JPWO2004031243A1 - Protein polymer and method for producing the same - Google Patents

Abstract

An object of the present invention is to provide a protein polymer that can exhibit various physiological activities and enzyme activities using the ubiquitin-like protein SUMO, and a system capable of producing protein conjugates and protein polymers in large quantities in host cells such as bacteria Is to provide. According to the present invention, there is provided a protein polymer containing a ubiquitin-like protein (SUMO) in which the target protein is bound directly or via an acceptor in the main chain.

Description

  The present invention relates to a novel protein polymer utilizing isopeptide binding of ubiquitin-like protein (SUMO) and a method for producing the same. Furthermore, the present invention relates to a recombinant vector for high-level expression of a protein conjugate or protein polymer in a host cell such as a bacterium and a method for high-level expression of a protein conjugate or protein polymer using the same. .

Protein molecules such as enzymes, antibodies, and receptors are immobilized on bioreactors and biosensors, and are used for production and separation / purification of useful substances, detection and function elucidation of unknown substances, and drug screening. Therefore, if these protein molecules can be polymerized, the functions and performances of existing bioreactors and biosensors can be improved, and they can be applied to drug discovery, regenerative medicine, food processing and the like.
Conventionally, a method using a crosslinking agent is generally used as a method for polymerizing protein molecules. However, this method has problems such as difficulty in controlling the direction of molecules to be polymerized, or binding of a crosslinking agent to the active center of the molecule to be polymerized, resulting in protein denaturation or loss of activity. there were. Therefore, there is no method for synthesizing a biochemically uniform polymer while maintaining the function of the protein.
Ubiquitin, on the other hand, consists of 76 amino acids and is a small protein that is well preserved from yeast to humans. Ubiquitin is covalently bound to the target protein by a complex enzyme system composed of an activating enzyme (E1), a binding enzyme (E2), and a ligase (E3). That is, the C-terminal carboxyl group of ubiquitin is isopeptide-bonded to the ε-amino group of the lysine residue of the target protein. Furthermore, a polyubiquitin chain is formed when the C-terminal of another ubiquitin repeats isopeptide bonds with the 48th lysine residue of ubiquitin bound to the lysine residue of the target protein.
In addition, SUMO (small ubiquitin-related modifier) is a small ubiquitin-like protein that is well preserved in evolution from yeast to humans, plays an important role in cell growth control, and close to the nuclear pore complex. There are many. SUMO is also reported to bind to other proteins via isopeptide bonds through a mechanism similar to that of ubiquitin, resulting in polymerization (MH Tatham et al., J. Biol. Chem. Vol. 276). A. Pichier et al., Cell, Vol.108, p.109, 2002; and ES Johnson & AA Gupta, Cell, Vol.106, p. 2001). In addition, it is known that a protein fused with ubiquitin is easily degraded, whereas a protein fused with SUMO is stable (JM Desterro et al., Mol. Cell, Vol. 2, p. 8660, 1998; and T. Buschmann st al., Cell, Vol. 101, p. 753, 2000). However, there has been no report on an example of polymerizing SUMO fused with a target protein.

（式中、ＳＵＭＯはユビキチン様タンパク質、Ｘは目的タンパク質、太線はイソペプチド結合、ａは２〜１００の平均重合度を示す。）
で表されるタンパク質ポリマー。
（７） 下記式（ＩＩ）：

（式中、ＳＵＭＯはユビキチン様タンパク質、Ｘ、Ｙ、及びＺは目的タンパク質、太線はイソペプチド結合、ｂ、ｃ、及びｄは０から１００の平均重合度を示す。但し、ｂ、ｃ、及びｄが同時に０の場合は除く）
で表されるタンパク質ポリマー。
（８） 下記式（ＩＩＩ）：

（式中、ＳＵＭＯはユビキチン様タンパク質、Ａはアクセプター、Ｘは目的タンパク質、太線はイソペプチド結合、ｅは２〜１００の平均重合度を示す。）
で表されるタンパク質ポリマー。
（９） 下記式（ＩＶ）：

（式中、ＳＵＭＯはユビキチン様タンパク質、Ｘ、Ｙ、及びＺは目的タンパク質、Ａはアクセプター、太線はイソペプチド結合、ｆ、ｇ、及びｈは０から１００の平均重合度を示す。但し、ｆ、ｇ、及びｈが同時に０の場合は除く）。
で表されるタンパク質ポリマー。
（１０） 下記式：Ｘ−ＳＵＭＯ−Ａ−Ｙ
（式中、ＳＵＭＯはユビキチン様タンパク質、Ａはアクセプター、Ｘ及びＹは目的タンパク質を示す。）
で表されるタンパク質コンジュゲイト。
（１１） 下記の工程：
（ａ）ユビキチン様タンパク質（ＳＵＭＯ）と目的タンパク質の融合体又は結合体を作製する工程；
（ｂ）前記融合体又は結合体をイソペプチド結合により重合する工程；
を含むタンパク質ポリマーの製造方法。
（１２） 下記の工程：
（ａ）ユビキチン様タンパク質（ＳＵＭＯ）とアクセプターと目的タンパク質の融合体又は結合体を作製する工程；
（ｂ）前記融合体又は結合体をイソペプチド結合により重合する工程；
を含むタンパク質ポリマーの製造方法。
（１３） ＳＵＭＯ活性化酵素Ｅ１、ＳＵＭＯ結合酵素Ｅ２、ＡＴＰ、及び金属イオンの存在下に重合反応を行うことを特徴とする、（１１）又は（１２）に記載の方法。
（１４） 反応促進物または基盤ペプチド、もしくはその両方の因子をさらに反応系に添加する、（１１）から（１３）のいずれかに記載の方法。
（１５） ユビキチン様タンパク質を活性化する酵素（Ｅ１）をコードするＤＮＡ、及びユビキチン様タンパク質とアクセプターの結合酵素（Ｅ２）をコードするＤＮＡを含み、活性化酵素（Ｅ１）と結合酵素（Ｅ２）とを宿主内で同時に発現できる組み換え発現ベクター。
（１６） ユビキチン様タンパク質を活性化する酵素（Ｅ１）がＡｏｓ１とＵｂａとの融合タンパク質であるＡＵであり、結合酵素（Ｅ２）がＵｂｃ９である、（１５）に記載の組み換え発現ベクター。
（１７） ユビキチン様タンパク質をコードするＤＮＡ及びアクセプターをコードするＤＮＡを含み、ユビキチン様タンパク質とアクセプターとを個別に宿主内で同時に発現できる組み換え発現ベクター。
（１８） ユビキチン様タンパク質をコードするＤＮＡ及び／又はアクセプターをコードするＤＮＡに隣接して１種以上の目的タンパク質をコードするＤＮＡをさらに含み、ユビキチン様タンパク質とアクセプター（このうちの両方又は片方には上記目的タンパク質が融合している）とを個別に宿主内で同時に発現できる、（１７）に記載の組み換え発現ベクター。
（１９） ユビキチン様タンパク質を活性化する酵素（Ｅ１）をコードするＤＮＡ；ユビキチン様タンパク質とアクセプターの結合酵素（Ｅ２）をコードするＤＮＡ；ユビキチン様タンパク質をコードするＤＮＡ；及び／又はアクセプターをコードするＤＮＡを含み、活性化酵素（Ｅ１）、結合酵素（Ｅ２）、ユビキチン様タンパク質及び／又はアクセプターとを個別に宿主内で同時に発現できる、組み換え発現ベクター。
（２０） （１）又は（２）に記載の組み換えベクターと、（１７）又は（１８）に記載の組み換えベクターとを組み合わせて使用することを含む、活性化酵素（Ｅ１）と結合酵素（Ｅ２）とユビキチン様タンパク質とアクセプターとを宿主内で同時に発現する方法。
（２１） （１９）に記載のベクターを使用し、必要に応じ、ユビキチン様タンパク質をコードするＤＮＡ又はアクセプターをコードするＤＮＡを含む組み換えベクターを組み合わせて使用することを含む、活性化酵素（Ｅ１）と結合酵素（Ｅ２）とユビキチン様タンパク質とアクセプターとを宿主内で同時に発現する方法。
（２２） ユビキチン様タンパク質を活性化する酵素（Ｅ１）、ユビキチン様タンパク質とアクセプターの結合酵素（Ｅ２）、目的タンパク質が結合していてもよいユビキチン様タンパク質、及び目的タンパク質が結合していてもよいアクセプターを発現できる形質転換体。
（２３） （１５）又は（１６）に記載の組み換えベクターと、（１７）又は（１８）に記載の組み換えベクターとを有する、（２２）に記載の形質転換体。
（２４） （２２）又は（２３）に記載の形質転換体を培養して、該形質転換体内においてユビキチン様タンパク質とアクセプターとの結合体を産生することを含む、ユビキチン様タンパク質とアクセプターとの結合体の製造方法。
（２５） ユビキチン様タンパク質とアクセプターとの融合タンパク質をコードするＤＮＡを含み、ユビキチン様タンパク質とアクセプターとの融合タンパク質を宿主内で発現できる、組み換え発現ベクター。
（２６） ユビキチン様タンパク質をコードするＤＮＡ及びアクセプターをコードするＤＮＡに隣接して１種以上の目的タンパク質をコードするＤＮＡをさらに含み、ユビキチン様タンパク質とアクセプターと目的タンパク質との融合タンパク質を宿主内で発現できる、（２５）に記載の組み換え発現ベクター。
（２７） （１５）又は（１６）に記載の組み換えベクターと、（２５）又は（２６）に記載の組み換えベクターとを組み合わせて使用することにより、活性化酵素（Ｅ１）、結合酵素（Ｅ２）及びユビキチン様タンパク質とアクセプターとの融合タンパク質を宿主内で同時に発現させることを含む、ユビキチン様タンパク質とアクセプターとの融合タンパク質のポリマーの製造方法。
（２８） ユビキチン様タンパク質を活性化する酵素（Ｅ１）をコードするＤＮＡ、ユビキチン様タンパク質とアクセプターの結合酵素（Ｅ２）をコードするＤＮＡ、及びユビキチン様タンパク質とアクセプターと所望により目的タンパク質との融合タンパク質をコードするＤＮＡを含み、上記の活性化酵素（Ｅ１）と結合酵素（Ｅ２）と融合タンパク質を宿主内で同時に発現できる組み換え発現ベクター。
（２９） ユビキチン様タンパク質を活性化する酵素（Ｅ１）、ユビキチン様タンパク質とアクセプターの結合酵素（Ｅ２）、及びユビキチン様タンパク質とアクセプターと所望により目的タンパク質との融合タンパク質を発現できる形質転換体。
（３０） （１５）又は（１６）に記載の組み換えベクターと、（２５）又は（２６）に記載の組み換えベクターとを有する、（２９）に記載の形質転換体。
（３１） （２９）又は（３０）に記載の形質転換体を培養して、該形質転換体内においてユビキチン様タンパク質とアクセプターと所望により目的タンパク質との融合タンパク質のポリマーを産生することを含む、タンパク質ポリマーの製造方法。A first object of the present invention is to provide a protein polymer that can exhibit various physiological activities and enzyme activities using the ubiquitin-like protein SUMO. The second object of the present invention is to provide a system capable of producing protein conjugates and protein polymers in large quantities in host cells such as bacteria.
As a result of intensive studies to solve the first object, the present inventors polymerize a ubiquitin-like protein (SUMO) in which the target protein is bound directly or via an acceptor by an isopeptide binding reaction by an enzyme. Succeeded. Furthermore, as a result of intensive studies to solve the above second object, the present inventors have found that a mammalian SUMO-enzyme (that is, two types of SUMO-ized E1 enzyme and E2 enzyme), SUMO, and an acceptor are transferred in Escherichia coli. By simultaneously expressing them, we succeeded in forming isopeptide bonds in the microbial cells, and developed a technology to produce large quantities of protein conjugates and polymers. The present invention has been completed based on these findings.
That is, according to the present invention, the following inventions (1) to (30) are provided.
(1) A protein polymer containing, in its main chain, a ubiquitin-like protein (SUMO) to which a target protein is bound directly or via an acceptor.
(2) The protein polymer according to (1), wherein the target protein is contained in the polymer in one or more kinds.
(3) The protein polymer according to (1) or (2), wherein the main chain is linear, branched, cyclic, or a self-assembled tube shape.
(4) The protein polymer according to any one of (1) to (3), wherein one or more acceptors are contained in the polymer.
(5) Acceptor is RanGAP1-C2 (Ran GTPase activating protein-C2), RanBP2-IR (Ran binding protein 2-Internal Repeat domain), TDG (Thymin DNA GlycosylSTP) The protein polymer according to any one of (1) to (3), wherein the protein polymer is (proneelotic leukemia).
(6) The following formula (I):

