WO2007069604A1

WO2007069604A1 - Method for production of modified protein

Info

Publication number: WO2007069604A1
Application number: PCT/JP2006/324761
Authority: WO
Inventors: Fuyuhiko Inagaki; Yoshihiro Kobashigawa; Masato Naito
Original assignee: National University Corporation Hokkaido University
Priority date: 2005-12-14
Filing date: 2006-12-12
Publication date: 2007-06-21
Also published as: JPWO2007069604A1

Abstract

Disclosed is a method for production of a modified protein comprising the step of modifying a substrate protein with an enzyme protein. In the method, the substrate protein and/or the enzyme protein is a chimeric protein having a protein-binding domain added thereto, wherein the protein-binding domain is capable of enhancing the affinity between the substrate protein and the enzyme protein. The method enables to achieve a protein-modification reaction with good efficiency by utilizing a protein-protein interaction. In the method, the affinity between a substrate protein and an enzyme can be increased by producing a protein-protein interaction between the substrate protein and the enzyme protein. In this manner, a substrate protein can be modified with a smaller amount of an enzyme with good efficiency.

Description

Specification

Method for producing modified protein

Technical field

[0001] The present invention relates to a method for modifying a protein using a substrate protein and an enzyme protein or a method for producing a modified protein.

The present invention relates to the above-described modification method or method for producing a modified protein, wherein Z or a chimeric protein to which a protein binding domain capable of increasing the affinity between the substrate protein and the enzyme protein is added as the enzyme protein.

[0002] The present invention also relates to a method of phosphorylating a substrate protein using a kinase (phosphorylating enzyme). More specifically, either the substrate protein or the kinase contains an SH3 domain, and the other is proline. It is characterized in that the SH3 domain binding sequence such as a rich sequence, that is, the protein binding domain contains an amino acid sequence showing affinity, and both molecules associate with each other by the interaction between the SH3 domain and the SH3 domain binding sequence, and the substrate protein. The present invention relates to a method for phosphorylating a substrate protein and a method for producing a phosphorylated protein, which effectively carry out phosphorylation reaction. The present invention also relates to a method for myristoylating a substrate protein using myristoyltransferase. Furthermore, the present invention relates to a method for dephosphorylating a substrate protein using a protein dephosphorylating enzyme.

Background art

[0003] Cell proliferation, differentiation, morphogenesis, and the like are basic processes for the establishment of all multicellular organisms. These processes are accomplished by the recognition of extracellular force signals by receptors on the cell surface, transmitting them to intracellular signaling factors, and processing them appropriately. Signal transmission is triggered by reactions directly under the biological membrane via receptors on the membrane, and the process is controlled by protein-protein interactions. In this process, the control of protein-protein interactions through phosphate and dephosphate plays an important role.

[0004] For example, in most cases, cell proliferation and cell responses to drugs are related to intracellular signal transduction mechanisms, and G protein and other phosphates are the actual state of cell signal transduction. It has been. In terms of cell proliferation, normal cells Although regulation is strictly performed, protein phosphate is increased in cancer cells, and the regulation becomes ineffective. For this reason, controlling intracellular phosphorylation can be expected to lead to maintenance of cell homeostasis and treatment of diseases. However, artificial manipulation and efficient use of regulatory molecules for the efficient phosphorylation of substrate proteins have hardly been realized, and the main focus is on tangle kinase typing and reaction mechanisms. .

[0005] Non-phosphate protein is prepared in large quantities using Escherichia coli, yeast, and insect cell systems, and has undergone basic research, drug discovery research, and applied research. It was.

[0006] With regard to a protein in a phosphoric acid state, the difficulty of large-scale preparation has hindered research and utilization.

[0007] Conventionally, in the case of conducting research using a large amount of sample (from several mg), a model that is a chemically synthesized peptide of about 10 residues that also consists of force only around the phosphate moiety is used as a model. Although it has been used (Non-patent Document 1, etc.), the chemical synthesis of phosphate peptides is more expensive than the chemical synthesis of ordinary peptides, and many signal transduction proteins are composed of multiple functional domains. However, studies using peptides have not been sufficient to elucidate the control mechanism of signal transduction through them.

[0008] Although attempts have been made to obtain a large amount of phosphate protein by co-expressing both in a host (Non-patent Document 2, etc.), phosphorylation is less efficient and further phosphorylation is performed. It was necessary to separate the protein from the non-phosphorylated protein. Although attempts have been made to obtain a phosphate protein by reacting a kinase and a substrate protein in a test tube (Non-patent Document 3, etc.), the efficiency of phosphate is low. Requires a large amount of expensive kinase!

[0009] Further, the present invention has an affinity for a protein having an amino acid sequence represented by SH3 domain strength PxxP (P is proline, X is an arbitrary amino acid, hereinafter referred to as Pro Rich Region: PRR). Is well known (see Patent Documents 1, 2, etc.). In this specification, amino acids are indicated by one letter or three letters as appropriate. Exceptionally, some SH3 domains have been reported to show affinity for amino acid sequences other than PRR. For example, SH3 domains such as Grb2, Gads, Hbp, and STAM2 are PX [V or I] [D or N] RXXKP However, X is an amino acid sequence containing any amino acid residue (Non-patent Documents 4, 5, 6, 13, 14), and the SH3 domain of Fyn, Fyb, and Lck is Nef (Non-patent Documents 7, 8, 9) ) And RKXXZXXZ (where X is any amino acid residue and Z is K or R) (Non-Patent Document 10), Pexl3p SH3 domain is WXXXFXXLE (where X is any amino acid residue) (Non-patent literature 6, 11, 12), and the SH3 domain of Eps8 is an amino acid sequence containing PXX DY (where X represents any amino acid residue) (non-patent literature 15) Each shows affinity. In the present invention, an amino acid sequence in which these SH3 domains that are protein binding domains show affinity is referred to as an SH3 domain binding sequence.

