per se.
  • an affinity label for example, biotin
  • an isotope label for example, 125 I
  • fusion protein comprising the Fc portion of IgG or a protein fragment containing the Fc portion and a fluorescent protein (eg, green fluorescent protein (GFP) or a mutant thereof) can also be used.
  • a fluorescent protein eg, green fluorescent protein (GFP) or a mutant thereof
  • Analysis such as the enzyme antibody method, immunohistochemical staining method, immunoplot method, direct fluorescent antibody method or indirect fluorescent antibody method using the labeled antibody described above can be performed by a method well known to those skilled in the art. Also, those skilled in the art can appropriately select.
  • Example 1 Amplification of ZZ domain showing specific affinity for Fc portion of IgG of protein A and construction of cell surface expression plasmid
  • Plasmid pEZZ18 was isolated from E. coli harboring plasmid pEZZ18 (Amersham Sharm Pharmacia, Fig. 1) grown according to a conventional method, and this was mirror-typed and subjected to PCR. In was prepared.
  • 5 '-gaattcatgcaactgttcaatttgcc-3 5 a (sequence number 1) and 5 5 -cttgttaaatcagtagtggttgag-3 5 (SEQ ID NO: 2) as primers one, 20 dNTPs mixture Zmol, each Buraima one 50 pmol, 1 n a 72 minutes at 94 ° C; 1 minute at 55 ° C in a 100 ⁇ 1 reaction system consisting of type ⁇ ⁇ plasmid DNA, pfu polymerase (manufactured by Stratagene) and PCR buffer of PCR polymerase. The PCR reaction was performed at 30 ° C. for 30 seconds at 30 ° C.
  • the gene (FIG. 2 or SEQ ID NO: 3) encoding the ZZ domain amplified in this manner is cut with SacII and Xhol, and then subjected to electrophoresis. It was purified and further extracted with a QIAEXII (QIAGEN Co.) gel extraction kit.
  • the present inventors have GAPDH promoter Isseki yeast Saccharomyces cerevisiae developed - from end edge - 3 5 of the gene coding for the gene and the multiple cloning site encoding a secretory signal Rhizopus oryzae Me glucoamylase and carry one Aguruchinin Multicopy plasmid pCAS1 (Murai et al., Appl.Environ. Microbiol., 64, 4857-4861 (1998 »and chromosomal integration plasmid pICAS1 (Murai et al., Appl. Environ. Microbiol., 64, 4857-4861 (1998)) was digested with SacII and Xhol (Fig. 3) .Each fragment was purified by electrophoresis and then QIAEXII (QIAGEN Co. .) Extracted with gel extraction kit.
  • the gene encoding the ZZ domain obtained above was ligated to a fragment obtained by cutting the multicopy plasmid pCAS1 and the chromosome-integrated plasmid pICAS1 with SacII and Xhol, to express the cell surface expression of the ZZ domain on the multicopi plasmid.
  • PCZZ and chromosomal integration plasmid pICZZ were constructed (Fig. 3).
  • Example 2 Preparation of yeast displaying the ZZ domain of protein A on the cell surface
  • pCZZ and pI CZZ were each introduced into the yeast Saccharomyces cerevisiae MT8-1 by the lithium acetate method.
  • SD-Trp solid medium was obtained (0.03% Leu, 0.02% His , 0.02% Ade, 0.02% Ura, 6.7% Yeast nitrogen base, 2% glucose 3 2% agar) in transformants comprising a Trp +.
  • the presence of the introduced gene in the host cell was confirmed by PCR and Southern blotting.
  • These transformants were cultured in SD-Trp liquid medium at 30 ° C. for 24 hours, washed with a PBS buffer, and then fixed with 3.7% formaldehyde.
  • Example 3 Secretion using yeast displaying one domain of protein A on the cell surface Separation and recovery of Fc-fused Green Fluorescent Protein (GFP)
  • a multicopy-type plasmid pCGFP-Fc (Fig. 5) was prepared and introduced into the yeast Saccharomyces cerevisiae MT8-l by the lithium acetate method.
  • the transformed yeast of the present invention can display the domain ZZ domain of protein A on the surface of yeast cells, specifically adsorbs antibody components having a compinatrial antibody library or Fc, and a catalytic antibody having an interesting activity. This is useful when performing stable supply of proteins, and is useful, for example, for screening new functional molecules.
  • the transformed yeast of the present invention can also be applied to the establishment of a new separation and purification method using secretory proteins with Fc.

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Abstract

It is intended to provide a yeast capable of expressing and presenting protein A or its fragment on the cell surface layer. A transformed yeast presenting protein A or its fragment on the cell surface layer which is obtained by transforming a yeast with the use of an expression vector for yeast having a DNA encoding protein A or its fragment.

Description

プロティン Aを細胞表層に提示する形質転換酵母 技術分野  Transformed yeast displaying protein A on cell surface
本発明は、 プロテイン A又はその断片を細胞表層に提示する形質転換酵母、 並 びにそれを用いた IgGの Fc部分の分析方法に関する。  The present invention relates to a transformed yeast which displays protein A or a fragment thereof on the cell surface, and a method for analyzing the Fc portion of IgG using the same.