(In the formula, SUMO is a ubiquitin-like protein, X is a target protein, a bold line is an isopeptide bond, and a is an average degree of polymerization of 2 to 100.)
A protein polymer represented by
(7) Formula (II) below:

(In the formula, SUMO is a ubiquitin-like protein, X, Y, and Z are target proteins, bold lines are isopeptide bonds, b, c, and d are average degrees of polymerization from 0 to 100, provided that b, c, and except when d is simultaneously 0)
A protein polymer represented by
(8) The following formula (III):

(In the formula, SUMO is a ubiquitin-like protein, A is an acceptor, X is a target protein, a bold line is an isopeptide bond, and e is an average degree of polymerization of 2 to 100.)
A protein polymer represented by
(9) The following formula (IV):

(In the formula, SUMO is a ubiquitin-like protein, X, Y, and Z are target proteins, A is an acceptor, thick lines are isopeptide bonds, f, g, and h are average degrees of polymerization from 0 to 100, where f is , G, and h are simultaneously 0).
A protein polymer represented by
(10) The following formula: X-SUMO-AY
(In the formula, SUMO represents a ubiquitin-like protein, A represents an acceptor, and X and Y represent target proteins.)
A protein conjugate represented by
(11) The following steps:
(A) producing a fusion or conjugate of a ubiquitin-like protein (SUMO) and a target protein;
(B) polymerizing the fusion or conjugate by isopeptide bonds;
A method for producing a protein polymer comprising:
(12) The following steps:
(A) producing a fusion or conjugate of ubiquitin-like protein (SUMO), acceptor and target protein;
(B) polymerizing the fusion or conjugate by isopeptide bonds;
A method for producing a protein polymer comprising:
(13) The method according to (11) or (12), wherein the polymerization reaction is performed in the presence of SUMO-activating enzyme E1, SUMO-binding enzyme E2, ATP, and metal ions.
(14) The method according to any one of (11) to (13), wherein a reaction accelerator or a base peptide, or both factors are further added to the reaction system.
(15) A DNA encoding an enzyme (E1) that activates a ubiquitin-like protein and a DNA encoding a binding enzyme (E2) of a ubiquitin-like protein and an acceptor, and includes an activating enzyme (E1) and a binding enzyme (E2) And a recombinant expression vector that can be expressed simultaneously in a host.
(16) The recombinant expression vector according to (15), wherein the enzyme (E1) that activates the ubiquitin-like protein is AU that is a fusion protein of Aos1 and Uba, and the binding enzyme (E2) is Ubc9.
(17) A recombinant expression vector comprising a DNA encoding a ubiquitin-like protein and a DNA encoding an acceptor, wherein the ubiquitin-like protein and the acceptor can be separately expressed simultaneously in a host.
(18) A DNA further encoding a ubiquitin-like protein and / or a DNA encoding one or more target proteins adjacent to the DNA encoding the acceptor, wherein the ubiquitin-like protein and the acceptor (both or one of them are The recombinant expression vector according to (17), wherein the protein of interest is fused separately in the host at the same time.
(19) DNA encoding an enzyme (E1) that activates a ubiquitin-like protein; DNA encoding a binding enzyme (E2) of a ubiquitin-like protein and an acceptor; DNA encoding a ubiquitin-like protein; and / or an acceptor A recombinant expression vector comprising DNA and capable of simultaneously expressing an activating enzyme (E1), a binding enzyme (E2), a ubiquitin-like protein and / or an acceptor individually in a host.
(20) An activating enzyme (E1) and a binding enzyme (E2), comprising using the recombinant vector according to (1) or (2) and the recombinant vector according to (17) or (18) in combination. ), A ubiquitin-like protein and an acceptor are simultaneously expressed in a host.
(21) An activating enzyme (E1) comprising using the vector according to (19) and, if necessary, a combination of a recombinant vector containing a DNA encoding a ubiquitin-like protein or a DNA encoding an acceptor And a conjugating enzyme (E2), a ubiquitin-like protein and an acceptor are simultaneously expressed in a host.
(22) Enzyme (E1) that activates ubiquitin-like protein, ubiquitin-like protein and acceptor binding enzyme (E2), ubiquitin-like protein to which the target protein may be bound, and target protein may be bound A transformant capable of expressing an acceptor.
(23) The transformant according to (22), comprising the recombinant vector according to (15) or (16) and the recombinant vector according to (17) or (18).
(24) Binding of ubiquitin-like protein and acceptor, comprising culturing the transformant according to (22) or (23) to produce a conjugate of ubiquitin-like protein and acceptor in the transformant Body manufacturing method.
(25) A recombinant expression vector comprising DNA encoding a fusion protein of a ubiquitin-like protein and an acceptor, wherein the fusion protein of the ubiquitin-like protein and the acceptor can be expressed in a host.
(26) A DNA encoding a ubiquitin-like protein and a DNA encoding one or more target proteins adjacent to the DNA encoding the acceptor, and further comprising a fusion protein of the ubiquitin-like protein, the acceptor and the target protein in the host The recombinant expression vector according to (25), which can be expressed.
(27) By using the recombinant vector according to (15) or (16) in combination with the recombinant vector according to (25) or (26), an activating enzyme (E1) and a binding enzyme (E2) And a method for producing a polymer of a fusion protein of a ubiquitin-like protein and an acceptor, comprising simultaneously expressing a fusion protein of the ubiquitin-like protein and an acceptor in a host.
(28) DNA encoding an enzyme (E1) that activates a ubiquitin-like protein, DNA encoding a ubiquitin-like protein-acceptor binding enzyme (E2), and a fusion protein of a ubiquitin-like protein, an acceptor, and, if desired, a target protein A recombinant expression vector comprising a DNA encoding, and capable of simultaneously expressing the activating enzyme (E1), the binding enzyme (E2) and the fusion protein in a host.
(29) A transformant capable of expressing an enzyme that activates a ubiquitin-like protein (E1), a binding enzyme (E2) of a ubiquitin-like protein and an acceptor, and a fusion protein of a ubiquitin-like protein, an acceptor, and, if desired, a target protein.
(30) The transformant according to (29), comprising the recombinant vector according to (15) or (16) and the recombinant vector according to (25) or (26).
(31) A protein comprising culturing the transformant according to (29) or (30), and producing a polymer of a fusion protein of a ubiquitin-like protein, an acceptor, and optionally a target protein in the transformant. A method for producing a polymer.

FIG. 1 shows the various structures that the protein polymer of the present invention can take.
FIG. 2 shows the structure of the GST-SUMO fusion expression vector.
FIG. 3 shows the results of separation of GST-SUMO polymerization reaction products by SDS-denatured polyacrylamide gel electrophoresis and detection by Western method.
FIG. 4 shows the structure of the C2-SUMO fusion expression vector.
FIG. 5 shows the structure of the GST-C2-SUMO fusion expression vector.
FIG. 6 shows the results of GST-C2-SUMO polymerization reaction using E1 and E2 enzymes, and the reaction products separated by SDS-modified polyacrylamide gel electrophoresis and detected by Western method.
FIG. 7 shows a structural schematic diagram of the modified SUMO-E1 enzyme (AU). An enzyme that forms an isopeptide bond between SUMO and an acceptor is modified to facilitate expression in E. coli. In the present specification, this modified enzyme is referred to as AU. Conventional SUMO-E1 is an enzyme composed of two subunit proteins, Aos1 and Uba2. A protein AU was prepared by fusing Aos1 and Uba2 by genetic engineering techniques. This AU protein is our original design, in which Aos1 and Uba2 I, II, III, and IV are fused in a manner that preserves the domain structure. Cys indicates the position of a cysteine residue essential for enzyme activity.
FIG. 8 schematically shows a method for synthesizing a conjugate of the target proteins X and Y in E. coli. A specific experimental example is shown in Example 3. In Example 3, X is T7-domain and Y is HIS-domain. Moreover, C2 arrangement | sequence is used as acceptor A.
FIG. 9 is a diagram of the expression vector used in Example 3.
FIG. 10 shows the results of Example 3.
FIG. 11 schematically shows a method of synthesizing a target protein X polymer in E. coli. A specific experimental example is shown in Example 4. In Example 4, X is GST-domain, and the C2 sequence is used as acceptor A.
FIG. 12 is a diagram of the expression vector used in Example 4.
FIG. 13 shows the results of Example 4.
FIG. 14 shows the results of Example 5 (analysis using various acceptor sequences).