Non-Patent Document 1: J Mol Biol., 1999, 289, 439-445

Non-Patent Document 2: Protein Expr Purif., 2005, 41st pp. 162-169

Non-Patent Document 3: Cell, 2000, 102, 625-633

Non-Patent Document 4: Berry et al., (Berry DM, et al.), J. Curr Biol., 2002, 12: 1336-1341

Non-Patent Document 5: Liu Q "et al., Mol. Cell., 2003, Vol. 11, 471-481 Non-Patent Document 6: Kami et al. (Kami, K., et al.), EMBO J., 2002, 21st, 4268- 4276 Non-patent document 7: Lee et al., Cell, 1996, 85-931-942 Non-patent document 8: Gurz Ziek (Grzesiek S, et al.), Nat. Struct. Biol., 1996, III, 340-345.

Non-patent document 9: Lee et al. (Lee CH, et al.), EMBO, 1995, pp. 14, 5006-5015 Non-patent document 10: Kang H, et al., EMBO J., 2000 19th, 2889-2899 Non-patent literature 11: Barnett P, et al., EMBO, 2000, 19th, 6382-6391

Non-Patent Document 12: Urquhart AJ, et al., J. Biol. Chem., 2000, 275, 4127-4136

Non-Patent Document 13: Kato M, et al., J. Biol. Chem., 2000, 275, 37481-37487

Non-Patent Document 14: Kaneko T, et al., J. Biol. Chem., 2003, 278, 4816 2-48168 Non-Patent Document 15: Mongiovi AM, et al., EMBO J., 1999, VIII, 5 300-5309

Patent Document 1: JP 2003-185655 A

Patent Document 2: Pamphlet of International Publication No. 03/006060

Disclosure of the invention

[0010] Conventional methods for obtaining a phosphate protein have been carried out using a large amount of kinase because the efficiency of the phosphorylation reaction is mainly low. In order to solve such a problem, the present invention includes co-expression of a substrate protein and a kinase having enhanced affinity with each other in a host cell or mixing in vitro, or in vitro mixing. The aim is to provide an efficient method for obtaining proteins.

[0011] The present inventors have used a protein-binding domain and an amino acid sequence in which the protein-binding domain has an affinity, for example, SH3 domain and SH3 domain-binding sequence, and either substrate protein or kinase. By forming a structure with the SH3 domain in the other and the SH3 domain binding sequence in the other, and increasing the affinity between the substrate protein and the kinase, modification reactions such as the phosphorylation reaction can be efficiently performed. We found out what we could do and completed the following inventions.

[0012] (1) A method for producing a modified protein comprising modifying a substrate protein with an enzyme protein, wherein the substrate protein and Z or the enzyme protein increases the affinity between the substrate protein and the enzyme protein. A method for producing the modified protein, wherein the protein is a chimeric protein to which a protein binding domain is added.

[0013] (2) In the above (1), the substrate protein or enzyme protein that binds to the chimeric protein to which the protein binding domain is added is a chimeric protein to which an amino acid sequence to which the protein binding domain has affinity is added. The manufacturing method as described.

[0014] (3) The protein-binding domain and Z or the amino acid sequence to which the protein-binding domain has an affinity to the substrate protein and Z or the enzyme protein via an amino acid sequence that can be cleaved enzymatically or chemically The production method according to (1) or (2), which is added.

[0015] (4) The method for producing a modified protein according to any one of (1) to (3), wherein the enzyme protein is a kinase. [0016] (5) The method for producing a modified protein according to any one of (1) to (4) above, wherein the protein binding domain is an H3 domain.

(6) The SH3 domain is a peptide of the following (a) or (b):

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1

(b) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having affinity for an SH3 domain binding sequence, wherein the SH3 The domain binding sequence is PXXPXZ or ZXXPXXP! /, Has a proline-rich sequence represented by PXXXRXXK (wherein X represents any amino acid and Z represents K or R), as described in (5) above Manufacturing method.

[7] (7) The SH3 domain is a peptide of the following (c) or (d),

(c) Peptide having the amino acid sequence of SEQ ID NO: 26

(d) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 26, and having affinity for an SH3 domain binding sequence, wherein the SH3 The production method according to (5), wherein the domain binding sequence has a sequence represented by PX [V or I] [D or N] RXXKP (wherein X represents an arbitrary amino acid).

[0019] (8) The production method according to any one of (6) and (7), wherein the substrate protein has an SH3 domain, and the kinase has an SH3 domain binding sequence.

[0020] (9) The production method of any one of (6) or (7), wherein the substrate protein has an SH3 domain binding sequence, and the kinase has an SH3 domain.

[0021] (10) The production method according to (3), further comprising the step of recovering the modified protein by cleaving an amino acid sequence that can be cleaved enzymatically or chemically after the reaction between the substrate protein and the enzyme protein .

[0022] (11) Transforming a host using a vector that expresses a substrate protein and Z or enzyme protein that is a chimeric protein and the substrate protein or enzyme protein to which the chimeric protein binds, and the substrate protein and the enzyme protein The production method according to any one of the above (1) to (3), which comprises co-expressing and in a host cell.

[0023] (12) In any one of (1) to (3), the enzyme protein is myristoyltransferase A method for producing the modified protein as described.

[0024] (13) The method for producing a modified protein according to any one of (1) to (3), wherein the enzyme protein is a protein dephosphorylating enzyme.

Brief Description of Drawings

[0025] FIG. 1 is a diagram showing the domain structure of Abl (SK) composed of an SH2 domain and a kinase domain.

FIG. 2 is a diagram showing the domain structure and amino acid sequence of Abl (SKPl) composed of SH2 domain, kinase domain, and Proline Rich Region.

FIG. 3 is a diagram showing the domain structure of CrkII.

FIG. 4 is a diagram showing the domain structure and amino acid sequence of Abl.

FIG. 5 shows the procedure for constructing pProEX-Crkll plasmid.

FIG. 6 is a diagram showing SDS-PAGE of purified Crkll. Lane 1: Molecular weight marker, Lane 2: CrkII before 6 X His tag cleavage, Lane 3: Crkll after 6 X His tag cleavage, Lane 4: Fractio n number 8, Lane 5: Fraction number 16, Lane 6: Fraction number 17, Lane 7: Fraction No. 18, Lane 8: Fraction No. 19

FIG. ⁷ is a diagram showing a plasmid construction procedure for pET-Duet-SK.