より詳細には、 本発明は、 IgGの Fc部分と親和性を有するプロテイン A又はそ の断片を含む酵母用発現ベクターを酵母に導入して得られる該プロティン Aまた はその断片を細胞表層に発現する形質転換酵母、 並びにそれを用いた IgGの Fc 部分の分析方法に関する。 背景技術  More specifically, the present invention relates to a method of expressing, on a cell surface, protein A or a fragment thereof obtained by introducing a yeast expression vector containing protein A or a fragment thereof having an affinity for the Fc portion of IgG into yeast. And a method for analyzing the Fc portion of IgG using the same. Background art
遺伝子組換え技術を応用した異種タンパク質の生産は様々な産業に用いられて おり、 その宿主として主に大腸菌、 酵母、 昆虫細胞、 動物細胞等が用いられてい る。 異種タンパク質、 特に真核生物由来のタンパク質を生産するためには、 真核 微生物である酵母が好適であると考えられている。酵母は大量培養方法が確立し ており、 これまでに種々の酵母を宿主とした発現系が開発されてきた。 酵母を宿 主として使用した場合、 生産されてくるタンパク質のァセチル化、 リン酸化およ び糖鎖の付加などの翻訳後修飾も動物細胞の場合と似ていると考えられている。 このため、 動物細胞由来のタンパク質を発現させる宿主としては、 酵母を用いた 異種タンパク質生産法を用いることが有利である。 これに対して、 大腸菌などの 原核生物を用いる方法では必ずしも全てのタンパク質について有効であるわけで はなく、 真核生物由来のタンパク質の複雑な翻訳後修飾あるいは天然体と同じ立 体構造を再現することは必ずしも容易ではない。 また大腸菌には特有のェンドト キシンが存在し、 最終製品の夾雑物になる可能性がある。 一方、 動物細胞ないし 昆虫細胞を用いる方法は、 取扱が微生物より難しく、 培養にコストがかかり、 か つ生産効率も悪い。 そこで、 酵母を宿主とした蛋白質ディスプレイ法を確立する ことができれば、 所望の機能又は新たな機能を有する蛋白質のスクリーニングな どにおいて非常に有用である。 The production of heterologous proteins using genetic recombination technology is used in various industries, and Escherichia coli, yeast, insect cells, animal cells, and the like are mainly used as hosts. In order to produce a heterologous protein, particularly a protein derived from a eukaryote, yeast, which is a eukaryotic microorganism, is considered to be suitable. Yeast mass culture methods have been established, and expression systems using various yeasts as hosts have been developed. When yeast is used as a host, post-translational modifications such as acetylation, phosphorylation and addition of sugar chains of the produced protein are considered to be similar to those of animal cells. For this reason, it is advantageous to use a heterologous protein production method using yeast as a host for expressing a protein derived from animal cells. On the other hand, prokaryotic methods such as E. coli are not always effective for all proteins, and reproduce complex post-translational modifications of eukaryotic proteins or the same conformational structure as the natural form. It is not always easy. Escherichia coli also has a specific endotoxin, which may be a contaminant in the final product. On the other hand, methods using animal cells or insect cells are more difficult to handle than microorganisms, require more cultivation, and have lower production efficiency. Therefore, we will establish a protein display method using yeast as a host. If it is possible, it will be very useful in screening for a protein having a desired function or a new function.
グラム陽性菌 Staphylococcus aureusのプロテイン Aは、 液性免疫における産 生抗体 IgGの Fc部分と選択的かつ特異的に強い親和性を有する。従って、プロテ イン Aを細胞表層に発現することができる酵母を作製することができれば、 コン ビナトリアル抗体ライブラリ一や Fcをもつ抗体成分の特異的吸着、並びに興味深 い活性を持つ触媒抗体の安定供給などを行うことが可能になり、 新規な機能分子 のスクリーニング等に有用である。 発明の開示  Protein A of Gram-positive bacterium Staphylococcus aureus has a selective and specific affinity for the Fc portion of IgG produced in humoral immunity. Therefore, if yeast capable of expressing protein A on the cell surface can be prepared, specific adsorption of combinatorial antibody libraries and antibody components with Fc, and stable supply of catalytic antibodies with interesting activities will be possible. This is useful for screening new functional molecules. Disclosure of the invention
本発明が解決しょうとする課題は、 プロテイン A又はその断片を細胞表層に発 現及び提示させることができる酵母を提供することである。  An object of the present invention is to provide a yeast capable of expressing and displaying protein A or a fragment thereof on the cell surface.
本発明が解決しょうとする別の課題は、 プロテイン A又はその断片を細胞表層 に発現及び提示させることができる酵母を用いて IgGの Fc部分を検出又は単離す る方法を提供することである。  Another problem to be solved by the present invention is to provide a method for detecting or isolating the Fc portion of IgG using yeast capable of expressing and displaying protein A or a fragment thereof on the cell surface.
本発明者らは鋭意検討した結果、 ある一定の酵母用発現ベクターを用いてプロ ティン Aの Z Zドメインをコードする遺伝子を酵母に形質転換することにより、 該プロテイン Aの Z Zドメインを酵母の細胞表層に発現及び提示できることを見 出し、 本発明を完成するに至った。  The present inventors have conducted intensive studies and found that by transforming the gene encoding the ZZ domain of protein A into yeast using a certain yeast expression vector, the ZZ domain of protein A was transferred to the yeast cell surface. The present inventors have found that they can be expressed and presented in the present invention, and have completed the present invention.
即ち、 本発明によれば以下の発明が提供される。  That is, according to the present invention, the following inventions are provided.
( 1 ) プロティン A又はその断片を細胞表層に提示する形質転換酵母。  (1) A transformed yeast displaying protein A or a fragment thereof on the cell surface.
( 2 ) プロテイン A又はその断片が I g Gの; c部分と親和性を有する部分で ある、 （ 1 ) に記載の形質転換酵母。  (2) The transformed yeast according to (1), wherein the protein A or a fragment thereof is a portion having an affinity for the c portion of IgG.
( 3 ) プロテイン A又はその断片が少なくとも Z Zドメインを含む断片である、 ( 1 ) 又は （ 2 ) に記載の形質転換酵母。  (3) The transformed yeast according to (1) or (2), wherein the protein A or a fragment thereof is a fragment containing at least a ZZ domain.
( 4 ) プロティン A又はその断片をコードする D N Aを有する酵母用発現べク 夕一を酵母に形質転換することにより得られる、 （1 ) から （3 ) の何れかに記 載の形質転換酵母。 (4) The method according to any of (1) to (3), which is obtained by transforming a yeast expression vector having a DNA encoding protein A or a fragment thereof into yeast. The transformed yeast described above.
(5) 酵母用発現べクタ一が、 プロモー夕一、 分泌シグナル配列、 プロテイン A又はその断片をコードする DNA、 酵母由来のァグルチニン遺伝子又はその断 片をこの順序で 55 から 35 方向に含むことを特徴とする、 （4) に記載の形質 (5) Kuta one base expression yeast, promoter evening including primary, secretion signal sequence, DNA coding for protein A or a fragment thereof, a Aguruchinin gene or its fragment derived from yeast from 5 5 in this order in 35 directions The trait according to (4), characterized in that:
( 6 ) 酵母用発現ベクターがマルチコピー型プラスミド又は染色体組み込み型 プラスミドである、 （4)又は （5) に記載の形質転換酵母。 (6) The transformed yeast according to (4) or (5), wherein the yeast expression vector is a multicopy plasmid or a chromosome-integrated plasmid.
(7) プロモ一夕一、 分泌シグナル配列、 プロテイン A又はその断片をコード する DNA、 酵母由来のァグルチニン遺伝子又はその断片をこの順序で 5' から 3' 方向に含む酵母用発現ベクター。  (7) A yeast expression vector comprising a promoter, a secretory signal sequence, a DNA encoding protein A or a fragment thereof, a yeast-derived aglutinin gene or a fragment thereof in this order in the 5 'to 3' direction.