（式中、ＳＵＭＯはユビキチン様タンパク質、Ａはアクセプター、Ｘは目的タンパク質、太線はイソペプチド結合、ｅは２〜１００の平均重合度を示す。）
で表されるタンパク質ポリマーが挙げられる。
タンパク質ポリマーの別の例としては、下記式（ＩＶ）：

（式中、ＳＵＭＯはユビキチン様タンパク質、Ｘ、Ｙ、及びＺは目的タンパク質、Ａはアクセプター、太線はイソペプチド結合、ｆ、ｇ、及びｈは０から１００の平均重合度を示す。但し、ｆ、ｇ、及びｈが同時に０の場合は除く）。
で表されるタンパク質ポリマーが挙げられる。
本発明のタンパク質ポリマーが、前記式（ＩＩＩ）で表される場合、ｅで示される平均重合度は２〜１００、好ましくは２〜５０、より好ましくは３〜１０である。
本発明のタンパク質ポリマーが、前記式（ＩＶ）で表される場合、ｆ，ｇ，及びｈで表される平均重合度は、０〜１００、好ましくは０〜５０、より好ましくは０〜１０である（但し、ｆ，ｇ，及びｈが同時に０の場合は除く）。
また、前記式（ＩＶ）で表されるタンパク質ポリマーにおいて、構成単位であるＸ−Ａ−ＳＵＭＯ、Ｙ−Ａ−ＳＵＭＯあるいはＺ−Ａ−ＳＵＭＯは、それぞれランダムに結合してもよく、またブロック的に結合していてもよい。式（ＩＩＩ）のタンパク質ポリマーを製造する場合は、Ｘ−Ａ−ＳＵＭＯ、Ｙ−Ａ−ＳＵＭＯあるいはＺ−Ａ−ＳＵＭＯをコードするＤＮＡを同一又は異なる発現ベクターに組み込んで組み換え発現ベクターを作製し、それを宿主に導入して発現させることにより行なうことができる。形質転換宿主内に最初からＸ−Ａ−ＳＵＭＯ、Ｙ−Ａ−ＳＵＭＯ及び／又はＺ−Ａ−ＳＵＭＯを発現させるとランダムなポリマーが得られ、最初にＸ−Ａ−ＳＵＭＯ又はＹ−Ａ−ＳＵＭＯ又はＺ−Ａ−ＳＵＭＯのいずれかを発現させ、次いで他の構成単位を順次発現させていくとブロック的なポリマーを製造することができる。
本発明を実施例によりさらに具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。  Hereinafter, embodiments of the present invention will be described in detail.
1. Protein polymer of the present invention
  The protein polymer of the present invention is a protein polymer mainly containing a ubiquitin-like protein (SUMO) to which a target protein is bound directly or via an acceptor.
  “SUMO” used in the protein polymer of the present invention means a ubiquitin-like protein, and includes all of the modifier groups having high homology with ubiquitin. Moreover, the origin is not ask | required. A mammal-derived one is preferable, but a non-mammal one such as yeast, insect, amphibian, reptile, plant may be used. Examples of SUMO that can be used in the present invention include SUMO-1 having the amino acid sequence of SEQ ID NO: 1, SUMO-2 having the amino acid sequence of SEQ ID NO: 2, and SUMO-3 having the amino acid sequence of SEQ ID NO: 3. However, the present invention is not limited thereto. For example, as long as it has SUMO protein activity / function, one to several amino acids may be deleted, substituted and / or inserted in the amino acid sequence.
  The “acceptor” in the protein polymer of the present invention is a protein having an amino acid sequence containing a lysine residue capable of isopeptide binding to the above-mentioned SUMO, and is generally -F-Lys-H-Glu- (F: hydrophobic amino acid) , H: any amino acid). Examples of the acceptor include RanGAP1 (Ran GTPase activating protein), RanBP2-IR (Ran binding protein 2-Internal Repeat domain), and PML (pronelolytic leukemia). RanGAP1 (Ran GTPase activating protein) preferably used in the present invention has the amino acid sequence of SEQ ID NO: 4, and the 121st lysine residue is isopeptide-bound to SUMO (note that the total length of RanGAP1 is 580 amino acids, Among these, the amino acid sequence from the 397th to the 580th in the C-terminal region is the C2 sequence).
  The above acceptors may be used alone or in combination of two or more. When two or more acceptors are used, the main chain of the polymer can take a branched structure. Further, when an acceptor having a structural arrangement with a bend of 60 degrees or 120 degrees is used, the main chain of the polymer can take a cyclic structure such as a triangle or a hexagon. Further, in the case of taking a cyclic structure, when proteins that interact with each other are bound to SUMO via an acceptor, self-assembly occurs due to the interaction of the proteins, and the main chain of the polymer can take a tubular structure. FIG. 1 shows the various structures that the protein polymer of the present invention can take.
  The “target protein” in the protein polymer of the present invention is not particularly limited and may be any protein. For example, enzymes, cytokines, hormones, antibodies, receptors, bioactive peptides and the like can be used. These target proteins contained in the polymer may be one type or two or more types.
  Examples of the enzyme include lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucanase, and lactase.
  Cytokines include, for example, interferon-α, -β, -γ, Tsumore necrosis factor-α, -β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, interleukin, growth factor (epidermal growth factor, Fibroblast growth factor, nerve cell growth factor) and the like.
  Examples of hormones include insulin, growth hormone, prolactin, erythropoietin, follicle stimulating hormone and the like.
  Examples of the antibody include human immunoglobulin and the like, and F (ab ') of the antibody₂, Fab ', or an active fragment such as Fab. Further, it may be a substance having a similar function as an antibody, for example, a substance having a single-chain antigen-binding activity, specifically a single-chain Fab peptide.
  Examples of receptors include hormone receptors, cytokine receptors, receptors for growth factors, receptors for neurotransmitters, antibody receptors (Fc receptors), complement receptors, and the like.
  Examples of the physiologically active peptide include β-amyloid, angiotensin, endothelin, calcitocin, neuropeptide Y, neurokinin A, oxytocin, PACAP and the like.
  The molecular weight (kDa) of the target protein is in the range of 2 to 1000 kDa, preferably 2 to 100 kDa, particularly 10 to 50 kDa. If the molecular weight of the target protein is in this range, the fusion with SUMO causes steric hindrance. It is preferable because it is not present.
  When the protein polymer of the present invention is represented by the formula (I), the average degree of polymerization represented by a is 2 to 100, preferably 2 to 50, more preferably 3 to 10.
  When the protein polymer of the present invention is represented by the formula (II), the average degree of polymerization represented by b, c and d is 0 to 100, preferably 0 to 50, more preferably 0 to 10. Yes (except when b, c, and d are 0 at the same time).
  When the protein polymer of the present invention is represented by the formula (III), the average degree of polymerization represented by e is 2 to 100, preferably 2 to 50, more preferably 3 to 10.
  When the protein polymer of the present invention is represented by the formula (IV), the average degree of polymerization represented by f, g, and h is 0 to 100, preferably 0 to 50, more preferably 0 to 10. Yes (except when f, g, and h are 0 at the same time).
  In the protein polymer represented by the formula (IV), XA-SUMO, YA-SUMO, and ZA-SUMO, which are structural units, may be bonded at random or block-like. May be bonded to. When a reaction system is initially charged with X-A-SUMO, Y-A-SUMO and / or Z-A-SUMO, a random polymer is obtained. First, X-A-SUMO or Y-A-SUMO or Z When any one of -A-SUMO is charged and then the other structural units are sequentially charged, a block polymer can be produced.
  Furthermore, a protein conjugate represented by the formula: X-SUMO-AY in which a fusion protein of ubiquitin-like protein SUMO and target protein X and acceptor A and target protein Y is bound is also within the scope of the present invention. The type and number of X and Y are not limited. In particular, the target protein (Y) to be bound to the acceptor may be bound to either the amino terminal side or the carboxy terminal side of the acceptor, or may be bound to both. it can.
2. Process for producing protein polymer of the present invention
(1) Preparation of reaction substrate
  Next, the manufacturing method of the protein polymer of this invention is demonstrated. In order to produce the protein polymer of the present invention, a reaction substrate to be a monomer is first prepared.
  The reaction substrate can be prepared by linking ubiquitin-like protein (SUMO), target protein, acceptor-encoding gene and introducing the gene into an appropriate expression system, or expressing each protein separately. Any of the methods of producing and subsequently chemically bonding using a crosslinking agent or the like may be used.
  In the present specification, the term “fusion” refers to a reaction substrate prepared by a genetic engineering expression method as a fusion protein, and the term “conjugate” refers to a reaction substrate prepared by chemical bonding. To do. Any reaction substrate prepared by genetic engineering or chemical bonding is a substrate with good polymerization.
  In order to prepare a fusion, after constructing DNA fragments each containing a gene encoding SUMO, a target protein, or an acceptor, the DNA fragment is inserted downstream of the promoter of the expression vector, and then the expression vector is appropriately used. It is introduced into the host cell.
  The target protein is linked downstream of a reporter gene such as GST (glutathione S transferase), MBP (maltose binding protein), histidine hexamer, thioredoxin, or a fluorescent protein such as green fluorescent protein (GFP), It is preferably expressed as a fusion protein with these. The target protein itself may be GST, MBP, histidine hexamer, thioredoxin, or fluorescent protein.
  Gene engineering of ubiquitin-like protein (SUMO) and target protein fusion, or ubiquitin-like protein (SUMO), acceptor and target protein fusion (hereinafter collectively referred to as SUMO-protein fusion) In this case, the host cell to be used is not particularly limited as long as it can express the target gene, and examples thereof include bacteria, yeast, animal cells, plant cells, and insect cells.
  As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the target DNA can be transcribed is used.
  The introduction of the recombinant vector may be performed by a method conventionally used depending on the host cell to be used.
  A transformant introduced with the recombinant vector is cultured in a medium, a SUMO-protein fusion is produced and accumulated in the culture, and the SUMO-protein fusion is collected from the culture. Can be isolated.
  The method of culturing the transformant can be performed according to a usual method. When the transformant is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism, For culture of the body, either a natural medium or a synthetic medium may be used. The culture conditions, culture temperature, culture time, and culture medium pH may be those usually employed depending on the host cell used.
  In order to isolate and purify the SUMO-protein fusion of the present invention from the culture of the transformant, a normal enzyme isolation and purification method may be used. For example, when the SUMO-protein fusion is expressed in a dissolved state in the cell, the cell is recovered by centrifugation after culturing, suspended in an aqueous buffer, and then disrupted by an ultrasonic disrupter or the like. A cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl ( DEAE) Anion exchange chromatography using a resin such as Sepharose, Cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), Hydrophobic chromatography using a resin such as butyl Sepharose A purified sample can be obtained by using a single method or a combination of methods such as gel filtration using a molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing.
  In addition, when the SUMO-protein fusion is expressed in the form of an insoluble substance in the cell, the cell is similarly collected, disrupted, and centrifuged from the precipitate fraction obtained by the usual method. After recovering the fusion, the insoluble form of the fusion is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed into a solution that does not contain a protein denaturant or the protein denaturant has a concentration that does not denature the protein, and the fusion is formed into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method as described above.
  When the SUMO-protein fusion used in the present invention is secreted extracellularly, the fusion can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. Can do.
  Alternatively, the synthesis of the SUMO-protein fusion may be performed using a cell-free protein synthesis system in which components having protein synthesis ability are extracted from appropriate cells, and the target protein is synthesized using the extract. Such a cell-free protein synthesis system includes elements necessary for a transcription / translation system such as a ribosome, an initiation factor, an elongation factor, and tRNA.
(2) Production of protein polymer
  Next, in order to produce a protein polymer from the obtained SUMO-protein fusion, the SUMO-protein fusion is prepared by using SUMO activating enzyme E1 (Aso1 / Uba2), SUMO-binding enzyme H2 (Ubc9), ATP, and metal ions. Is polymerized by isopeptide bonds in the reaction solution.
  In general, the polymerization reaction is preferably performed using purified enzyme components. However, since the E1 and E2 enzymes involved in the SUMO polymerization reaction have high substrate specificity, they can be used even if their purification purity is not particularly high.
  The concentration of the substrate in the reaction solution may be any concentration as long as the reaction is not hindered, but is preferably 0.01 mM to 1M. The enzyme concentration used for the reaction is not particularly limited, but is preferably 0.01 mg / ml to 100 mg / ml. The ATP concentration is preferably in the range of 0.01 to 10 mM.
  As the metal ions, divalent metal ions such as magnesium ions and calcium ions are preferably used. When adding magnesium ions to the reaction solution, the concentration is preferably in the range of 0.01 to 10 mM.
  In addition, a reaction promoting substance may be appropriately added to the reaction system. Examples of the reaction promoting substance include, but are not limited to, a group of protein families called SP-RING protein or SIZ / PIAS, RanBP2 / Nup358, and the like.
  The reaction temperature should just be the temperature range which does not inhibit the activity of E1 and E2, and the temperature of the range containing optimal temperature is preferable. The reaction temperature is usually 10 ° C to 60 ° C, preferably 30 ° C to 40 ° C.
  Moreover, in order to perform said polymerization reaction efficiently, it is preferable to carry out on a base protein. In the present invention, the “base protein” refers to a protein having an amino acid sequence including a lysine residue capable of isopeptide bonding with the C-terminus of SUMO. An internal repeat domain (PML), PML (proneelotic leukemia), Sp100, p53, etc. can be used preferably.
  Examples of the method for separating and quantifying the target substance include high performance liquid chromatography (HPLC) as necessary. Alternatively, after the reaction products are separated by polyacrylamide gel electrophoresis (SDS-PAGE) or the like, a decrease in substrate and an increase in reaction products can be detected by performing immunoblot analysis using an appropriate antibody. .
  The SUMO protein polymer of the present invention can be used in various applications as it is, but it is supported on various inorganic compound carriers or organic compound carriers in order to facilitate handling or to improve the reactivity per unit weight. It is preferable to use in the state.