FIG. 8 is a diagram showing a plasmid construction procedure of pET-Duet-SK-Crkll.

FIG. 9 is a diagram showing a plasmid construction procedure of pET-Duet-SKP1.

FIG. 10 is a diagram showing a procedure for constructing a plasmid of pET-Duet-SKPl-Crkll.

FIG. 11 shows SDS-PAGE of Crkll co-expressed with SKP1. Lane 1: Molecular weight marker 1, Lane 2: CrkII, Lane 3: 0 «1 co-expressed with 31 ^ 1.

FIG. 12 shows immunostaining of CrkII co-expressed with CrkII and SK and Crkll co-expressed with SKPI after a phosphorylation reaction with a commercially available kinase. Lane 1: molecular weight marker, lane 2: CrkII phosphorylated with a commercially available Abl, lane 3: CrkII co-expressed with SK, lane 4: Crkll co-expressed with SKP1

FIG. 13 shows Crkll SDS-PAGE before and after phosphorylation with a commercially available kinase. Lane 1: Molecular weight marker, Lane 2: CrkII before phosphorylation, Lane 3: Crkll after phosphate-reaction FIG. 14 is a view showing immunostaining of Crkll after carrying out a phosphorylation reaction using purified SKP1. Lane 1: Molecular weight marker, Lane 2: Crkll phosphorylated in vitro using purified SKP1

FIG. 15 shows the domain structure of Crk-Vav (170-375).

FIG. 16 is a view showing a procedure for constructing a plasmid of pET22-Crk-Vav (170-375).

[Fig. 17] SD showing phosphorylation of Crk-Vav (170-375) and Vav (170-375) by SKP1.

It is a figure which shows S-PAGE. Lane 1: Molecular weight marker, Lane 2: Crk before phosphorylation

-Vav (170-375), Lane 3: Crk-Vav (170-375) after phosphorylation, Lane 4: Vav before phosphorylation (170-375), Lane 5: Vav after phosphorylation (170- 375)

FIG. 18 shows the domain structure and amino acid sequence of Vav (170-375).

FIG. 19 shows the domain structure and amino acid sequence of SK-SLP.

FIG. 20 is a diagram showing the domain structure of Grb2-CrkII.

FIG. 21 is a view showing a procedure for constructing a plasmid of pET-Duet-SK-SLP.

FIG. 22 is a diagram showing a plasmid construction procedure for pET22-Grb2-Crkll.

FIG. 23 shows SDS-PAGE of Grb2-CrkII phosphorylated by SK-SLP and SK. Left figure Lane 1: Molecular weight marker, Lane 2: Grb-Crkll before phosphorylation, Lane 3: S

Grb-CrkII phosphorylated with K-SLP, right side Lane 1: Molecular weight marker, Lane 2: Grb-CrkII before phosphorylation, Lane 3: Grb-Crkll phosphorylated with SK

[24] Plasmid pTYR and its partial structure.

FIG. 25 is a drawing showing an SDS-PAGE of Crk-Vav (170-375) phosphorylated by pTYR-Vav (l 70-375). Lane 1: molecular weight marker, lane 2: Crk-Vav (170-375) before phosphorylation, lane 3: Crk-Vav (170-375) expressed in pTYR-Vav (170-375).

[26] It is a diagram showing pTYR (-) and its partial structure. .

FIG. 27 is a diagram showing pSH3-NMT and its partial structure.

FIG. 28 is a diagram showing pGBl-Myr and its partial structure.

FIG. 29 is a diagram showing an SDS-PAGE of GBl-Myr that is myristoylated using SH3-NMT. Lane 1 shows a 10 μ 1 SH3— 1, Lane 2 shows a 1 μ 1 SH3—ΝΜΤ, and Lane 3 shows a 0 μ1 SH3—ΝΜΤ. FIG. 30 is a diagram showing ρΡΤΡ1Β-PxxP and its partial structure.

FIG. 31 is a diagram showing an SDS-PAGE of phosphorylated Crkll dephosphorylated using PTP-1B-PxxP. Lane 1 shows 10 µ 1 PTP-IB-ΡχχΡ, Lane 2 shows 1 µ 1 PTP-IB-ΡχχΡ, and Lane 3 shows 0 µ 1 PTP-1B-PxxP.

FIG. 32 shows immunostaining of phosphate Crkll dephosphorylated using PTP-IB-PxxP. Lane 1 shows dephosphorylation with 10 μ 1 PTP-IB-ΡχχΡ, Lane 2 with 1 μ1 PTP-IB-ΡχχΡ, and Lane 3 with 0 μ1 PTP-IB-PxxP.

BEST MODE FOR CARRYING OUT THE INVENTION

[0026] Hereinafter, the modification of the substrate protein of the present invention will be described using protein phosphorylation as an example, but the modification of the substrate protein in the present invention is not limited to this. Dephosphorylation, myristylation, palmitoylation It is effective for post-translational modification of proteins with enzyme proteins, including ubiquitination, ubiquitin-like protein modification, ADP ribosylation, PE formation, and glycosylation. Therefore, the protein binding domain added to the substrate and Z or the enzyme protein is not limited to the SH3 domain and SH3 domain binding sequence, and the combination of the amino acid sequences to which the domain has affinity is not limited to other sequences of calmodulin and calmodulin interaction. Any protein-binding domain, such as a PDZ domain and a PDZ domain interaction sequence, and an amino acid sequence that exhibits affinity for the domain can be used. It is also possible to use an amino acid sequence that has binding properties to any protein obtained by phage display or the like.

[0027] The present invention also relates to a method for producing a phosphate protein by phosphorylating a substrate protein with a kinase, the SH3 domain and SH3 domain complementary to the substrate protein and Z or kinase. By having a binding sequence, the affinity of both molecules is increased and the phosphorylation reaction is efficiently performed.

[0028] "Substrate protein" in the phosphorylation reaction means a protein that is phosphorylated and has a phosphorylation site (Thr, Ser or Tyr). Such proteins may be in any form such as those present in vivo or in cells, or isolated. In addition, such a substrate protein may originally have an SH3 domain or may not have such an SH3 domain. A little.