(8) ( 1 )から （ 6 )の何れかに記載の形質転換酵母の作製における、 （ 7 ) に記載の酵母用発現ベクターの使用。  (8) Use of the yeast expression vector according to (7) in producing the transformed yeast according to any one of (1) to (6).
(9) ( 1 )から （ 6 )の何れかに記載の形質転換酵母と IgGの Fc部分又はそ れを含む蛋白質断片を含む試料とを混合することにより、 IgGの Fc部分を該形質 転換酵母の表層に提示されたプロティン A又はその断片に結合させることを含む、 IgGの Fc部分を検出又は単離する方法。  (9) By mixing the transformed yeast according to any one of (1) to (6) with a sample containing the Fc portion of IgG or a protein fragment containing the same, the Fc portion of IgG is transformed into the transformed yeast. A method for detecting or isolating the Fc portion of IgG, which comprises binding to protein A or a fragment thereof displayed on the surface of the above.
(10) IgGの Fc部分又はそれを含む蛋白質断片を含む試料がコンピナトリァ ル抗体ライブラリーである、 （9) に記載の方法。  (10) The method according to (9), wherein the sample containing the Fc portion of IgG or a protein fragment containing it is a companion antibody library.
(11) IgGの Fc部分又はそれを含む蛋白質断片が標識されている、 （9)又 は （ 10 ) に記載の方法。 図面の簡単な説明  (11) The method according to (9) or (10), wherein the Fc portion of IgG or a protein fragment containing the Fc portion is labeled. BRIEF DESCRIPTION OF THE FIGURES
図 1はプラスミド pEZZ 18の構造を示す図である。  FIG. 1 shows the structure of plasmid pEZZ18.
図 2は ZZドメインをコードする遺伝子の配列を示す。  FIG. 2 shows the sequence of the gene encoding the ZZ domain.
図 3は、 ドメインの細胞表層発現マルチコピープラスミド pCZZと染色体 組み込みプラスミド p I C Z Zの構築を示す図である。  FIG. 3 is a diagram showing the construction of the cell surface expression multicopy plasmid pCZZ and the chromosome integration plasmid pICZZ of the domain.
図 4は、 ヒト IgG 1の Fcをコードする断片遺伝子の配列を示す。 図 5は、 マルチコピー型プラスミド p C G F P— F cの構造を示す図である。 発明を実施するための最良の形態 FIG. 4 shows the sequence of a fragment gene encoding Fc of human IgG1. FIG. 5 is a diagram showing the structure of the multicopy plasmid pCGFP-Fc. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施方法及び実施態様について詳細に説明する。  Hereinafter, a method and an embodiment of the present invention will be described in detail.
グラム陽性菌 Staphylococcus aureusのプロテイン Aは、 液性免疫における産 生抗体 IgGの Fc部分と選択的かつ特異的に強い親和性を有する。本発明者が開発 した酵母表層発現べクタ一を用いることにより、 このプロテイン Aの IgGの Fc 部分と反応する部分をコードする領域 ZZ ドメインを、酵母の細胞表層に提示させ ることに今回初めて成功した。このプロティン Aの領域 ZZ ドメインを酵母細胞の 表層に提示することにより、コンビナトリアル抗体ラィブラリ一や: Fcをもつ抗体 成分の特異的吸着、 並びに興味深い活性を持つ触媒抗体の安定供給などを行うこ とが可能になり、 新規な機能分子のスクリーニング等に有用である。 また、 本発 明の技術は、 Fcを持たせた分泌タンパク質を用いた新しい分離精製法の確立など にも応用が可能である。  Protein A of Gram-positive bacterium Staphylococcus aureus has a selective and specific affinity for the Fc portion of IgG produced in humoral immunity. By using the yeast surface expression vector developed by the present inventors, we succeeded for the first time to display the ZZ domain, which encodes the part of protein A that reacts with the Fc part of IgG, on the yeast cell surface. did. By displaying the domain ZZ domain of protein A on the surface of yeast cells, it is possible to perform specific adsorption of combinatorial antibody libraries and antibody components having Fc, and stable supply of catalytic antibodies having interesting activities. This is useful for screening new functional molecules. In addition, the technology of the present invention can be applied to the establishment of a new separation and purification method using a secreted protein having Fc.
本発明の酵母は、 プロティン A又はその断片をコードする D NAを有する酵母 用発現ベクターを酵母に形質転換することにより得られる形質転換酵母である。 本発明の酵母は、プロテイン A又はその断片を細胞表層に提示することができる。 本発明で用いる発現べクタ一は、 酵母由来のァグルチニン遺伝子又はその断片 を含む。 ァグルチニン遺伝子を取得するための酵母としては、 例えば、 長谷川武 治編著「微生物の分類と同定」 （学会出版セン夕一、 1975年） に酵母として記載 されている微生物群が挙げられる。 なかでも、 好適な酵母はサッカロミセス属に 属する酵母であり、 例として、 サヅカロミセス セレピシェ （ Saccharomyces cerevisiae) M T 8— 1などを挙げることができる。 酵母の詳細な性質について は、 Tajima, M. et al.3 Yeast 1， 67 - 77(1985)に記載されている。 The yeast of the present invention is a transformed yeast obtained by transforming a yeast expression vector having DNA encoding protein A or a fragment thereof into yeast. The yeast of the present invention can display protein A or a fragment thereof on the cell surface. The expression vector used in the present invention contains a yeast-derived aglutinin gene or a fragment thereof. Examples of yeast for obtaining the aglutinin gene include a group of microorganisms described as yeast in "Classification and Identification of Microorganisms", edited by Takeharu Hasegawa (Society Press, Sen-ichi, 1975). Among them, preferred yeasts are those belonging to the genus Saccharomyces, and include, for example, Saccharomyces cerevisiae MT8-1. Detailed properties of yeast are described in Tajima, M. et al. 3 Yeast 1, 67-77 (1985).
前記ァグルチ二ンは a—ァグルチニン及びひ—ァグルチ二ンの何れであっても よい。 なお、 ひ—ァグルチニンの分子構造は、 N末端から順に、 分泌シグナル配 列、 ァグルチニン活性領域、 細胞表層内在領域及び G P Iアンカー （グリコシル ホスファチジルイノシトール） 付着シグナル領域という構成を有する。 The agglutinin may be any of a-agglutinin and hyaglutinin. The molecular structure of hyaglutinin is, in order from the N-terminal, a secretory signal sequence, an agglutinin active region, a cell surface internal region, and a GPI anchor (glycosylation). (Phosphatidylinositol) It has a configuration of an adhesion signal region.