3. The protein conjugate of the present invention using a biosynthetic system in a fungus body Is a method for producing polymers
  Until now, it was not possible to overexpress or accumulate protein conjugates by isopeptide bonds in bacterial host cells. The biggest reason is that bacteria do not have enzymes in the body that catalyze isopeptide bonds. The present inventors succeeded in forming an isopeptide bond in the microbial cells by simultaneously expressing mammalian SUMOylase (E1 and E2 enzymes), SUMO and an acceptor in Escherichia coli. Developed technology for mass production.
  That is, the method of the present invention relates to a recombinant expression system that enables overexpression of a protein conjugate or polymer by using a biosynthetic system in Escherichia coli for a reaction that was conventionally performed in a test tube.
  In Examples 3 and 4 of the present invention, two types of expression vectors were used. The first expression vector is a vector that encodes SUMO, an acceptor, and a target protein, and the second expression vector encodes an enzyme (E1 and E2 enzyme) that catalyzes the reaction of isopeptide binding between SUMO and an acceptor. Yes. By incorporating these two types of vectors into the microbial cells and simultaneously expressing genes on the vectors, it is possible to express a conjugate or polymer containing the target protein at a high level.
  SUMO is a ubiquitin-like protein, and in the presence of ATP-Mg2 +, an enzyme called E1 (SUMO activating enzyme) and E2 (SUMO conjugating enzyme) causes amino acids of lysine residues in the acceptor sequence and the glycine residue at the C-terminal of SUMO The proximal side chain is an isopeptide bond.
  Originally, SUMO-E1 consists of a hetero-complex of two proteins called Aos1 and Uba2. In Example 3 described herein, Aos1 and Uba2 were cloned from mouse cDNA by PCR, and then the fusion protein was designed by ligating the two fragments in tandem. This Aos1-Uba2 fusion gene (AU gene) was introduced into the BamHI-EcoRI site of the pGEX expression vector to develop a system for expressing the GST-AU protein in E. coli. Independently of AU, Ubc9 which is SUMO-E2 was introduced into the XbaI site of this pGEX vector to prepare pGEX-AU / Ubc9 as shown in FIG. In this vector, GST-AU is linked downstream of the Taq promoter, Ubc9 is linked downstream of the T7 promoter, and the two gene products are designed to be inducible by IPTG. The vector used here has a replication origin derived from pBR322 and is resistant to ampicillin drug. The plasmid name was pGEX-AU / Ubc9. GST-AU and Ubc9 expressed from this plasmid vector have E1 and E2 enzyme activities, respectively, and have the ability to catalyze the SUMOylation reaction.
  On the other hand, the C-terminal domain C2 of a protein called RanGAP1 (RanGAP1 has a total length of 580 amino acids, of which the 397th to 580th amino acid sequences of the C-terminal region are C2 sequences) is specifically subjected to SUMOylation in cells. It is known. Coding two products, protein HIS-domain-C2 fused with amino acid sequence containing (His) 6 to C2 domain and protein T7-domain-SUMO-1 fused with amino acid sequence containing T7-tag sequence to SUMO-1. FIG. 9 shows expression vectors in which the genes to be introduced are respectively introduced downstream of the T7 promoter. SUMO-1 was obtained from human cDNA, and C2 was obtained from Xenopus cDNA by PCR. The vector used here is a modified version of pACYC, has a p15A origin of replication, and is resistant to chloramphenicol. Two gene expressions can be induced by IPTG. The plasmid name was pT-SUMO / C2.
  The above-mentioned pGEX-AU / Ubc9 and pT-SUMO / C2 are simultaneously introduced into a host (for example, E. coli etc.). Cells that have taken up these two plasmids simultaneously can be selected on a medium containing both ampicillin and chloramphenicol. The obtained colony is cultured in a separate liquid medium under suitable conditions, and further cultured with IPTG added to induce expression. The cells are precipitated with a centrifugal separator, and then the cells are crushed by ultrasonic treatment or the like. After centrifugation, the supernatant is removed, incubated with nickel agarose beads, and the conjugate of T7-domain-SUMO-1 and His-domain-C2 is recovered by operations such as elution of the protein bound to the beads. Can do.
  Various protein conjugates can be prepared by modifying the vectors used above. As shown in FIG. 8, when the target protein X is introduced instead of T7-domain, and the target protein Y is introduced instead of His-domain, an XY conjugate X-SUMO-C2-Y Can be produced.
  In this experiment, C2 is used as an acceptor, but acceptor sequences other than C2 can be used. Furthermore, in addition to SUMO-1, related sequences such as SUMO-2 and SUMO-3 can also be used. There are no restrictions on these combinations.
  Furthermore, in the past, it was not possible to polymerize proteins by covalently binding them in bacterial host cells. A technique for overexpressing and accumulating protein polymers has not been developed. In the present invention, expression of a mammalian SUMO-related factor in E. coli has succeeded in forming an isopeptide bond in the microbial cell, and it has become possible to provide a technique for producing a large amount of protein polymer. .
  In Example 4 described herein, a GST protein (27 kDa) is used as X of X-A-SUMO, and a C-terminal domain C2 of a protein called RanGAP1 is used as an A sequence (GST-C2-SUMO-1). ). GST-C2-SUMO-1 is incorporated into pACYC as a vector, and this plasmid is called pT-GST-C2-SUMO (see FIG. 12).
  The above-mentioned pGEX-AU / Ubc9 and pT-GST-C2-SUMO are simultaneously introduced into a host (for example, E. coli etc.). Cells that have taken up these two plasmids simultaneously can be selected on a medium containing both ampicillin and chloramphenicol. The obtained colony is cultured in a separate liquid medium under suitable conditions, and further cultured with IPTG added to induce expression. The cells are precipitated with a centrifugal separator, and then the cells are crushed by ultrasonic treatment or the like. After centrifugation, the supernatant is removed, incubated with glutathione sepharose beads, and the GST-C2-SUMO polymer obtained by polymerizing GST-C2-SUMO can be recovered by, for example, eluting the protein bound to the beads. it can.
  By modifying the vector used above, various proteins X can be introduced into the polymer instead of GST. That is, as shown in FIG. 11, by introducing various target proteins X as C2-SUMO fusion proteins, a large amount of X polymers based on C2-SUMO can be produced in E. coli.
  In this experiment, C2 is used as an acceptor, but acceptor sequences other than C2 can also be used. Furthermore, in addition to SUMO-1, related sequences such as SUMO-2 and SUMO-3 can also be used, and the shape of the base axis of the polymer can be modified. There is no restriction | limiting in particular in the combination of an acceptor and SUMO.
  Hereinafter, a method for carrying out the method for producing a protein conjugate or polymer of the present invention using a biosynthetic system in a fungus body (hereinafter also referred to as this method) will be described in more detail.
(1) E1 enzyme and E2 enzyme
  A group of proteins having a ubiquitin-like structure such as ubiquitin, SUMO, NEDD8 / Rub1, and Apg12 is called a ubiquitin family. These ubiquitin family proteins have an isopeptide bond to an acceptor site of another protein by an activating enzyme (E1) and a binding enzyme (E2). Uba1 is known as ubiquitin E1, Aos1 / Uba2 heterodimer as SUMO-E1, APP-BP1 / Uba3 heterodimer as NEDD8-E1, and Apg7 as E1 of Apg12. Ubc1-8, 10, 11, 13 as ubiquitin E2, Ubc9 as SUMO-E2, Ubc12 as NEDD8-E2, and Apg10 as E2 of Apg12 (Keiji Tanaka, Yoshinori Ohtsuki (edit), experimental medicine) (See "Proteolysis Frontline 2001" Vol. 19, No. 2, 2001).
  In this method, AU that is a fusion protein of Aos1 and Uba2 as shown in FIG. 7 is preferably used as the E1 enzyme, and Ubc9 is preferably used as the E2 enzyme.
(2) Ubiquitin-like protein, acceptor, and target protein,
  The ubiquitin-like protein used in this method means a ubiquitin-like protein and includes all of the modifier groups having high homology with ubiquitin. Specific examples thereof include ubiquitin, SUMO, NEDD8 / Rub1, and Apg12. It is done. Further, its origin is not limited, and those derived from mammals are preferable, but those other than mammals such as yeasts, insects, amphibians, reptiles, and plants may be used. Examples of SUMO that can be used in the present invention include SUMO-1 having the amino acid sequence of SEQ ID NO: 1, SUMO-2 having the amino acid sequence of SEQ ID NO: 2, and SUMO-3 having the amino acid sequence of SEQ ID NO: 3. However, the present invention is not limited thereto. For example, as long as it has SUMO protein activity / function, one to several amino acids may be deleted, substituted and / or inserted in the amino acid sequence.
  The “acceptor” used in the present method is a protein having an amino acid sequence containing a lysine residue capable of isopeptide bonding with the above-mentioned ubiquitin-like protein, and is generally -F-Lys-H-Glu- (F: hydrophobic amino acid) , H: any amino acid). As the acceptor, those described in the item “1. Protein polymer of the present invention” in the present specification can be used. There are roughly two groups of acceptors, one with a single SUMO binding site and the other with a plurality of acceptors. In one case (for example, RanGAP1-C2 or TDG), it is considered that a linear or tufted polymer is formed when there are a plurality of (for example, RanBP2-IR or TONAS).
  The “target protein” used in the present invention is not particularly limited, and may be any protein. As the “target protein”, those described in the item “1. Protein polymer of the present invention” in the present specification can be used.
(3) Expression vector
  The present invention includes:
  A DNA encoding an enzyme (E1) that activates a ubiquitin-like protein and a DNA encoding a binding enzyme (E2) of a ubiquitin-like protein and an acceptor, and an activating enzyme (E1) and a binding enzyme (E2) A recombinant expression vector that can be expressed simultaneously in
  A recombinant expression vector comprising a DNA encoding a ubiquitin-like protein and a DNA encoding an acceptor, wherein the ubiquitin-like protein and the acceptor can be separately expressed in the host simultaneously;
  DNA encoding an enzyme (E1) that activates a ubiquitin-like protein; DNA encoding a binding enzyme (E2) of a ubiquitin-like protein and an acceptor; DNA encoding a ubiquitin-like protein; and / or DNA encoding an acceptor A recombinant expression vector capable of individually and simultaneously expressing an activating enzyme (E1), a linking enzyme (E2), a ubiquitin-like protein and / or an acceptor in a host;
  A recombinant expression vector comprising DNA encoding a fusion protein of a ubiquitin-like protein and an acceptor, wherein the fusion protein of the ubiquitin-like protein and the acceptor can be expressed in a host; and
  DNA encoding the enzyme (E1) that activates the ubiquitin-like protein, DNA encoding the binding enzyme (E2) of the ubiquitin-like protein and acceptor, and a fusion protein of the ubiquitin-like protein, acceptor, and target protein, if desired. A recombinant expression vector comprising DNA and capable of simultaneously expressing the activating enzyme (E1), the binding enzyme (E2) and the fusion protein in a host;
It is about.
  Various recombinant expression vectors described above can be constructed by inserting a desired gene into an appropriate transgene insertion site in a normal expression vector. As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the target base sequence can be transcribed is used. As the expression vector, for example, various expression vectors commercially available from Promega, QIAGEN, Stratagene, Pharmacia, Takara Shuzo and the like can be used.
(4) Production of transformant, conjugate of ubiquitin-like protein and acceptor and protein polymer using the same
  In the present invention, the host cell used for introducing the expression vector described in (3) above is not particularly limited as long as it can express the target gene. For example, bacteria, yeast, animal cells, plant cells, insects A cell etc. can be mentioned. In the present invention, among the above, it is preferable to use bacteria (for example, bacteria belonging to the genus Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc.). It is particularly preferred to use
  Introduction of the recombinant expression vector may be performed by a method conventionally used depending on the host cell to be used, for example, calcium phosphate method, protoplast method, electroporation method, spheroblast method, lithium acetate method, lipofection method, etc. Is mentioned.
  A transformant introduced with the recombinant expression vector is cultured in a medium, a SUMO-protein fusion is produced and accumulated in the culture, and the ubiquitin-like protein-protein fusion is collected from the culture. Protein-protein fusions can be isolated.
  The method for culturing the transformant, the isolation and purification of the ubiquitin-like protein-protein fusion of the present invention from the culture of the transformant, and the separation and quantification of the target substance are described in 2. above. Can be carried out as described in.
(5) Ubiquitin-like protein / acceptor conjugate and ubiquitin-like protein / acceptor polymer (protein polymer)
  By the method (4) above, a ubiquitin-like protein / acceptor conjugate or a ubiquitin-like protein / acceptor polymer (protein polymer) can be produced and isolated.
  As a specific example of the ubiquitin-like protein / acceptor conjugate, a fusion protein in which the target protein X and Y are respectively bound to the acceptor (A) and SUMO can be used. As a result, a Conjugate of X-SUMO-YA can be formed. There are no restrictions on the type and number of X and Y to be bound to SUMO or acceptor, and the target protein to be bound to acceptor may be either on the amino terminal side or carboxy terminal side of the acceptor, and attached to both. It doesn't matter.
  The protein polymer produced by the method of the present invention is a protein polymer containing, in its main chain, a ubiquitin-like protein (for example, SUMO: small uiquitin-related modifier) to which a target protein is bound directly or via an acceptor.
  As an example of a protein polymer, the following formula (III):