[0029] Crkll, pl30Cas, etc. can be mentioned as those having an SH3 domain.

Moreover, paxillin, AKT, etc. are mentioned as those that do not have the SH3 domain.

[0030] In the present invention, the "SH3 domain" refers to an SH3 domain binding sequence comprising two small | 8 sheets composed of about 50 to 70 amino acid residues and packed into each other at almost right angles. Means functional domain.

[0031] Proteins containing such SH3 domains include Abl, Tec, Btk, Lyn, Frk, Srms, Blk, Hck, Txk ゝ Vav, Crk, CrkL ゝ JNK ゝ CSK ゝ CasL, pl30Cas ゝ p47phox, p40phox, Pexl 3, Grb2, RasGAP ゝ RhoGAP ゝ Fyb, Fyn, PLCganmma ゝ Intersectin, Ctk, ArlGAP, PI 3K ゝ PSD—95, Yes ゝ Srk, Fgr, Nck ゝ Gads ゝ Zo—1, Zo—2, Zo—3, Spectrin , Drk, Sem-5, etc. As the “SH3 domain” of the present invention, any functional domain of these proteins can be used.

[0032] Examples of the "SH3 domain" that can be used in the present invention are the following peptides (a) or (b).

[0033] (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1

(b) A peptide having an amino acid sequence ability in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and exhibiting affinity for the SH3 domain binding sequence.

[0034] Another example of the SH3 domain that can be used in the present invention is the following peptide (c) or (d).

[0035] (c) a peptide having the amino acid sequence ability of SEQ ID NO: 26

(d) A peptide having an amino acid sequence ability in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 26, and exhibiting affinity for the SH3 domain binding sequence.

[0036] The SH3 domain usable in the present invention is 60% homologous to the amino acid sequence of SEQ ID NO: 1 or 25, preferably 80% homologous, more preferably 90% homologous, and still more preferably 95% homologous. It may be a peptide having an amino acid sequence ability and having an affinity for the SH3 domain binding sequence.

[0037] In the SH3 domain binding sequence in the present invention, each of the above SH3 domains is specifically, Alternatively, it means an amino acid sequence that is commonly recognized and shows affinity. The SH3 domain binding sequence may differ depending on the sequence of the SH3 domain, or all SH3 domains may bind in common. Examples of amino acid sequences to which all SH3 domains bind include amino acid sequences of about 10 residues including PxxP (where X represents any amino acid residue). This amino acid sequence binds to 31 "[3 domains with a dissociation constant of 5-100 \ 1.

[0038] This SH3 domain binding sequence is further limited to??, K) X (X represents any amino acid residue, Φ is a hydrophobic amino acid residue, and more specifically I, L, V, P It represents one of the following.)

[0039] For example, the SH3 domain binding sequence in which the SH3 domain of a) b) shows affinity is PXXPXZ, ZXXPXXP, or PXXXRXXK (wherein X represents an arbitrary amino acid, and Z represents K or R). ). In addition, the SH3 domain binding sequence in which the SH3 domain of c) d) shows affinity is a sequence represented by PX [V or I] [D or N] RXXKP (wherein X represents any amino acid). It is.

[0040] In the present invention, "showing affinity" means the property that a protein binding domain binds to the domain binding sequence with a binding (dissociation) constant that causes a specific interaction between proteins and proteins. Say. This affinity can be determined by adding a peptide having an arbitrary amino acid sequence to a peptide having an SH3 domain, for example, nuclear magnetic resonance (NMR) spectrum, fluorescence vector, ultraviolet absorption spectrum, fluorescence polarization method, surface plasmon resonance, A pull-down assay can be examined by measuring the change and the like by measuring with ELISA or the like (Patent Documents 1 and 2) using FRET.

[0041] "Kinase", which is an example of the enzyme protein of the present invention, is an enzyme protein that catalyzes the reaction of adding a phosphate group to the OH group of T (Thr), S (Ser), or Y (Tyr) in the protein. It is. Depending on its specificity, it is classified into two types: SerZThr phosphate enzyme that phosphorylates Ser and Thr, and Tyr phosphate enzyme that phosphorylates Tyr. Tyr kinases include Abl, Lyn, Fyn, Hck, Jak, CSK, Flk, Syk, EGFR, VEGFR, ZAP-70, Blk, Ryk, Btk, Tec, Src, Frk, Itk, Lck, Yes Kit, Fes, Mer, Fgr, IGFR, FGFR, FAK, EPHR, Alk, Met, Trk, Eck, Hek, Sek, Mdk, Esk, Htk, Rek, erbB and the like. SerZThr phosphorylation enzymes include CK II, nPKC, aPKC, PKC ゝ PKA, PKB ゝ ROCK, STE20, CAMK, and MAPK. , Ark, Cdk ゝ Chk ゝ Drp, Zip ゝ MAPKK, MAPKKK, JNK ゝ MEKK, MLCK, KIN, YAK, SAT4, PAK, etc. All enzymes are composed of a plurality of functional domains, of which the kinase domain is responsible for the catalytic function, and it is known that the kinase domain alone is active. In the present invention, the whole kinase or a domain having phosphate activity can be used, and “kinase” in the present invention means both unless otherwise specified.

[0042] In one embodiment of the present invention, a substrate protein (including a phosphate chain site) that is a chimeric protein in which either the SH3 domain-binding sequence or the SH3 domain is added at the amino acid sequence level, and the SH3 of the substrate protein A kinase, which is a chimeric protein to which an amino acid sequence showing affinity for a domain binding sequence or SH3 domain is added, is used. Preferably, a chimeric protein in which the SH3 domain is added to the substrate protein and the SH3 domain binding sequence is added to the kinase at the amino acid level, respectively. Each chimeric protein has an appropriate amino acid sequence, preferably an amino acid sequence cleavable by a protease capable of recognizing and cleaving a specific amino acid sequence, between the substrate protein or enzyme protein and the amino acid sequence to be added. May be provided. In addition, there is no particular limitation on the order of the substrate protein or enzyme protein and the amino acid sequence to be added, which may be located on the N-terminal side.