ァグルチニン遺伝子領域断片は、 酵母の細胞表層に異種蛋白を発現できる部分 The fragment of the aglutinin gene region can express a heterologous protein on the yeast cell surface.
(細胞表層内在領域を含む部分） であればよいが、 特に好ましい例として、 サッ カロミセス属由来のひーァグルチニンの C末端から 320アミノ酸をコ一ドする 遺伝子配列と 446塩基からなる 3 ' —非翻訳領域とを含む D N A断片を挙げる ことができる。 この DNA断片には GP Iアンカ一付着シグナル領域が含まれて いる。 この DNA断片は、 Murai, T. et al. Applied and Environmental Microbiology, 63， 1362- 1366(1997)に記載されている該断片を含むプラスミ ド p GA 11を制限酵素により切り出すことによって得ることもできる。 (Including the cell surface internal region), but a particularly preferred example is a gene sequence encoding 320 amino acids from the C-terminal of Saccharomyces spp. And a DNA fragment containing the region. This DNA fragment contains the GPI anchor attachment signal region. This DNA fragment can also be obtained by cutting out a plasmid pGA11 containing the fragment described in Murai, T. et al. Applied and Environmental Microbiology, 63, 1366-1366 (1997) with a restriction enzyme. .
ァグルチニン遺伝子又はその断片およびプロティン A又はその断片をコードす る DNAを含む本発明の発現べクタ一は、 通常酵母で利用する発現ぺク夕一に前 記遺伝子を導入することにより得ることができる。 上記発現ベクターの好適な例 として、 例えば、 pICAS 1 (Murai et al., Appl. Environ. Microbiol., 6 4,4857-4861(1998))、 p CAS 1 (Murai etal.， Appl. Environ. Microbiol., 64,4857-4861(1998))等を挙げることができる。  The expression vector of the present invention containing the agglutinin gene or a fragment thereof and the DNA encoding protein A or a fragment thereof can be obtained by introducing the gene into an expression vector usually used in yeast. . Preferred examples of the expression vector include, for example, pICAS 1 (Murai et al., Appl. Environ. Microbiol., 64, 4857-4861 (1998)), pCAS1 (Murai et al., Appl. Environ. Microbiol. ., 64, 4857-4861 (1998)).
本発明で用いる発現ベクターは通常、プロモーターおよび分泌シグナル配列（例 えば、カビ由来の分泌シグナル配列）を含んでいる。前記プロモー夕一としては、 通常酵母での発現に利用されるプロモ一夕一であれば特に限定されないが、 好適 な列として、 サヅカロミセス 'セレビシェ (Saccharomyces cerevisiae) のグリ セロアルデヒド 3リン酸デヒ ドロゲナ一ゼ （GAPDH) のプロモ一夕一 ( Sawani-Hatanaka, H. , T. et al. , Biosci. Biotechnol. Biochem. , 59, 1221-1228(1995))、 キャンディダ' トロピカリス （Candida tropicalis)のイソ クェン酸リア一ゼ （ICL) のプロモ一夕一 （UPR—ICL) ( anai etal., Appl. Microbiol. Biotechnology, 44, 759-765 (1996)) などを挙げることができ る。  The expression vector used in the present invention usually contains a promoter and a secretory signal sequence (for example, a fungal-derived secretory signal sequence). The promoter is not particularly limited as long as it is a promoter generally used for expression in yeast, but a preferred column is glyceroaldehyde triphosphate dehydrogenase of Saccharomyces cerevisiae. Promote of Ichinose (GAPDH) (Sawani-Hatanaka, H., T. et al., Biosci. Biotechnol. Biochem., 59, 1221-1228 (1995)), Candida tropicalis And isopronate lyase (ICL) (UPR-ICL) (anai et al., Appl. Microbiol. Biotechnology, 44, 759-765 (1996)).
また、 前記分泌シグナル配列としては、 通常酵母での発現、 分泌に利用される シグナル領域、 例えば、 リゾブス オリザェ （Rhizopus oryzae) 由来のグルコア ミラ一ゼ遺伝子の分泌シグナル領域を使用できる。 上記プロモ一夕一および分泌 シグナル配列は、 既知の方法により、 例えば、 既知配列を基にして作製したブラ ィマ一を用いて、 上記微生物のゲノム D N Aを錶型にして P C R法を行うことな どから得ることができる。 Examples of the secretory signal sequence include a signal region usually used for expression and secretion in yeast, for example, glucoa derived from Rhizopus oryzae. The secretory signal region of the Mirase gene can be used. The above-mentioned promoter and secretory signal sequence can be obtained by a known method, for example, by using a primer prepared based on the known sequence and performing a PCR method using the genomic DNA of the microorganism as type III. From where you can get.
本発明においては、 プロティン A又はその断片をコードする D NAを細胞表層 に発現及び提示すべき蛋白質をコードする D N Aとして使用する。  In the present invention, DNA encoding protein A or a fragment thereof is used as DNA encoding a protein to be expressed and displayed on the cell surface.
プロテイン Aとは、 グラム陽性菌 Staphylococcus aureus (黄色ブドウ球華） 細胞壁成分の 5 %を占める分子量約 4 2 , 0 0 0の蛋白質であり、ヒト、マウス、 ゥサギなどの免疫グロブリン G ( I g G) の F cフラグメントと特異的に結合す る。 プロテイン Aのこの性質を利用することにより、 適当な担体に結合させ、 抗 体産生細胞の定量、 I g Gの精製、 また抗体を結合した細胞の分離などに用いる ことが可能である。  Protein A is a protein having a molecular weight of about 42,000, which accounts for 5% of the cell wall component of the Gram-positive bacterium Staphylococcus aureus (Yellow Staphylococcus aureus). ) Specifically binds to the Fc fragment. By utilizing this property of protein A, it can be bound to an appropriate carrier and used for quantification of antibody-producing cells, purification of IgG, separation of antibody-bound cells, and the like.