(In the formula, SUMO is a ubiquitin-like protein, A is an acceptor, X is a target protein, a bold line is an isopeptide bond, and e is an average degree of polymerization of 2 to 100.)
The protein polymer represented by these is mentioned.
  As another example of the protein polymer, the following formula (IV):

(In the formula, SUMO is a ubiquitin-like protein, X, Y, and Z are target proteins, A is an acceptor, thick lines are isopeptide bonds, f, g, and h are average degrees of polymerization from 0 to 100, where f is , G, and h are simultaneously 0).
The protein polymer represented by these is mentioned.
  When the protein polymer of the present invention is represented by the formula (III), the average degree of polymerization represented by e is 2 to 100, preferably 2 to 50, more preferably 3 to 10.
  When the protein polymer of the present invention is represented by the formula (IV), the average degree of polymerization represented by f, g, and h is 0 to 100, preferably 0 to 50, more preferably 0 to 10. Yes (except when f, g, and h are 0 at the same time).
  In the protein polymer represented by the formula (IV), XA-SUMO, YA-SUMO, and ZA-SUMO, which are structural units, may be bonded at random or block-like. May be bonded to. When producing a protein polymer of the formula (III), a DNA encoding X-A-SUMO, YA-SUMO or ZA-SUMO is incorporated into the same or different expression vector to produce a recombinant expression vector, It can be carried out by introducing it into a host and expressing it. When XA-SUMO, YA-SUMO, and / or ZA-SUMO is expressed from the beginning in the transformed host, a random polymer is obtained, and first X-A-SUMO or YA-SUMO is obtained. Alternatively, when either ZA-SUMO is expressed and then other structural units are expressed in sequence, a block polymer can be produced.
  The present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.

増幅したＰＣＲ断片をｐＧＥＸベクター（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）のＥｃｏＲＩ部位にクローニングし、得られたプラスミド構築物をそれぞれｐＧＥＸ−Ａｏｓ１及びｐＧＥＸ−Ｕｂａ２と命名した。Ｍｏｕｓｅ Ａｏｓ１ ｃＤＮＡの塩基配列を配列番号５に示し、Ｍｏｕｓｅ Ｕｂａ２の塩基配列を配列番号６に示す。
Ａｏｓ１−Ｕｂａ２キメラ構築物を作製するために、Ａｏｓ１及びＵｂａ２を以下のオリゴヌクレオチドを用いてＰＣＲにより増幅した。

増幅した断片をＢａｍＨＩ＋ＥｃｏＲＩ及びＥｃｏＲＩでそれぞれ切断し、ＢａｍＨＩ＋ＥｃｏＲＩで消化ｐＧＥＸベクターに挿入した。得られたプレスミドｐＧＥＸ−ＡＵはＧＳＴ−Ａｏｓ１−Ｕｂａ２融合タンパク質をコードしている。マウスＡＵ ｃＤＮＡの塩基配列を（ＧＡＡＴＴＣはＡｏｓ１とＵｂａ２の結合領域）を配列番号７に示す。
Ｘｅｎｏｐｕｓ（アフリカツメガエル）のＵｂｃ９（塩基配列を配列番号８に示す）をクローニングしｐＴ７−７ｖｅｃｔｏｒに挿入した（Ｓａｉｔｏｈ ｅｔ ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ．，Ｖｏｌ９４，３７３６−３７４１，１９９７）。ちなみに、アフリカツメガエルのＵｂｃ９のアミノ酸配列はヒトおよびマウスのＵｂｃ９のアミノ酸配列と１００％相同である。以下のプライマーを用いてＰＣＲを行った。

増幅したＤＮＡ断片をＮＨｅＩで切断後、ＸｈｏＩで処理したｐＧＥＸ−ＡＵとライゲーションした。作製されたプラスミドを解析したところ、図８に示すように、それぞれの配列がＮ末端からＣ末端に向かう形で順向きにタンデムに並ぶ構造をしていた。このプラスミドベクターをＰＧＥＸ−ＡＵ／Ｕｂｃ９と名付けた。ＧＳＴ−ＡＵタンパク質はｔａｃプロモーターの、Ｕｂｃ９はＴ７プロモーターの制御を受け、ＩＰＴＧにより誘導される。
（２）Ｔ７−ｄｏｍａｉｎ−ＳＵＭＯ−１を発現する発現ベクターのｐＴ−ＳＵＭＯ−１／Ｃ２プラスミドベクターの作製
ｈｕｍａｎ ＳＵＭＯ−１ ｃＤＮＡ（塩基配列を配列番号９に記載する）と以下のプライマーを用いて、Ｔ７−ＳＵＭＯ−１をコードするＤＮＡ断片をＰＣＲで増幅する。このタンパク質はＴ７−ｔａｇ配列（ｍｅｔ−ａｌａ−ｓｅｒ−ｍｅｔ−ｔｈｒ−ｇｌｙ−ｇｌｙ−ｇｌｎ−ｇｌｎ）（配列番号２０）がＳＵＭＯ−１のＮ末端にある融合タンパク質である。

増幅断片をＮｄｅＩ＋ＨｉｎｄＩＩＩで切断後、同じくＮｄｅＩ＋ＨｉｎｄＩＩＩで処理したｐＴ−Ｔｒｘ（Ｃｈａｎｇ，Ａ．Ｃ．Ｙ．＆ Ｃｏｈｅｎ，Ｓ．Ｎ．：Ｊ．Ｂａｃｔｅｒｉｏｌ．，１３４，１１４１−１１５６，１９７８；Ｙａｓｕｋａｗａ，Ｔ．ｅｔ ａｌ．：Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７０，２５３２８−２５３３１，１９９５）とライゲーションする。得られたプラスミドをｐＴ−Ｔ７−ＳＵＭＯ−１と呼ぶ。
アクセプターとしてＣ２と呼ばれるＲａｎＧＡＰ１（ＲａｎＧＴＰａｓｅ Ａｃｔｉｖａｔｉｎｇ Ｐｒｏｔｅｉｎ １）のＣ末端領域を選択した。ＲａｎＧＡＰ１は低分子量ＧＴＰ結合タンパク質Ｒａｎの加水分解を促進する活性を持つタンパク質で、核−細胞質間の物質輸送に関わる。Ｃ２−ＳＵＭＯ融合体を得るために、Ｃ２、及びＳＵＭＯをコードするＤＮＡ断片をそれぞれＰＣＲを用いて合成し、発現ベクターｐＥＴ２８（ノバジェン）に連結した。このプラスミドベクターをｐＥＴ２８−Ｈｉｓ−Ｃ２と称する。このプラスミドはＨｉｓ−Ｃ２融合タンパク質をコードしている。Ｃ２配列のＮ末端上流に以下の配列が融合している。