[0043] There is no particular limitation on the method of performing phosphoric acid using the method of the present invention, but the following methods are exemplified:

(1) A chimeric protein is prepared by adding the SH3 domain to the substrate protein. The SH3 domain may use the endogenous domain of the substrate protein or enzyme protein, but may be a chimeric protein with an SH3 domain derived from another protein. In this case, as described above, the amino acid sequence that can be cleaved between the amino acid sequence of the target substrate protein and the amino acid sequence of the SH3 domain by an enzymatic method such as a protease or a chemical method such as CNBr. For example, if a proteolytic enzyme cleavage sequence such as Tev Protease or Thrombin Protease is inserted, the target amino acid can be digested or cleaved with a protein degrading enzyme after the completion of the phosphorylation reaction. It is possible to obtain a phosphoric acid protein in which only the sequence force is composed. [0044] On the other hand, a chimeric protein having an SH3 domain binding sequence on the C-terminal side of the amino acid sequence of the kinase may be prepared and used to act on a substrate protein, or a vector that expresses this peptide. Etc. may be prepared and expressed or co-expressed in the vicinity of the substrate protein. In the latter case, any vector including pET, pGEX, pPRO, etc. can be used as a vector, and an expression vector should be constructed according to a conventional method.

[0045] (2) When the substrate protein originally has an SH3 domain, a chimeric protein in which an SH3 domain binding sequence is added to the kinase at the amino acid level is allowed to act on the substrate protein in the same manner as described above. Good.

[0046] (3) A transformant in which a kinase, particularly a DNA encoding a peptide in which an SH3 domain-binding sequence is added to the C-terminal side of the kinase at the amino acid level and a DNA encoding a substrate protein containing the SH3 domain, are introduced. To co-express them. Any host including E. coli, yeast, mammalian cells, insect cells and the like can be used, and any promoter including T7, Taq, lac, etc. can be used as the promoter.

[0047] In addition to the combination of the substrate protein containing the SH3 domain and the kinase containing the SH3 domain binding sequence, the combination of the kinase containing the SH3 domain and the substrate protein containing the SH3 domain binding sequence may be similarly used. And the affinity of the kinase can be increased.

[0048] The phosphorylated substrate protein can be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). When the substrate protein is phosphorylated, two negative charges increase at each phosphate site, and the mobility in electrophoresis changes before and after phosphate. Therefore, phosphate can be confirmed by comparing the mobility of non-phosphorylated substrate protein and phosphorylated substrate protein. Furthermore, after SDS-PAGE, immunostaining with an antibody specific for a phosphorylated target protein or an antibody that specifically recognizes a phosphorylated amino acid is more reliable. It is possible to detect a substrate protein that has been phosphorylated. Normal SDS-PAGE detects both phosphorylated and unreacted substrate protein, but immunostaining detects only phosphorylated substrate protein, so to determine the phosphorylation reaction efficiency. It is preferred to do a normal SDS-PAGE. [0049] In addition, as shown in the Examples of the present invention, myristoyltransferase or protein dephosphorylating enzyme can be used as the enzyme protein. Myristoyl candy is one of the protein modification reactions with fatty acids, and myristoyl transferase recognizes common sequences present in proteins that receive myristoy candy, such as G (E, etc.), XX (S, etc.), and Myristoyl CoA To a rearrangement of the myristoyl group to the amino group of the N-terminal glycine of the protein to form an acid amide bond. As for myristoyltransferase (NMT), an enzyme that catalyzes this myristoy potato, 19 NMTs have been identified and reported from more than 15 eukaryotes including humans. The presence of NMT1 and NMT2 has been reported in humans, and NMT2 is detected as a single 65 kDa band on SDS-PAGE, while NMT1 exhibits various isoforms. In the present invention, it is preferable to use human NMT1 or NMT2 which can appropriately use these NMTs.

[0050] Myristoli is considered to be involved in intracellular signal transduction, determination of intracellular localization of protein, particularly targeting of protein to the cell membrane, stabilization of protein three-dimensional structure, and the like. Yes. In addition, in HIV-1, it is suggested that it is an essential modification for the virus protein formation for myristoylation of viral structural protein pi 7gag and viral regulatory protein p27nef. In particular, NMT is expected to discover a number of molecules that are controlled by Myristoyl sputum in the future as research on the cranial nervous system, which has the highest expression level in the brain and nerves, progresses. Thus, there are a wide variety of proteins that receive myristoy buds, that is, those having myristoy bud sequences, and the present invention can appropriately use myristoy bud sequences possessed by these proteins.

[0051] Further, the present invention can easily provide not only the phosphorylated substrate protein as described above but also the dephosphorylated substrate protein. Protein dephosphorylation works in conjunction with intracellular protein phosphate enzymes, thereby transmitting intracellular information such as cell growth and differentiation, cell shape and movement, cell cycle, metabolism, and transcriptional activity. It is widely known that it is precisely controlled, and it can be applied to gene therapy as a basic technology for controlling both the positive and negative phosphate signals.

[0052] In addition, the method of the present invention combines an enzyme protein that performs a modification reaction of a protein other than phosphorylation, dephosphorylation, and myristoyl koji, and a specific sequence recognized by the enzyme protein. By using it, the modified protein can be easily and efficiently produced.

The following examples illustrate the invention, but are not intended to limit the invention.

Example 1

[0053] In this example, the following three plasmids were prepared.

1. Plasmid pProEX-Crkll: A plasmid for expressing the substrate protein Crkll (SEQ ID NO: 2).

2. Plasmid pET- Duet-SK-Crkll: A plasmid containing a DNA fragment (SEQ ID NO: 4) encoding SK that contains the kinase shown in Fig. 1 but does not contain a proline-rich sequence, and Crkll. Are co-expressed.

3. Plasmid pET- Duet-SKP1-Crkll: A plasmid containing the DNA fragment (SEQ ID NO: 5) encoding SKP1 containing the kinase and proline-rich sequence shown in Fig. 2, and Crkll. To express.