本発明では、 プロティン Aの全長から成る完全蛋白質を使用する場合だけでは なく、 プロテイン Aの断片を使用することもできる。 プロテイン Aを酵母の細胞 表層に効率よく提示させるという観点から言うと、 プロテイン Aの断片を使用す ることが好ましい。 本発明で使用できるプロティ Aの断片は好ましくは、 、 少 なくとも IgGの Fc部分と親和性を有する部分を含む。より具体的には、 IgGの Fc 部分と反応する部分をコードする領域としては ZZ ドメインが挙げられる。  In the present invention, not only a case where a complete protein consisting of the full length of protein A is used, but also a fragment of protein A can be used. From the viewpoint of efficiently displaying protein A on the surface of yeast cells, it is preferable to use a fragment of protein A. Fragments of Protein A that can be used in the present invention preferably include at least a portion that has an affinity for the Fc portion of an IgG. More specifically, a region encoding a portion that reacts with the Fc portion of IgG includes a ZZ domain.
本発明で用いる発現べクタ一の好ましい態様では、 プロモー夕一、 分泌シグナ ル配列、 プロテイン A又はその断片をコードする D N A、 酵母由来のァグルチニ ン遺伝子又はその断片 （例えば、 α—ァグルチニンの C末端断片をコードする D ΝΑ断片） をこの順序で有している。  In a preferred embodiment of the expression vector used in the present invention, promoter, secretory signal sequence, DNA encoding protein A or a fragment thereof, yeast-derived aglutinin gene or a fragment thereof (for example, C-terminal of α-agglutinin) D ΝΑ fragment) that encodes the fragment in this order.
このような発現べクタ一は、 例えば、 前記したプラスミ ドぺク夕一に、 前記プ ロモ—夕—、 分泌シグナル配列、 マルチクローニングサイ ト及びァグルチニン遺 伝子又はその断片を、 この順に配列するように導入した後、 前記マルチクロー二 ングサイ トにプロティン Α又はその断片をコードする D N Αを導入することによ り構築できる。 このような組換え発現べク夕一の構築には慣用の組換え D N A技 術を利用できる。 In such an expression vector, for example, the promoter, secretory signal sequence, multicloning site, and agglutinin gene or a fragment thereof are arranged in this order in the plasmid vector. After the introduction as above, it can be constructed by introducing a protein or a DN thereof encoding a fragment thereof into the multicloning site. Conventional recombinant DNA techniques are used to construct such a recombinant expression vector. Technique is available.
また、 本発明で用いる発現ベクターには抗生物質耐性遺伝子などの選択マーカ —遺伝子が含まれていてもよい。  The expression vector used in the present invention may contain a selection marker gene such as an antibiotic resistance gene.
本発明において、 上記した組換えべクタ一による形質転換の対象とされる酵母 としては、例えば、長谷川武治編著「微生物の分類と同定」 （学会出版センター、 In the present invention, examples of yeast to be transformed by the above-described recombinant vector include, for example, "Classification and Identification of Microorganisms" (edited by Takeharu Hasegawa).
1975年） に酵母として記載されている微生物群が挙げられる。酵母の種類は特に は限定されず、 例えば、 サッカロミセス属、 シゾサヅカロミセス属、 キャンディ ダ属、 ピキア属、 ハンセヌラ属などが挙げられる。 本発明では、 サッカロミセス 属に属する酵母を使用することが好ましく、 好ましい具体例としては、 サッカロ ミセス 'セレピシェ （Saccharomyces cerevisiae) などを挙げることができる。 酵母の詳細な性質については、 Tajima, M. et al . , Yeast 1， 67-77( 1985)に記載 されている。 1975). The type of yeast is not particularly limited, and includes, for example, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula and the like. In the present invention, it is preferable to use yeast belonging to the genus Saccharomyces, and specific examples thereof include Saccharomyces cerevisiae. The detailed properties of yeast are described in Tajima, M. et al., Yeast 1, 67-77 (1985).
発現ベクターによる酵母の形質転換は、 慣用の手法により行うことができ、 例 えば、 酢酸リチウム法、 電気パルス法又はプロトプラスト法等によって形質転換 体を得ることができる。形質転換体の培養方法もまた公知であり、例えば、「M. D. Rose et al. ,，- Methods In Yeast Genetics"， Cold SprmgHarbor Laboratory Press (1990)」等に記載されている。  Transformation of yeast with an expression vector can be performed by a conventional method, and a transformant can be obtained by, for example, a lithium acetate method, an electric pulse method, a protoplast method, or the like. Methods for culturing transformants are also known and described, for example, in "MD Rose et al.,-Methods In Yeast Genetics", Cold Sprmg Harbor Laboratory Press (1990).
本発明によれば、 上記した本発明の酵母を用いる蛋白質のスクリーニング方法 が提供される。 本発明の酵母は細胞表層にプロティン A又はその断片を提示する ことができるため、 提示されたプロティン A又はその断片と相互作用する任意の 物質 （例えば、 IgGの Fc部分など） をスクリーニングすることができる。  According to the present invention, there is provided a method for screening a protein using the above-described yeast of the present invention. Since the yeast of the present invention can display protein A or a fragment thereof on the cell surface, it is possible to screen for any substance that interacts with the displayed protein A or a fragment thereof (for example, the Fc portion of IgG). it can.
具体的には、 本発明のプロティン A又はその断片を細胞表層に提示できる形質 転換酵母と IgGの Fc部分又はそれを含む蛋白質断片を含む試料（例えば、コンビ ナトリアル抗体ライブラリ一など） を混合することにより、 IgGの Fc部分を該形 質転換酵母の表層に提示されたプロティン A又はその断片に結合させ、 結合した IgGの Fc部分を検出したり、 あるいは結合した IgGの Fc部分を単離することが できる。試料中の IgGの Fc部分又はそれを含む蛋白質断片を標識しておくことに より、酵母細胞表層のプロテイン A又はその断片に結合した IgGの Fc部分を容易 に検出することが可能である。 Specifically, mixing a transformed yeast capable of displaying the protein A of the present invention or a fragment thereof on the cell surface with a sample containing the Fc portion of IgG or a protein fragment containing the same (for example, a combinatorial antibody library). By binding the Fc portion of IgG to protein A or a fragment thereof displayed on the surface of the transformed yeast, and detecting the bound Fc portion of IgG or isolating the bound Fc portion of IgG. Can be done. To label the Fc portion of IgG or the protein fragment containing it in the sample Thus, it is possible to easily detect the Fc portion of IgG bound to protein A or a fragment thereof on the surface of yeast cells.