このプラスミドを鋳型として、以下のプライマーを用いてＰＣＲを行った。

増幅断片をＢｇｌＩＩで切断後、同じくＢｇｌＩＩで処理した上述のｐＴ−Ｔ７−ＳＵＭＯ−１とライゲーションした。得られたプラスミドｐＴ−ＳＵＭＯ−１／Ｃ２は、Ｔ７プロモターをＩＰＴＧで誘導することで、Ｈｉｓ−Ｃ２とＴ７−ＳＵＭＯ−１の２種類の融合タンパク質を同時に発現するようにデザインされている。
（３）大腸菌での発現とタンパク質の部分精製
上述のｐＧＥＸ−ＡＵ／Ｕｂｃ９およびｐＴ−ＳＵＭＯ／Ｃ２を同時に大腸菌ＢＬ２１ＤＥ３株に導入する。２つのプラスミドを同時に取り込んだ菌体はアンピシリンとクロランフェニコールの両方を含む培地で選別できる。これとは独立に、ｐＴ−ＳＵＭＯ／Ｃ２のみを導入したものをクロランフェニコール培地で選別する。
それぞれの培地で得られたコロニーを別個のＬＢ液体培地１Ｌで３７℃で１６時間震盪培養する。０．０２ｍＭ ＩＰＴＧを加え、２５℃でさらに１６時間震盪培養する。
菌体を遠心分離器で沈澱させ、その後、１０ｍｌのＰＢＳバッファーに懸濁した後、超音波処理により菌体を破砕する。遠心分離の後、上澄み液を取り出し、ニッケルアガロースビーズ１ｍｌと１時間４℃でインキュベーションする。ビーズをＰＢＳで洗浄後、２５０ｍＭイミダゾールを含むＰＢＳでビーズに結合したタンパク質を溶出する。タンパク質は１２％ＳＤＳ−ＰＡＧＥおよびウエスタン法で解析する。
図１０のレーン１にはｐＧＥＸ−ＡＵ／Ｕｂｃ９およびｐＴ−ＳＵＭＯ／Ｃ２を同時に大腸菌ＢＬ２１ＤＥ３株に導入したもの、レーン２にはｐＴ−ＳＵＭＯ／Ｃ２を導入したものを示してある。前者では４０ｋｄａのタンパク質が、後者では２５ｋｄａのタンパク質がクマシ−ブル−染色で明瞭に観察される。前者のバンドはＨｉｓ抗体およびＳＵＭＯ−１抗体に反応性を示すが、後者はＨｉｓ抗体のみに反応性がある。４０ｋｄａタンパク質は１Ｌの培養から約０．２ｍｇ得ることができる。
これらの結果は、前者において、Ｔ７−ｄｏｍａｉｎ−ＳＵＭＯ−１とＨｉｓ−ｄｏｍａｉｎ−Ｃ２のコンジュゲイトが大腸菌体内で大量に生産されたことを示している。
（実施例４）ＳＵＭＯとアクセプターを構成単位とするポリマーの生産
（１）ＧＳＴ−Ｃ２−ＳＵＭＯ−１を発現するｐＴ−ＧＳＴ−Ｃ２−ＳＵＭＯ−１プラスミドベクターの作製
Ｃ２−ＳＵＭＯ融合体、及びＧＳＴ（グルタチオン結合蛋白質：２７ｋＤａ）、をコードするＤＮＡ断片をそれぞれＰＣＲを用いて合成し、発現ベクターｐＧＥＸ（アマシャムファルマシア）に連結した。得られたプラスミドをｐＧＥＸ−Ｃ２−ＳＵＭＯ−１と称する。このｐＧＥＸ−Ｃ２−ＳＵＭＯ−１を鋳型ＤＮＡとして、以下のプライマーを用いてＰＣＲで、ＧＳＴ−Ｃ２−ＳＵＭＯ−１の断片を増幅した。

増幅断片をＮｏｔＩで切断後、同じくＮｏｔＩで処理したｐＴ−Ｔｒｘ（Ｃｈａｎｇ，Ａ．Ｃ．Ｙ．＆ Ｃｏｈｅｎ，Ｓ．Ｎ．：Ｊ．Ｂａｃｔｅｒｉｏｌ．，１３４，１１４１−１１５６，１９７８；Ｙａｓｕｋａｗａ，Ｔ．ｅｔ ａｌ．：Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７０，２５３２８−２５３３１，１９９５）とライゲーションする。得られたプラスミドｐＴ−ＧＳＴ−Ｃ２−ＳＵＭＯ−１は、Ｔ７プロモーターをＩＰＴＧで誘導することで、ＧＳＴとＣ２とＳＵＭＯ−１の融合タンパク質を発現するようにデザインされている。
（２）大腸菌での発現とタンパク質の部分精製
上述のｐＧＥＸ−ＡＵ／Ｕｂｃ９およびｐＴ−ＧＳＴ−Ｃ２−ＳＵＭＯを同時に大腸菌ＢＬ２１ＤＥ３株に導入する。２つのプラスミドを同時に取り込んだ菌体はアンピシリンとクロランフェニコールの両方を含む培地で選別できる。これとは独立に、ｐＴ−ＧＳＴ−Ｃ２−ＳＵＭＯのみを導入したものをクロランフェニコール培地で選別する。
それぞれの培地で得られたコロニーを別個のＬＢ液体培地１Ｌで３７℃で１６時間震盪培養する。０．０２ｍＭ ＩＰＴＧを加え、２５℃でさらに６時間または１２時間震盪培養する。
菌体を遠心分離器で沈澱させ、その後、１０ｍｌのＰＢＳバッファーにけんだく後、超音波処理により菌体を破砕する。遠心分離の後、上澄み液を取り出し、グルタチオンセファロースビーズ１ｍｌと１時間４℃でインキュベーションする。ビーズをＰＢＳで洗浄後、２０ｍＭグルタチオンを含むＰＢＳでビーズに結合したタンパク質を溶出する。タンパク質は１０％ＳＤＳ−ＰＡＧＥおよびウエスタン法で解析する。
図１３のレーン１にはｐＧＥＸ−ＡＵ／Ｕｂｃ９およびｐＴ−ＧＳＴ−Ｃ２−ＳＵＭＯを同時に大腸菌ＢＬ２１ＤＥ３株に導入し１２時間発現誘導したもの、レーン２には６時間発現誘導したものを示してある。コントロールとして、レーン３にはｐＴ−ＧＳＴ−Ｃ２−ＳＵＭＯのみを１２時間誘導発現したものを示してある。レーン１および２では約６０ｋｄａの階段状のバンドがクマシ−ブル−染色で明瞭に観察されるが、レーン３ではこれらが認められない。前者のバンドはＳＵＭＯ−１抗体に反応性を示すことから、ＧＳＴ−Ｃ２−ＳＵＭＯがポリマー化したバンドであると考えられる。こうしたタンパク質は１Ｌの培養から約０．１ｍｇ得ることができる。以上の結果は、ＧＳＴ−Ｃ２−ＳＵＭＯポリマーが大腸菌体内で大量に生産されたことを示している。
（実施例５）各種アクセプター配列を用いた解析
アクセプター配列Ｃ２に加えて、Ｔｈｙｍｉｎｅ ＤＮＡ Ｇｌｙｃｏｓｙｌａｓｅ（ＴＤＧ），ＴＯＮＡＳ１，ＲａｎＢＰ２−ＩＲのアクセプター配列としての性質を解析した。即ち、ＴＤＧ（Ｔｈｙｍｉｎｅ ＤＮＡ Ｇｌｙｃｏｓｙｌａｓｅ）（アミノ酸配列は配列番号２８に示す）、ＲａｎＢＰ２−ＩＲ（ｉｎｔｅｒｎａｌ ｒｅｐｅａｔ ｄｏｍａｉｎ ｉｎ Ｒａｎ ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ ２）（アミノ酸配列は配列番号２９に示す）、ＴＯＮＡＳ（Ｔｏｎａｌｌｉ ｒｅｌａｔｅｄ ＳＰ−ｒｉｎｇ ｐｒｏｔｅｉｎ）（アミノ酸配列は配列番号３０に示す）をＧＳＴのＣ末端に結合した形の融合タンパク質（ＧＳＴ−ＴＤＧ，ＧＳＴ−ＢＰ２−ＩＲ，ＧＳＴ−ＴＯＮＡＳ）をＳＵＭＯ酵素Ｅ１およびＥ２，ＡＴＰ，ＳＵＭＯ−１と３０℃で３０分反応させた。コントロールとして酵素を加えずに反応させた。反応後ＧＳＴビーズを加えて反応物を精製し、電気泳動に供し、染色し、タンパク質を検出した。結果を図１４に示す。具体的には以下の通り行った。
（１）ＧＳＴ−ＴＤＧ
ＧＳＴ−ＴＤＧ（７０ｋＤａ）１μｇを１μｇ ＳＵＭＯ−１、１μｇ Ｅ１、１μｇ Ｅ２、５ｍＭ ＡＴＰ存在下、１００μｌの２０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ７．５，２０ｍＭ ＮａＣｌ，０．１ｍＭ ＤＴＴ，５ｍＭ ＭｇＣｌ_２溶液中で３０℃にて３０分間反応させた。反応後、反応溶液中に３０μｌのグルタチオンビーズを加え、室温にて３０分間反応させた。ビーズに結合した蛋白質をＳＤＳポリアクリルアミド変性ゲル電気泳動で解析したところ、９０ｋＤａの位置にバンドが確認された（図１４のＡ）。このバンドと反応前のＧＳＴ−ＴＤＧの分子量の差は約２０ｋＤａで一分子のＳＵＭＯ−１にほぼ一致するものであった。このことから、ＴＤＧはＣ２と同様に１分子のＳＵＭＯ−１を結合するアクセプターと考えられる。
（２）ＧＳＴ−ＲａｎＢＰ２−ＩＲ
ＧＳＴ−ＲａｎＢＰ２−ＩＲ（７０ｋＤａ）１μｇを１μｇ ＳＵＭＯ−１、１μｇ Ｅ１、１μｇ Ｅ２、５ｍＭ ＡＴＰ存在下、１００μｌの２０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ７．５，２０ｍＭ ＮａＣｌ，０．１ｍＭ ＤＴＴ，５ｍＭ ＭｇＣｌ_２溶液中で３０℃にて３０分間反応させた。反応後、反応溶液中に３０μｌのグルタチオンビーズを加え、室温にて３０分間反応させた。ビーズに結合した蛋白質をＳＤＳポリアクリルアミド変性ゲル電気泳動で解析したところ、階段状にバンドが確認された（図１４のＢ）。このことから、ＲａｎＢＰ２−ＩＲは複数分子のＳＵＭＯ−１を結合するアクセプターと考えられる。このアクセプターは枝分かれタイプあるいは積層タイプのポリマーを構築する可能性が高い。
（３）ＧＳＴ−ＴＯＮＡＳ１ｄｅｌｔａ
ＧＳＴ−ＴＯＮＡＳ１ｄｅｌｔａ（６０ｋＤａ）１μｇを１μｇ ＳＵＭＯ−１、１μｇ Ｅ１、１μｇ Ｅ２、５ｍＭ ＡＴＰ存在下、１００μｌの２０ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ７．５，２０ｍＭ ＮａＣｌ，０．１ｍＭ ＤＴＴ，５ｍＭ ＭｇＣｌ_２溶液中で３０℃にて３０分間反応させた。反応後、反応溶液中に３０μｌのグルタチオンビーズを加え、室温にて３０分間反応させた。ビーズに結合した蛋白質をＳＤＳポリアクリルアミド変性ゲル電気泳動で解析したところ、階段状にバンドが確認された（図１４のＣ）。このことから、ＴＯＮＡＳは複数分子のＳＵＭＯ−１を結合するアクセプターと考えられる。このアクセプターは枝分かれタイプあるいは積層タイプのポリマーを構築する可能性が高い。(Example 1) Production of protein polymer having SUMO-target protein fusion as a structural unit (1) Preparation of SUMO-target protein fusion GST (glutathione binding protein: 27 kDa) and DNA fragment encoding SUMO were each PCR. Was ligated to an expression vector, pGEX (Amersham Pharmacia) (FIG. 2). E. coli transformed with recombinant plasmids. An extract was prepared from E. coli, placed on a glutathione-binding column and equilibrated with phosphate buffered saline. After washing the column with the same buffer, the GST-SUMO fusion was eluted with the same buffer containing 20 mM reduced glutathione and gel filtered through a Sephadex G-75 column.
(2) Polymerization of SUMO-target protein fusion The GST-SUMO fusion obtained above was about 50 kDa. 0.2 μg of this GST-SUMO fusion was added in 20 μl of 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.1 mM DTT, 5 mM MgCl ₂ solution in the presence of 0.02 μg E1, 0.02 μg E2, 5 mM ATP. The reaction was carried out at 37 ° C. for 10 minutes. After separating the reaction product by SDS polyacrylamide denaturing gel electrophoresis, the protein was transferred to a nitrocellulose membrane, and GST-SUMO was detected with a GST antibody. As a result, a plurality of stepped bands were detected toward the polymer region. (Figure 3). This band existed at intervals of about 50 kDa, and this difference in molecular weight almost coincided with one molecule of GST-SUMO. From this, the step-like band shows that the C-terminus of the GST-SUMO molecule (n = 1) is isopeptide-bonded to a lysine residue in another GST-SUMO molecule (n = 2), and this reaction is 2 The reaction product was considered to be repeated (n = 3) and 3 times (n = 4). That is, it was shown that a novel protein polymer having a GST-SUMO repeating structure was synthesized by this reaction. Under this reaction condition, it was shown that a polymer with n = 4 can be synthesized.
(Example 2) Production of protein polymer having SUMO-acceptor-target protein fusion as a structural unit (1) Preparation of target protein (GST) -acceptor (C2) -SUMO fusion RanGAP1 (RanGTase Activating) called C2 as an acceptor The C-terminal region of Protein 1) was selected. RanGAP1 is a protein having an activity to promote hydrolysis of the low molecular weight GTP-binding protein Ran, and is involved in mass transport between the nucleus and the cytoplasm. In order to obtain a C2-SUMO fusion, DNA fragments encoding C2 and SUMO were respectively synthesized using PCR and ligated to an expression vector, pET28 (Novagen) (FIG. 4). Extracts were prepared from E. coli transformed with the recombinant plasmid, placed on a Ni (nickel) column and equilibrated with phosphate buffered saline. After washing the column with the same buffer, the C2-SUMO fusion was eluted with the same buffer containing 250 mM imidazole and gel filtered through a Sephadex G-75 column.
DNA fragments encoding the C2-SUMO fusion obtained above and GST (glutathione binding protein: 27 kDa) were respectively synthesized using PCR and ligated to an expression vector, pGEX (Amersham Pharmacia) (FIG. 5). . E. coli transformed with recombinant plasmids. An extract was prepared from E. coli, placed on a glutathione-binding column and equilibrated with phosphate buffered saline. After washing the column with the same buffer, the GST-C2-SUMO fusion was eluted with the same buffer containing 20 mM reduced glutathione and gel filtered through a Sephadex G-75 column.
(2) Polymerization of target protein (GST) -acceptor (C2) -SUMO fusion The GST-C2-SUMO fusion obtained above was about 60 kDa. 0.2 μg of GST-C2-SUMO fusion in 20 μl of 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.1 mM DTT, 5 mM MgCl ₂ solution in the presence of 0.1 μg E1, 0.1 μg E2, 5 mM ATP For 10 minutes at 37 ° C. When the reaction product was analyzed by SDS polyacrylamide denaturing gel electrophoresis, a plurality of stepped bands were detected toward the polymer region (FIG. 6). This band existed at intervals of about 60 kDa, and the difference in molecular weight was almost identical to one molecule of GST-C2-SUMO. From this, the stepped band is isopeptide-bonded to the acceptor lysine residue in the C2 sequence of another GST-C2-SUMO molecule at the C-terminus of the GST-C2-SUMO molecule (n = 1) (n = 2) Further, this reaction was considered to be a reaction product that was repeated twice (n = 3) and three times (n = 4). Under this reaction condition, it was shown that a polymer with n = 11 can be synthesized.
Example 3 Production of Conjugate of SUMO and Acceptor (1) Construction of Expression Vector that Expresses E1 and E2 Enzymes To clone mouse Aos1 and Uba2 cDNA, first, BLAST (www.ncbi.nlm. nih.gov) and mouse Aos1 and Uba2 cDNAs were found (Genebank accession numbers: NM-0197748 (mouse Aos1) and NM-016682 (mouse Uba2)). The coding regions of Aos1 and Uba2 were amplified by PCR using a BALB / MK mouse epithelial keratinocyte cDNA library as a template and the following oligonucleotides as primers.