[0054] In this example, human Crkll (SEQ ID NO: 2) was used as a phosphate substrate. Figure 3 shows the domain structure. As shown in FIG. 3, Crkll is composed of one SH2 domain (positions 5 to 120 of SEQ ID NO: 2) followed by two SH3 domains (positions 134 to 191 and 238 to 293 of SEQ ID NO: 2). In the region between these two SH3 domains, there is a phosphate chain (221th Tyr of SEQ ID NO: 2).

[0055] The N-terminal SH3 domain of Crkll (134th to 191st of SEQ ID NO: 2) is specific to — PXXPXK— (where X represents any amino acid residue, K may be R ヽ) Join.

On the other hand, in this example, human Abl (SEQ ID NO: 3) was used as the kinase. Figure 4 shows the domain structure. As shown in Fig. 4, Abl consists of one SH3 domain (80th to 140th positions in SEQ ID NO: 3), followed by one SH2 domain (141st to 239th positions in SEQ ID NO: 3), and kinase domain (from 247 of SEQ ID NO: 3). 253), SH3 binding sequence (545 to 550, 589 to 594 and 778 to 783 of SEQ ID NO: 3), a DNA binding domain, and an actin binding domain. The Crkll N-terminal SH3 domain recognizes and binds to this SH3 domain binding sequence. [0057] (1) Preparation of plasmid pProEX-Crkll

Crkll cDNA was prepared by the method described previously (Mol. Cell. Biol., No. 12, pages 3482-3489, 1992). Crkll cDNA, dNTP 200 Μ, magnesium chloride lmM, 2 primers (SEQ ID NOs: 6 and 7), 300 nM each, buffer (1 X) attached to KOD-Plus-, reaction containing KOD-Plus-polymerase 1U PCR reaction was performed with 100 μl of the solution.

[0058] The obtained DNA fragment was cleaved with Ncol (NEB) and Xhol (NEB) and purified with a PCR purification kit (Quiagen). Both the pProEX Htb vector fragment obtained by cutting pProEXCrkll Htb (Quiagen) with Ncol and Xhol and the PCR fragment containing the Crkll gene (SEQ ID NO: 2) obtained above were combined with Takara DNA Ligation kit (Takara). ), And this vector was used to transform E. coli DH5a. After culturing this, the plasmid (pProEX-Crkll) shown in FIG. 5 was obtained using MiniPrep kit (Quiagen).

[0059] Crkll was expressed in E. coli using the obtained plasmid pProEX-Crkll, and Crkll was purified using the 6 X His tag. It was confirmed by SDS-PAGE that Crkll with a molecular weight of 34 kDa was purified with high purity (Fig. 6).

[0060] (2) Preparation of plasmid pET- Duet-SK-Crkll

Abl cDNA was prepared by the method described previously (Proc. Natl. Acad. Sci. U.S.A., 84, 8200-8204, 1987). Using the Abl cDNA and primers (SEQ ID NOs: 10 and 11), PCR was performed in the same manner, and a PCR fragment containing only the SH2 domain and kinase domain (Fig. 1, SK) was incorporated into pET Duet (Novagen). A plasmid (FIG. 7, pET-Duet-SK) was obtained. Crkll obtained by PCR using primers (SEQ ID NOs: 8 and 9) was similarly inserted into this plasmid (pET-Duet-SK) to obtain a plasmid (pET-Duet-SK-Crkll) (Fig. 8).

[0061] (3) Preparation of plasmid pET- Duet-SKP1-Crkll

Using the Abl cDNA and primers (SEQ ID NOs: 12 and 13), a PCR reaction was carried out in the same manner, and a PCR fragment containing only the SH2 domain, kinase domain and the most SH3 domain binding sequence at the N-terminal side (Figure 2, SKP1 ) Was incorporated into pET Duet (Novagen) to obtain a plasmid (pET-Duet-SKPl) (FIG. 9). Crkll obtained by PCR using primers (SEQ ID NOs: 8 and 9) was similarly inserted into this plasmid to obtain a plasmid (pET-Duet-SKP1-Crkll) (FIG. 10). Example 2

[0062] In this example, CrkllZAbl was co-expressed in E. coli, and Crkll phosphate was observed.

Escherichia coli BL-21 (DE3) was transformed with the plasmid pET-Duet-SKP1-Crkll. Transformants were selected by spreading the colon bacteria on LB agar medium containing ampicillin and incubating at 37 ° C. One of the colonies formed was planted in 20 ml of LB medium containing 100 g / ml ampicillin and cultured at 37 ° C. This culture solution was transferred to 1 L of 2 XYT medium containing 100 μg Zml of ampicillin, and the final concentration of 0.5 mM isopropyl-β-D (—)-thiogalatatopyrano was measured at a turbidity of 600 nm between 0.4 and 1.0. The sid was added and further cultured at 25 ° C. The culture solution was centrifuged at 3600 × g at 4 ° C. for 15 minutes to recover the cells. Dissolve the cells in PBS buffer solution (NaCl 8g, KC1 0.2g, Na HPO-12H O 14.5g ゝ KH PO lg in 1L of distilled water in total.

2 4 2 2 4

And was sonicated in ice. This was centrifuged at 11900 Xg and 4 ° C for 60 minutes to separate into soluble and insoluble fractions.

[0063] This was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.). The results are shown in Fig. 11. As a control, the band was shifted to the higher molecular weight side compared to Crkll, which was run as a control, confirming that the phosphorylation reaction was performed. In addition, no band was observed at the molecular weight of non-phosphorylated Crkll, and it was confirmed that almost all expressed Crkll was phosphorylated!