蛋白質断片の標識の種類及び標識方法は当業者に知られているものから適宜選 択することができる。 標識として酵素を使用する場合には、 例えば、 西洋ヮサビ ペルォキシダ一ゼ、 アルカリホスファタ一ゼ、 グルコースォキシダ一ゼ、 ?ーガ ラクトシダ一ゼ、 グルコアミラーゼ、 炭酸アンヒドラ一ゼ、 アセチルコリンエス テラーゼ、 リゾチーム、 マレ一トデヒドロゲナ一ゼ、 グルコース一 6—ホスフエ —トデヒドロゲナ一ゼ等を標識として使用することができる。 標識として酵素を 使用する場合、 上記の標識酵素の基質を作用させて発色等で反応を測定すること によって標識抗体を検出することができる。 例えば、 ペルォキシダ一ゼで標識さ れる場合には、 基質として過酸化水素、 発色試薬としてジァミノべンジジンまだ は 0—フエ二レンジァミンと組み合わさって褐色または黄色を生じる。 グル.コ一 スォキシダ一ゼで標識される場合には、 基質として、 たとえば 2， 2 ' —ァシド —ジ一（3—ェチルベンゾチアゾリン— 6—スルホン酸（A B T S )等を用いる。 標識として蛍光色素を使用する場合には、 例えば、 F I T C (フルォレセイン イソチオシァネート） 又は T R I T C (テトラメチルローダミン Bイソチオシァ ネート） 等の蛍光色素で抗体又はその断片を標識することができる。  The type and labeling method for labeling the protein fragment can be appropriately selected from those known to those skilled in the art. When an enzyme is used as a label, for example, horseradish peroxidase, alkaline phosphatase, glucose oxidase,? -Galactosidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme , Maletodehydrogenase, glucose-6-phospho-todehydrogenase and the like can be used as labels. When an enzyme is used as the label, the labeled antibody can be detected by reacting the substrate of the above-mentioned labeled enzyme and measuring the reaction by color development or the like. For example, when labeled with peroxidase, it produces a brown or yellow color in combination with hydrogen peroxide as a substrate and diaminobenzidine or 0-phenylenediamine as a chromogenic reagent. In the case of labeling with glucosidase, a substrate such as 2,2'-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is used. When a dye is used, for example, the antibody or a fragment thereof can be labeled with a fluorescent dye such as FITC (fluorescein isothiosinate) or TRITC (tetramethylrhodamine B isothiosinate).
標識として呈色標識物質を使用する場合には、 例えば、 コロイ ド金属および着 色ラテックスなどを標識として使用できる。 コロイ ド金属の代表例としては、 金 ゾル、 銀ゾル、 セレンゾル、 テルルゾルおよび白金ゾルなどのそれそれの分散粒 子である金属コロイ ド粒子を挙げることができる。 コロイ ド金属の粒子の大きさ は、 通常は、 直径 3〜6 0皿程度とされる。 また、 着色ラテックスの代表例とし ては、 赤色および青色などのそれそれの顔料で着色されたポリスチレンラッテク スなどの合成ラテックスを挙げることができる。 ラテックスとして天然ゴムラテ ックスのような天然ラッテクスを使用することができる。 着色ラテックスの大き さは、 直径数十而〜数百 nm程度から選択することができる。 これらの呈色標識 物質は市販品をそのまま使用することができるが、 場合によりさらに加工し、 ま たは、 それ自体公知の方法で製造することもできる。 When a colored labeling substance is used as a label, for example, colloid metal and colored latex can be used as the label. Representative examples of the colloid metal include metal colloid particles which are dispersed particles of gold sol, silver sol, selenium sol, tellurium sol and platinum sol. The size of colloidal metal particles is usually about 3 to 60 dishes in diameter. As a typical example of the colored latex, a synthetic latex such as polystyrene latex colored with respective pigments such as red and blue can be given. Natural latex such as natural rubber latex can be used as the latex. The size of the colored latex can be selected from several tens to several hundreds of nm in diameter. Commercially available products can be used for these coloring markers as they are. Alternatively, it can be produced by a method known per se.
なお、標識としては、上記以外にもァフィ二ティ一標識（例えば、ビォチン等）、 又は、 同位体標識 （例えば、 125 I等） 等を使用することもできる。 In addition, as a label, an affinity label (for example, biotin) or an isotope label (for example, 125 I) can be used in addition to the above.
あるいは、 IgGの Fc部分又はそれを含む蛋白質断片と蛍光蛋白質（例えば、 緑 色蛍光タンパク質 （GFP) またはその変異体） とから成る融合蛋白質を使用す ることもできる。  Alternatively, a fusion protein comprising the Fc portion of IgG or a protein fragment containing the Fc portion and a fluorescent protein (eg, green fluorescent protein (GFP) or a mutant thereof) can also be used.
上記したような標識抗体を用いた酵素抗体法、 免疫組織染色法、 免疫プロット 法、 直接蛍光抗体法又は間接蛍光抗体法等の分析は当業者に周知の方法で行なう ことができ、 その実験条件も当業者ならば適宜選択することができる。  Analysis such as the enzyme antibody method, immunohistochemical staining method, immunoplot method, direct fluorescent antibody method or indirect fluorescent antibody method using the labeled antibody described above can be performed by a method well known to those skilled in the art. Also, those skilled in the art can appropriately select.
本出願の優先権主張の基礎となる出願である特願 2000— 2.06689号の 明細書に開示した内容は全て引用により本明細書に開示したものとする。  The contents disclosed in the specification of Japanese Patent Application No. 2000-2.06689, which is the application on which the priority of the present application is based, are all disclosed herein by reference.