The amplified PCR fragment was cloned into the EcoRI site of the pGEX vector (Amersham Pharmacia Biotech), and the resulting plasmid constructs were named pGEX-Aos1 and pGEX-Uba2, respectively. The base sequence of Mouse Aos1 cDNA is shown in SEQ ID NO: 5, and the base sequence of Mouse Uba2 is shown in SEQ ID NO: 6.
To create the Aos1-Uba2 chimeric construct, Aos1 and Uba2 were amplified by PCR using the following oligonucleotides.

The amplified fragment was cut with BamHI + EcoRI and EcoRI, respectively, and inserted into a digested pGEX vector with BamHI + EcoRI. The resulting pressmid pGEX-AU encodes a GST-Aos1-Uba2 fusion protein. The nucleotide sequence of mouse AU cDNA (GAATTC is the binding region between Aos1 and Uba2) is shown in SEQ ID NO: 7.
Xenopus (Xenopus) Ubc9 (base sequence shown in SEQ ID NO: 8) was cloned and inserted into pT7-7 vector (Saitoh et al. Proc. Natl. Acad. Sci. USA., Vol94, 3736-3741, 1997). . By the way, the amino acid sequence of Xenopus Ubc9 is 100% homologous to the amino acid sequence of human and mouse Ubc9. PCR was performed using the following primers.

The amplified DNA fragment was cleaved with NHeI and ligated with pGEX-AU treated with XhoI. When the prepared plasmid was analyzed, as shown in FIG. 8, each sequence had a structure arranged in tandem in a forward direction from the N-terminal to the C-terminal. This plasmid vector was named PGEX-AU / Ubc9. The GST-AU protein is under the control of the tac promoter and Ubc9 is under the control of the T7 promoter and is induced by IPTG.
(2) Preparation of expression vector pT-SUMO-1 / C2 plasmid vector expressing T7-domain-SUMO-1 Using human SUMO-1 cDNA (base sequence is described in SEQ ID NO: 9) and the following primers: A DNA fragment encoding T7-SUMO-1 is amplified by PCR. This protein is a fusion protein in which the T7-tag sequence (met-ala-ser-met-thr-gly-gly-gln-gln) (SEQ ID NO: 20) is at the N-terminus of SUMO-1.

The amplified fragment was cleaved with NdeI + HindIII and then treated with NdeI + HindIII (Chang, ACY & Cohen, SN: J. Bacteriol., 134, 1141-1156, 1978; Yasukawa, T .; et al .: J. Biol.Chem., 270, 25328-25331, 1995). The resulting plasmid is called pT-T7-SUMO-1.
The C-terminal region of RanGAP1 (RanGTPase Activating Protein 1) called C2 was selected as an acceptor. RanGAP1 is a protein having an activity to promote hydrolysis of the low molecular weight GTP-binding protein Ran, and is involved in mass transport between the nucleus and the cytoplasm. In order to obtain a C2-SUMO fusion, DNA fragments encoding C2 and SUMO were respectively synthesized using PCR and ligated to the expression vector pET28 (Novagen). This plasmid vector is called pET28-His-C2. This plasmid encodes a His-C2 fusion protein. The following sequences are fused upstream of the N-terminus of the C2 sequence.

PCR was performed using this plasmid as a template and the following primers.

The amplified fragment was cleaved with BglII and then ligated with the above-described pT-T7-SUMO-1 treated with BglII. The resulting plasmid pT-SUMO-1 / C2 is designed to simultaneously express two fusion proteins, His-C2 and T7-SUMO-1, by inducing the T7 promoter with IPTG.
(3) Expression in E. coli and partial purification of protein The above-described pGEX-AU / Ubc9 and pT-SUMO / C2 are simultaneously introduced into E. coli BL21DE3 strain. Cells that have taken up two plasmids simultaneously can be selected on media containing both ampicillin and chloramphenicol. Independently of this, those into which only pT-SUMO / C2 has been introduced are selected on a chloramphenicol medium.
The colonies obtained with each medium are cultured with shaking in 1 L of separate LB liquid medium at 37 ° C. for 16 hours. Add 0.02 mM IPTG and incubate for an additional 16 hours at 25 ° C.
The cells are precipitated with a centrifuge, then suspended in 10 ml of PBS buffer, and then disrupted by sonication. After centrifugation, the supernatant is removed and incubated with 1 ml of nickel agarose beads for 1 hour at 4 ° C. After washing the beads with PBS, the protein bound to the beads is eluted with PBS containing 250 mM imidazole. Protein is analyzed by 12% SDS-PAGE and Western methods.
Lane 1 in FIG. 10 shows pGEX-AU / Ubc9 and pT-SUMO / C2 simultaneously introduced into E. coli BL21DE3 strain, and Lane 2 shows that pT-SUMO / C2 has been introduced. A protein of 40 kda is clearly observed in the former and a protein of 25 kda is clearly observed in the latter by Coomassie-staining. The former band is reactive to the His antibody and the SUMO-1 antibody, while the latter is reactive only to the His antibody. About 0.2 mg of 40 kda protein can be obtained from 1 L of culture.
These results indicate that in the former case, a conjugate of T7-domain-SUMO-1 and His-domain-C2 was produced in large quantities in E. coli.
(Example 4) Production of polymer having SUMO and acceptor as building blocks (1) Preparation of pT-GST-C2-SUMO-1 plasmid vector expressing GST-C2-SUMO-1 C2-SUMO fusion and GST DNA fragments encoding (glutathione binding protein: 27 kDa) were synthesized using PCR, and ligated to an expression vector pGEX (Amersham Pharmacia). The resulting plasmid is referred to as pGEX-C2-SUMO-1. Using this pGEX-C2-SUMO-1 as a template DNA, a GST-C2-SUMO-1 fragment was amplified by PCR using the following primers.