[0064] Next, the polyacrylamide gel and the PVDF membrane after electrophoresis were immersed in a transfer buffer and shaken for about 5 minutes. The transfer buffer used was a mixture of 15.14 g of tris (hydroxymethyl) aminomethane and 72.07 g of glycine dissolved in a total of 1 L of distilled water and 4 L and 1 L of methanol. Next, the filter paper is immersed in a transfer buffer solution and placed on a transfer device (Bio-Rad). Next, PVDF membrane, gel, and filter paper were stacked in this order, and transfer was performed at a constant voltage of 10V. The voltage was adjusted so that the current at this time was 0.1 to 0.15 A, and transfer was performed for about 1 hour. Next, the PVDF membrane after transfer was immersed in a blocking buffer and shaken for about 1 hour. Then, it was washed 3 times with TBS-T buffer. During this time, mouse anti-phosphorylated Crkll antibody (Zymed) was diluted 1000 times with blocking buffer, PVDF membrane was immersed in 5 ml of this solution, shaken for 1 hour, and washed 3 times with TBS-T. During this period, the PVDF membrane was immersed in 5 ml of a secondary antibody solution diluted with a TBS-T buffer 5000 times (anti-mouse antibody), shaken for 1 hour, and washed 3 times with TBS-T buffer. Secondary antibody is Pero xidase Labeled Anti-Mouse Antibody (Santa Cruz) was used. Thereafter, staining was performed using a peroxidase staining DAB kit (Nacalai Testa).

As a result, as shown in FIG. 12, a band in which Crkll was phosphorylated was confirmed.

[0066] Comparative Example 1

Using a commercially available Abl (New England Biolabs, which has only the SH2 domain and kinase domain), Crkll's phosphorylation reaction was attempted.

Phosphorylation was performed at 10 mg / ml CrkIl / 20 mM Tris-HCl / lmM EDTA / 150 mM NaCl (pH 7.5) at 100 / zl, attached lO XAbl buffer at 100 / zl, and ATP at a final concentration of O.lmM. And adjust the volume of the solution to 1 ml with HO, and leave at 25 ° C for 4 days.

2

went. This was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.). As a result, it was confirmed from FIG. 13 that the position of the band was the same as that of Crkll subjected to electrophoresis as a control, and that the phosphorylation reaction was hardly performed.

The polyacrylamide gel after electrophoresis was confirmed to be phosphorylated by the same immunostaining as in Example 2. The result is shown in FIG. It was confirmed that no phosphorylated Crkll band was detected.

[0067] Comparative Example 2

Escherichia coli BL-21 (DE3) was transformed with the plasmid pET-Duet-SK-Crkll, and a fraction containing Crkll was obtained in the same manner as in Example 2.

Example 3

In this example, SKP1 (FIG. 2) was expressed in E. coli as a chimeric protein (SEQ ID NO: 14) with a base sequence encoding a 6 X His tag. Escherichia coli BL-21 (DE3) was transformed with the plasmid pET-Duet-SKPl (FIG. 9), a fraction containing SKP1 was obtained in the same manner as in Example 2, and SKP1 was further purified. The same volume of glycerol was added to the elution fraction containing purified SKP1.

[0069] Using SKP1 obtained in this way, Crkll's phosphate reaction was attempted in a test tube. This The Crkll obtained in Example 1 was used as the Crkll. lOmgZml CrkIl / 20mM Tris-HCl / lm M EDTA / 150mM NaCl (pH7.5) 100 / z 1, lO XAbl buffer used in Comparative Example 1 is 10 01, ATP is final concentration O.lmM Add HO so that the volume of the solution is lml.

2 Prepared and allowed to stand at 25 ° C for 6 hours or more.

[0070] Crkll subjected to the phosphorylation reaction was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.).

The polyacrylamide gel after electrophoresis was confirmed to be phosphorylated by the same immunostaining as in Example 2. The result is shown in FIG. A band in which Crkll was phosphorylated was confirmed.

Example 4

[0071] In this example, the following plasmids were prepared.

1.Plasmid pET22-Crk-Vav (170-375): N-terminal SH3 domain of Crkll and His-tag and precision protease cleavage sequence shown in Fig. 15 and amino acid residues 170-375 of Vav's amino acid sequence ( (Hereinafter referred to as Vav (170-375)) is a plasmid containing the gene (SEQ ID NO: 15) of the chimeric protein that also has the power. In this example, phosphorylation was attempted using Vav (170-375) (SEQ ID NO: 16) as a substrate.

[0072] (1) Preparation of plasmid pET22- Crk-Vav (170-375)

The PCR fragment of the N-terminal SH3 domain of Crkll (residue numbers 130-198) obtained using the following primers is inserted into pET22 in the same manner as in Example 1.

CCTCTAGACATATGAGGCAGGAGGAGGCGGAG (F) (SEQ ID NO: 17)

CCCTCGAGGAGCTCCGATACTGAGGCGGAGGC (R) (SEQ ID NO: 18)

The Tev protease cleavage site of pProEX Htb is first mutated to the precision protease cleavage site using Quick Change (Quiagene). This vector is hereinafter referred to as p ProEX Htb-Pres. Insert a PCR fragment of Vav (170-375) obtained using the following primers into pProEX Htb-Pres.

[0073] GCGCCATGGGGGACGAGATCTACGAGG (F) (SEQ ID NO: 19)

GCGCTCGAGTCACCTCTTGACCTCGTTCACG (R) (SEQ ID NO: 20)

The inserted fragment is prepared using the following primers. CCGAGCTCATGTCGTACTACCATCACC (F) (SEQ ID NO: 21) GCGCTCGAGTCACCTCTTGACCTCGTTCACG (R) (SEQ ID NO: 22)

From this, a DNA fragment (SEQ ID NO: 23) encoding Vav (170-375) with a His tag derived from pProEX Htb-Pres and a precision protease cleavage site is obtained.

[0074] By inserting the Vav DNA fragment attached with the His tag and precision protease cleavage site obtained for pET22 into which the DNA fragment encoding the Crkll SH3 domain created above was inserted, Crkll Construction of plasmid pET-22- Crk-Vav (l 70-375) (Fig. 16) encoding chimeric protein Crk-Vav (170-375) of SH3-His-tag precision protease cleavage site Vav (170-375) Is done.

[0075] Vav cDNA was prepared by the method described previously (Coppola J., Bryant SKoda T., Conway D., Barbacid M .; "Mechanism of activation of the vav protooncogene .; Cell Growt h Differ. 2 pp. 95-105, 1991).