以下の実施例により本発明を具体的に説明するが、 本発明は実施例によって限 定されることはない。 実施例  The present invention will be specifically described by the following examples, but the present invention is not limited to the examples. Example
実施例 1 ：プロテイン Aの IgGの Fc部分に特異的親和性を示す ZZドメインの増 幅と細胞表層発現プラスミドの構築 Example 1: Amplification of ZZ domain showing specific affinity for Fc portion of IgG of protein A and construction of cell surface expression plasmid
常法に従って増殖させたプラスミド pEZZ 18 (アマ一シャムフアルマシア 社製、 図 1) を保持した大腸菌から、 プラスミ ド pEZZ 18を単離し、 これを 鏡型に して P C Rを行い、 1 ド メ イ ン を作製 した。 即ち、 5' -gaattcatgcaactgttcaatttgcc-35 ( 配 列 番 号 1 ) と 55 -cttgttaaatcagtagtggttgag-35 (配列番号 2) をプライマ一として、 20 zmol の dNTP混合液、 50pmolの各ブラィマ一、 1 n の錶型ブラスミド D N Aと pfuポ リメラ一ゼ （ストラタジーン社製） と P CRpfuポリメラ一ゼのバッファ一から なる 100〃 1の反応系により、 94°Cで 1分； 55°Cで 1分； 72°Cで 30秒の 30サイク ル反応により P CR反応を行った。このようにして増幅させた ZZドメインをコ一 ドする遺伝子（図 2又は配列番号 3)を SacIIと Xholで切断した後、電気泳動で 精製し、 さらに、 QIAEXII (QIAGEN Co.)ゲル抽出キットで抽出した。 Plasmid pEZZ18 was isolated from E. coli harboring plasmid pEZZ18 (Amersham Sharm Pharmacia, Fig. 1) grown according to a conventional method, and this was mirror-typed and subjected to PCR. In was prepared. That, 5 '-gaattcatgcaactgttcaatttgcc-3 5 a (sequence number 1) and 5 5 -cttgttaaatcagtagtggttgag-3 5 (SEQ ID NO: 2) as primers one, 20 dNTPs mixture Zmol, each Buraima one 50 pmol, 1 n a 72 minutes at 94 ° C; 1 minute at 55 ° C in a 100〃1 reaction system consisting of type ブ ラ plasmid DNA, pfu polymerase (manufactured by Stratagene) and PCR buffer of PCR polymerase. The PCR reaction was performed at 30 ° C. for 30 seconds at 30 ° C. The gene (FIG. 2 or SEQ ID NO: 3) encoding the ZZ domain amplified in this manner is cut with SacII and Xhol, and then subjected to electrophoresis. It was purified and further extracted with a QIAEXII (QIAGEN Co.) gel extraction kit.
次に、本発明者が開発した酵母 Saccharomyces cerevisiaeの GAPDHプロモ一夕 —と Rhizopus oryzaeめグルコアミラーゼの分泌シグナルをコードする遺伝子と マルチクローニングサイトと ひ一ァグルチニンをコードする遺伝子の 35 —末 端からなる遺伝子を含む細胞表層発現用マルチコピー型プラスミ ド p CAS 1 (Murai et al., Appl.Environ. Microbiol., 64,4857-4861(1998» と染色体組み 込み型プラスミ ド p I CAS 1 (Murai et al., Appl.Environ. Microbiol., 6 4,4857-4861(1998)) を SacIIと Xholで切断した （図 3)。それそれの断片を電 気泳動で精製した後、 QIAEXII (QIAGEN Co. )ゲル抽出キットで抽出した。 Then, the present inventors have GAPDH promoter Isseki yeast Saccharomyces cerevisiae developed - from end edge - 3 5 of the gene coding for the gene and the multiple cloning site encoding a secretory signal Rhizopus oryzae Me glucoamylase and carry one Aguruchinin Multicopy plasmid pCAS1 (Murai et al., Appl.Environ. Microbiol., 64, 4857-4861 (1998 »and chromosomal integration plasmid pICAS1 (Murai et al., Appl. Environ. Microbiol., 64, 4857-4861 (1998)) was digested with SacII and Xhol (Fig. 3) .Each fragment was purified by electrophoresis and then QIAEXII (QIAGEN Co. .) Extracted with gel extraction kit.
先に得た ZZ ドメインをコードする遺伝子を、マルチコピー型プラスミド pCA S 1と染色体組み込み型プラスミド p I CAS 1を SacIIと Xholで切断した断片 にライゲーシヨンして、 ZZ ドメインの細胞表層発現マルチコビ一プラスミ ド pC ZZと染色体組み込みプラスミ ド p I CZZを構築した （図 3)。 実施例 2 ：プロテイン Aの ZZ ドメインを細胞表層に提示した酵母の作製  The gene encoding the ZZ domain obtained above was ligated to a fragment obtained by cutting the multicopy plasmid pCAS1 and the chromosome-integrated plasmid pICAS1 with SacII and Xhol, to express the cell surface expression of the ZZ domain on the multicopi plasmid. PCZZ and chromosomal integration plasmid pICZZ were constructed (Fig. 3). Example 2: Preparation of yeast displaying the ZZ domain of protein A on the cell surface
pCZ Zと p I CZZは、 それそれ酵母 Saccharomyces cerevisiae MT8-1に酢 酸リチウム法で導入した。 SD-Trp 固体培地 （0.03%Leu, 0.02%His, 0.02%Ade, 0.02%Ura， 6.7%Yeast nitrogen base, 2%glucose32%agar) で Trp+となる形質転 換体を得た。 導入された遺伝子の宿主細胞内における存在は、 PCRならびにサ ザンブロット法により確認した。これらの形質転換体を SD- Trp液体培地で、 30°C で 24時間培養した後、 PBSバッファ一で洗浄し、 次に、 3.7%ホルムアルデヒド で固定した。 この試料にラビット IgG (ジャクソンィムノリサ一チラボラトリー 社製） とフルォレセイン標識したャギの抗ラビット IgG (モレキュラープロ一ブ 社製）を用いて、細胞表層に提示されたプロテイン の ドメインを蛍光顕微鏡 により検出した。 実施例 3 ：プロテイン Aの 1 ドメインを細胞表層に提示した酵母を用いた分泌 Fc融合 Green Fluorescent Protein (GFP)の分離回収 pCZZ and pI CZZ were each introduced into the yeast Saccharomyces cerevisiae MT8-1 by the lithium acetate method. SD-Trp solid medium was obtained (0.03% Leu, 0.02% His , 0.02% Ade, 0.02% Ura, 6.7% Yeast nitrogen base, 2% glucose 3 2% agar) in transformants comprising a Trp +. The presence of the introduced gene in the host cell was confirmed by PCR and Southern blotting. These transformants were cultured in SD-Trp liquid medium at 30 ° C. for 24 hours, washed with a PBS buffer, and then fixed with 3.7% formaldehyde. Using a rabbit IgG (Jackson Imnorisa Laboratory) and fluorescein-labeled goat anti-rabbit IgG (Molecular Probes), the protein domain presented on the cell surface was analyzed with a fluorescence microscope. Detected by Example 3: Secretion using yeast displaying one domain of protein A on the cell surface Separation and recovery of Fc-fused Green Fluorescent Protein (GFP)
GAPDHプロモ一夕一と Rhizopus oryzaeのグルコアミラーゼの分泌シグナルをコ —ドする遺伝子、 さらに、 GFPをコードする遺伝子とヒト IgG lの Fcをコードす る断片遺伝子（図 4、 三菱化学より供与）を含むマルチコピー型プラスミド p C G F P - F c (図 5 ) を作製し、 これを酵母 Saccharomyces cerevisiae MT8-lに酢 酸リチウム法で導入した。  A gene encoding the secretion signal of glucoamylase of GAPDH Promoter and Rhizopus oryzae, a gene encoding GFP and a fragment gene encoding Fc of human IgG1 (Fig. 4, donated by Mitsubishi Chemical) A multicopy-type plasmid pCGFP-Fc (Fig. 5) was prepared and introduced into the yeast Saccharomyces cerevisiae MT8-l by the lithium acetate method.