After cutting the amplified fragment with NotI, pT-Trx (Chang, A.C.Y. & Cohen, SN: J. Bacteriol., 134, 1141-1156, 1978) treated with NotI was also used. et al .: J. Biol.Chem., 270, 25328-25331, 1995). The obtained plasmid pT-GST-C2-SUMO-1 is designed to express a fusion protein of GST, C2, and SUMO-1 by inducing the T7 promoter with IPTG.
(2) Expression in E. coli and partial purification of protein The above-described pGEX-AU / Ubc9 and pT-GST-C2-SUMO are simultaneously introduced into E. coli BL21DE3 strain. Cells that have taken up two plasmids simultaneously can be selected on media containing both ampicillin and chloramphenicol. Independently of this, those into which only pT-GST-C2-SUMO has been introduced are selected on a chloramphenicol medium.
The colonies obtained with each medium are cultured with shaking in 1 L of separate LB liquid medium at 37 ° C. for 16 hours. Add 0.02 mM IPTG and incubate for an additional 6 or 12 hours at 25 ° C.
The bacterial cells are precipitated with a centrifuge, and then the cells are crushed by sonication after being poured into 10 ml of PBS buffer. After centrifugation, the supernatant is removed and incubated with 1 ml of glutathione sepharose beads for 1 hour at 4 ° C. After washing the beads with PBS, the protein bound to the beads is eluted with PBS containing 20 mM glutathione. Protein is analyzed by 10% SDS-PAGE and Western methods.
Lane 1 in FIG. 13 shows pGEX-AU / Ubc9 and pT-GST-C2-SUMO introduced into E. coli BL21DE3 strain at the same time and induced expression for 12 hours, and lane 2 shows that induced for 6 hours. As a control, in Lane 3, only pT-GST-C2-SUMO is induced and expressed for 12 hours. In lanes 1 and 2, a stepped band of about 60 kda is clearly observed by Coomassie-stain, but in lane 3, these are not observed. Since the former band shows reactivity with the SUMO-1 antibody, it is considered that GST-C2-SUMO is a polymerized band. About 0.1 mg of such protein can be obtained from a 1 L culture. The above results indicate that GST-C2-SUMO polymer was produced in large quantities in E. coli.
(Example 5) Analysis using various acceptor sequences In addition to acceptor sequence C2, the properties of Thymine DNA Glycosylas (TDG), TONAS1, and RanBP2-IR as acceptor sequences were analyzed. That is, TDG (Thymine DNA Glycosylate) (amino acid sequence is shown in SEQ ID NO: 28), RanBP2-IR (internal repeat domain in Ran binding protein 2) (amino acid sequence is shown in SEQ ID NO: 29), TONAS (Tonally related SP) protein) (amino acid sequence shown in SEQ ID NO: 30) bound to the C-terminus of GST (GST-TDG, GST-BP2-IR, GST-TONAS) was added to SUMO enzymes E1 and E2, ATP, SUMO- The reaction was performed at 1 and 30 ° C. for 30 minutes. The reaction was carried out without adding enzyme as a control. After the reaction, GST beads were added to purify the reaction product, subjected to electrophoresis, and stained to detect the protein. The results are shown in FIG. Specifically, it was performed as follows.
(1) GST-TDG
1 μg of GST-TDG (70 kDa) in the presence of 1 μg SUMO-1, 1 μg E1, 1 μg E2, 5 mM ATP, 100 μl in 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.1 mM DTT, 5 mM MgCl ₂ solution The reaction was carried out at 0 ° C. for 30 minutes. After the reaction, 30 μl of glutathione beads were added to the reaction solution and reacted at room temperature for 30 minutes. When the protein bound to the beads was analyzed by SDS polyacrylamide denaturing gel electrophoresis, a band was confirmed at a position of 90 kDa (A in FIG. 14). The difference in molecular weight between this band and GST-TDG before the reaction was about 20 kDa, which almost coincided with one molecule of SUMO-1. From this, TDG is considered to be an acceptor that binds one molecule of SUMO-1 like C2.
(2) GST-RanBP2-IR
1 μg of GST-RanBP2-IR (70 kDa) in the presence of 1 μg SUMO-1, 1 μg E1, 1 μg E2, 5 mM ATP in 100 μl of 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.1 mM DTT, 5 mM MgCl ₂ solution For 30 minutes at 30 ° C. After the reaction, 30 μl of glutathione beads were added to the reaction solution and reacted at room temperature for 30 minutes. When the protein bound to the beads was analyzed by SDS polyacrylamide denaturing gel electrophoresis, a band was confirmed in a stepped shape (B in FIG. 14). Therefore, RanBP2-IR is considered as an acceptor that binds multiple molecules of SUMO-1. This acceptor is likely to build a branched or laminated polymer.
(3) GST-TONAS1delta
1 μg of GST-TONAS1delta (60 kDa) in the presence of 1 μg SUMO-1, 1 μg E1, 1 μg E2, 5 mM ATP, in 100 μl of 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 0.1 mM DTT, 5 mM MgCl ₂ solution 30 The reaction was carried out at 0 ° C for 30 minutes. After the reaction, 30 μl of glutathione beads were added to the reaction solution and reacted at room temperature for 30 minutes. When the protein bound to the beads was analyzed by SDS polyacrylamide denaturing gel electrophoresis, a band was confirmed in a step shape (C in FIG. 14). From this, TONAS is considered to be an acceptor that binds multiple molecules of SUMO-1. This acceptor is likely to build a branched or laminated polymer.

Industrial applicability

According to the present invention, a protein polymer capable of introducing various types of proteins and exhibiting various physiological activities and enzyme activities is provided. When the protein polymer of the present invention is used in a bioreactor, biosensor, etc., it can efficiently produce useful proteins and recognize trace chemical substances, and is useful in the fields of food production, pharmaceutical production, and the like. In addition, the protein polymer of the present invention can be used for basic research in molecular biology such as clarification and control of the functions of molecules involved in various biological mechanisms such as immunity, information transmission, neurotransmission, and cancer cell growth, or regenerative medicine / It can be applied to cancer treatment. Furthermore, according to the present invention, it is possible to provide a system that can produce protein conjugates and protein polymers in large quantities in host cells such as bacteria.
The contents described in Japanese Patent Application Nos. 2002-288766 and 2003-64713, which are Japanese patent applications serving as the basis of the priority claimed by the present application, are all cited in this specification as part of the disclosure of this specification. It shall be taken in by.

[Sequence Listing]

Claims (31)

A protein polymer containing, in the main chain, a ubiquitin-like protein (SUMO) to which a target protein is bound directly or via an acceptor. The protein polymer according to claim 1, wherein the target protein is contained in the polymer in one or more kinds. The protein polymer according to claim 1 or 2, wherein the main chain is linear, branched, cyclic, or a self-assembled tube shape. The protein polymer according to any one of claims 1 to 3, wherein one or more acceptors are contained in the polymer. The acceptors are RanGAP1-C2 (Ran GTPase activating protein-C2), RanBP2-IR (Ran binding protein 2-ical repetitive domain), TDG (Thymin DNA Glycosylase), TDG (Thin DNA Glycosylase) The protein polymer according to any one of claims 1 to 3, wherein The following formula (I):

(In the formula, SUMO is a ubiquitin-like protein, X is a target protein, a bold line is an isopeptide bond, and a is an average degree of polymerization of 2 to 100.)
A protein polymer represented by Formula (II) below:

(In the formula, SUMO is a ubiquitin-like protein, X, Y, and Z are target proteins, bold lines are isopeptide bonds, b, c, and d are average degrees of polymerization from 0 to 100, provided that b, c, and except when d is simultaneously 0)
A protein polymer represented by Formula (III) below:

(In the formula, SUMO is a ubiquitin-like protein, A is an acceptor, X is a target protein, a bold line is an isopeptide bond, and e is an average degree of polymerization of 2 to 100.)
A protein polymer represented by Formula (IV) below:

(In the formula, SUMO is a ubiquitin-like protein, X, Y, and Z are target proteins, A is an acceptor, thick lines are isopeptide bonds, f, g, and h are average degrees of polymerization from 0 to 100, where f is , G, and h are simultaneously 0).
A protein polymer represented by The following formula: X-SUMO-AY
(In the formula, SUMO represents a ubiquitin-like protein, A represents an acceptor, and X and Y represent target proteins.)
A protein conjugate represented by The following steps:
(A) producing a fusion or conjugate of a ubiquitin-like protein (SUMO) and a target protein;
(B) polymerizing the fusion or conjugate by isopeptide bonds;
A method for producing a protein polymer comprising: The following steps:
(A) producing a fusion or conjugate of ubiquitin-like protein (SUMO), acceptor and target protein;
(B) polymerizing the fusion or conjugate by isopeptide bonds;
A method for producing a protein polymer comprising: The method according to claim 11 or 12, wherein the polymerization reaction is carried out in the presence of SUMO activating enzyme E1, SUMO binding enzyme E2, ATP, and metal ions. The method according to any one of claims 11 to 13, wherein a reaction accelerator and / or a base peptide are further added to the reaction system. A DNA encoding an enzyme (E1) that activates a ubiquitin-like protein and a DNA encoding a binding enzyme (E2) of a ubiquitin-like protein and an acceptor, and comprising an activating enzyme (E1) and a binding enzyme (E2) Recombinant expression vectors that can be expressed simultaneously in The recombinant expression vector according to claim 15, wherein the enzyme (E1) that activates a ubiquitin-like protein is AU that is a fusion protein of Aos1 and Uba, and the binding enzyme (E2) is Ubc9. A recombinant expression vector comprising a DNA encoding a ubiquitin-like protein and a DNA encoding an acceptor, wherein the ubiquitin-like protein and the acceptor can be separately expressed in the host simultaneously. It further comprises DNA encoding one or more target proteins adjacent to DNA encoding ubiquitin-like protein and / or DNA encoding acceptor, and ubiquitin-like protein and acceptor (both or one of them includes the target protein) The recombinant expression vector according to claim 17, which can be simultaneously expressed in a host individually. DNA encoding an enzyme (E1) that activates a ubiquitin-like protein; DNA encoding a binding enzyme (E2) of a ubiquitin-like protein and an acceptor; DNA encoding a ubiquitin-like protein; and / or DNA encoding an acceptor , A recombinant expression vector capable of separately expressing an activating enzyme (E1), a linking enzyme (E2), a ubiquitin-like protein and / or an acceptor individually in a host. An activating enzyme (E1), a binding enzyme (E2), and a ubiquitin-like protein, comprising using the recombinant vector according to claim 1 or claim 2 in combination with the recombinant vector according to claim 17 or 18. And acceptor are simultaneously expressed in a host. An activating enzyme (E1) and a binding enzyme, comprising using the vector according to claim 19 and, if necessary, combining a DNA encoding a ubiquitin-like protein or a recombinant vector including an acceptor-encoding DNA. A method of simultaneously expressing (E2), a ubiquitin-like protein and an acceptor in a host. Expresses an enzyme that activates a ubiquitin-like protein (E1), a ubiquitin-like protein and acceptor binding enzyme (E2), a ubiquitin-like protein to which the target protein may be bound, and an acceptor to which the target protein may be bound A transformant that can. The transformant of Claim 22 which has the recombinant vector of Claim 15 or Claim 16, and the recombinant vector of Claim 17 or 18. A method for producing a conjugate of a ubiquitin-like protein and an acceptor, comprising culturing the transformant according to claim 22 or 23 and producing a conjugate of the ubiquitin-like protein and the acceptor in the transformant. A recombinant expression vector comprising DNA encoding a fusion protein of a ubiquitin-like protein and an acceptor, and capable of expressing the fusion protein of the ubiquitin-like protein and the acceptor in a host. A DNA encoding a ubiquitin-like protein and a DNA encoding one or more target proteins adjacent to the DNA encoding the acceptor, and a fusion protein of the ubiquitin-like protein, acceptor and target protein can be expressed in the host; The recombinant expression vector according to claim 25. By using a combination of the recombinant vector according to claim 15 or claim 16 and the recombinant vector according to claim 25 or 26, an activating enzyme (E1), a binding enzyme (E2) and a ubiquitin-like protein A method for producing a polymer of a fusion protein of a ubiquitin-like protein and an acceptor, comprising simultaneously expressing a fusion protein with an acceptor in a host. DNA encoding the enzyme (E1) that activates the ubiquitin-like protein, DNA encoding the binding enzyme (E2) of the ubiquitin-like protein and acceptor, and a fusion protein of the ubiquitin-like protein, acceptor, and target protein, if desired. A recombinant expression vector comprising DNA and capable of simultaneously expressing the activating enzyme (E1), the binding enzyme (E2) and the fusion protein in a host. A transformant capable of expressing an enzyme that activates a ubiquitin-like protein (E1), a ubiquitin-like protein-acceptor binding enzyme (E2), and a fusion protein of a ubiquitin-like protein, an acceptor, and, if desired, a target protein. The transformant of Claim 29 which has the recombinant vector of Claim 15 or Claim 16, and the recombinant vector of Claim 25 or 26. A method for producing a protein polymer, comprising culturing the transformant according to claim 29 or 30, and producing a polymer of a fusion protein of a ubiquitin-like protein, an acceptor, and optionally a target protein in the transformant.

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