この GFP- Fc融合タンパクを分泌する細胞と、 実施例 2で作製した ZZ ドメイン を細胞表層に提示した酵母とを混合培養した場合、並びに、分泌 OTP- Fc融合タン パク質を含む溶液に実施例 2で作製した ZZ ドメインを細胞表層に提示した酵母 細胞を浸漬した場合の 2通りの場合において、 11 ドメイン提示細胞の表層に分泌 Fc融合タンパクが特異的に、 かつ選択的に吸着したことを、 GFPの蛍光を蛍 光顕微鏡で捉えて確認できた。 産業上の利用の可能性  When the cells secreting this GFP-Fc fusion protein and the yeast displaying the ZZ domain prepared in Example 2 on the cell surface were mixed and cultured, and the example was prepared in a solution containing the secreted OTP-Fc fusion protein. In two cases, when the yeast cells displaying the ZZ domain prepared in 2 on the cell surface were immersed, the secretory Fc fusion protein was specifically and selectively adsorbed on the surface of the 11 domain-presenting cells. The fluorescence of GFP was confirmed with a fluorescence microscope. Industrial applicability
本発明の形質転換酵母は、プロティン Aの領域 ZZ ドメインを酵母細胞の表層に 提示することができ、コンピナトリアル抗体ラィブラリ一や Fcをもつ抗体成分の 特異的吸着、 並びに興味深い活性を持つ触媒抗体の安定供給などを行う際に有用 であり、 例えば、 新規な機能分子のスクリーニング等に有用である。 また、 本発 明の形質転換酵母は、 Fcを持たせた分泌夕ンパク質を用いた新しい分離精製法の 確立などにも応用が可能である。  The transformed yeast of the present invention can display the domain ZZ domain of protein A on the surface of yeast cells, specifically adsorbs antibody components having a compinatrial antibody library or Fc, and a catalytic antibody having an interesting activity. This is useful when performing stable supply of proteins, and is useful, for example, for screening new functional molecules. The transformed yeast of the present invention can also be applied to the establishment of a new separation and purification method using secretory proteins with Fc.

Claims

1 . プロティン A又はその断片を細胞表層に提示する形質転換酵母。 1. A transformed yeast displaying protein A or a fragment thereof on the cell surface.
2 . プロティン A又はその断片が I g Gの F c部分と親和性を有する部分で ある、 請求項 1に記載の形質転換酵母。  2. The transformed yeast according to claim 1, wherein the protein A or a fragment thereof has affinity with the Fc portion of IgG.
3 . プロテイン A又はその断片が少なくとも Z Zドメインを含む断片である、 請求項 1又は 2に記載の形質転換酵母。  3. The transformed yeast according to claim 1, wherein the protein A or a fragment thereof is a fragment containing at least a ZZ domain.
4 . プロティン A又はその断片をコ一ドする D NAを有する酵母用発現べク 夕一を酵母に形質転換することにより得られる、 請求項 1から 3の何れかに記載 の形質転換酵母。  4. The transformed yeast according to any one of claims 1 to 3, which is obtained by transforming a yeast expression vector having a DNA encoding protein A or a fragment thereof into yeast.
5 . 酵母用発現べクタ一が、 プロモ一夕一、 分泌シグナル配列、 プロテイン A又はその断片をコ一ドする D NA、 酵母由来のァグルチニン遺伝子又はその断 片をこの順序で 5 5 から 3 ' 方向に含むことを特徴とする、 請求項 4に記載の形 質転換酵母。 5. Yeast for expression base Kuta one is, promoter Isseki one secretory signal sequence, 3 protein A or a fragment thereof co one de to D NA, the Aguruchinin gene or its fragment derived from yeast in this order 5 5 ' The transformed yeast according to claim 4, wherein the yeast is contained in a direction.
6 . 酵母用発現べク夕一がマルチコピー型プラスミド又は染色体組み込み型 プラスミドである、 請求項 4又は 5に記載の形質転換酵母。  6. The transformed yeast according to claim 4, wherein the yeast expression vector is a multicopy plasmid or a chromosome-integrated plasmid.
7 . プロモー夕一、分泌シグナル配列、プロテイン A又はその断片をコードす る D NA、酵母由来のァグルチニン遺伝子又はその断片をこの順序で 5，から 3 ' 方向に含む酵母用発現べクタ一。  7. An expression vector for yeast containing a promoter, a secretory signal sequence, a DNA encoding protein A or a fragment thereof, a yeast-derived aglutinin gene or a fragment thereof in this order in the 5 to 3 ′ direction.
8 . 請求項 1から 6の何れかに記載の形質転換酵母の作製における、 請求項 7に記載の酵母用発現ぺク夕一の使用。  8. Use of the yeast expression vector according to claim 7 for producing the transformed yeast according to any one of claims 1 to 6.
9 . 請求項 1から 6の何れかに記載の形質転換酵母と IgGの Fc部分又はそれ を含む蛋白質断片を含む試料とを混合することにより、 IgGの Fc部分を該形質転 換酵母の表層に提示されたプロティン A又はその断片に結合させることを含む、 IgGの Fc部分を検出又は単離する方法。  9. By mixing the transformed yeast according to any one of claims 1 to 6 with a sample containing an IgG Fc portion or a protein fragment containing the IgG, the IgG Fc portion is added to the surface of the transformed yeast. A method for detecting or isolating the Fc portion of an IgG, comprising binding to the displayed protein A or a fragment thereof.
1 0 . IgGの Fc部分又はそれを含む蛋白質断片を含む試料がコンビナトリア ル抗体ライプラリーである、 請求項 9に記載の方法。 10. The method according to claim 9, wherein the sample containing the Fc portion of IgG or a protein fragment containing it is a combinatorial antibody library.
1 1 . IgGの Fc部分又はそれを含む蛋白質断片が標識されている、 請求項 9 又は 1 0に記載の方法。 11. The method according to claim 9 or 10, wherein the Fc portion of IgG or a protein fragment containing the Fc portion is labeled.
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US10138477B2 (en) 2013-01-03 2018-11-27 Merck Patent Gmbh Method of producing secretable antibodies by expression in saccharomyces cerevisiae

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