In this example, phosphorylation of Cr k-Vav (170-375) was attempted in a test tube using SKP1 purified by the same method as in Example 3. This Crk-Vav (170-375) was prepared in the same manner as in Example 1. Phosphate reaction was carried out in the same manner as in Example 3 with SKP1 of 101, 10 mg / ml Crk-Vav (170-375) / 20 mM Tris-HCl / lmM EDTA / 150 mM NaCl (pH 7.5) 100 / zl, lO XAbl buffer used in Comparative Example 1 was added at 100 / zl, and ATP was added to a final concentration of O.lmM, and the volume of the solution was adjusted to HO with HO, 25 ° C Over 4 hours

2

It was done by leaving still.

Crk-Vav (170-375) subjected to phosphorylation reaction was run on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.). A change in mobility was observed compared to Crk-Vav (170-375) before the phosphoric acid reaction, and the phosphoric acid of Vav (170-375) was confirmed (Fig. 17).

[0077] Comparative Example 4

Vav (170-375) (FIG. 18) was subjected to phosphorylation by the same method as in Example 4, and was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nihon Aid Co., Ltd.). No change in mobility was observed compared to Vav (170-375) before phosphorylation, confirming that Vav (170-375) was not phosphorylated (Fig. 17).

Example 5 In this example, the following two types of plasmids were prepared.

1. Plasmid pET-Duet-SK-SLP: A plasmid containing the kinase shown in FIG. 19 and a DNA fragment (SEQ ID NO: 24) encoding SK-SLP containing SLP76-derived RxxK-containing sequence.

2. Plasmid pET22-Grb2—Crkll: The Grb2 C-terminal SH3 domain and His-tag shown in Figure 20 and the DNA encoding the precision protease cleavage sequence and Vav (170-375) chimeric protein (SEQ ID NO: 25) Containing plasmid.

[0079] In this example, phosphoric acid was tried using Crkll as a substrate. Furthermore, Grb2 C-terminal SH3 domain (SEQ ID NO: 26) binds specifically to PxxxRxxK (where x represents any amino acid) sequence contained in proteins such as SLP76 and Gabl. An SLP76-derived Pxx xRxxK sequence was included, and the substrate protein side containing the C-terminal SH3 domain of Grb2 was prepared, and phosphorylation was attempted.

[0080] (1) Preparation of plasmid pET— Duet— SK- SLP

Using the Abl cDNA and the following primers, perform a PCR reaction in the same way, and add the PCR fragment (Fig. 19) encoding SK-SLP containing the SH2 domain, kinase domain and SLP76-derived sequence to pET Duet (Novagen). The integrated plasmid (Fig. 21, pET-Duet-SK-SLP) was obtained.

CCGGATCCGAACAGCCTGGAGAAACATTCC (F) (SEQ ID NO: 27)

CCTGCAGC (R) (SEQ ID NO: 28)

In this example, SK-SLP (FIG. 2) was expressed in E. coli as a chimeric protein (SEQ ID NO: 14) with a base sequence encoding a 6 X His tag. E. coli BL-21 (DE3) was transformed with the plasmid pET-Duet-SK-SLP, a fraction containing SK-SLP was obtained in the same manner as in Example 2, and SK-SLP was further purified. The same volume of glycerol was added to the elution fraction containing purified SK-SLP.

[0081] (2) Preparation of plasmid pET22-Grb2-—Crkll

A gene fragment encoding the C3 terminal SH3 domain (residue numbers 159-217) of Grb2 was amplified using the following primers.

CCTCTAGACATATGACATACGTCCAGGCC (F) (SEQ ID NO: 29) GGGAGCTCGACGTTCCGGTTCACGGGGG (R) (SEQ ID NO: 30) For the vector fragment obtained by cleaving pET22 with NdelZSacI, the DNA fragment of the Grb2 C-terminal SH3 domain prepared above was digested with NdelZSacI in the same manner as in Example 1. To insert pET22-Grb2. Next, PCR was performed using the following primers to amplify the Cr kll gene, which was then incorporated into the vector fragment of pET22-Grb2 treated with SaclZXhoI to obtain plasmid pET22-Grb2-Crkll (FIG. 22).

[0082] CCGAGCTCATGTCGTACTACCATCACC (F) (SEQ ID NO: 31)

GGGCTCGAGTCAGCTGAAGTCCTCTTCGGG (R) (SEQ ID NO: 32)

Using the obtained SK-SLP, the phosphorylation reaction of Grb2-CrkII was attempted in a test tube. Phosphorylation was performed at 100 mg / zl of lOmgZml of Grb2-CrkIlZ20 mM Tris-HCl / lmM EDTA / 150 mM NaCl (pH 7.5), 100 / zl of the lO XAbl buffer used in Comparative Example 1, and ATP at the final concentration. Add O.lmM, add SK-SLP to a final concentration of 0.001 μΜ, and adjust the volume of the solution to 1 ml with HO. It was done by leaving it to stand for more than an hour.

2

[0083] Grb2-CrkII that had undergone the phosphorylation reaction was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.). Compared with Grb2-CrkII before the phosphoric acid reaction, a change in mobility was observed, confirming phosphorylation (Fig. 23).

[0084] Comparative Example 5

Grb2-CrkII was phosphorylated with SK in the same manner as in Example 5. However, SK was added so that the final concentration was 0.1 μΜ. Electrophoresis was performed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.). Compared to Grb2-CrkII before the phosphoric acid reaction, no change in mobility was observed, confirming that Grb2-Crkll was phosphorylated! /! (Fig. 23). Example 6

In this example, the following plasmids were prepared.

1. Plasmid pTYR (Figure 24): DNA fragment (SEQ ID NO: 5) that encodes SKP1 containing the kinase and proline-rich sequences shown in Figure 2 and Crkll SH3 domain—His tag-precision protease cleavage site Multicloning A plasmid containing a site. It is possible to insert genes encoding arbitrary peptides and proteins into the multicloning site. [0086] (1) Preparation of plasmid pTYR

The NdelZXhoI fragment of pET22-Crk-Vav (170-375) prepared in Example 4 is incorporated into the vector fragment of pET-Duet-SKP1 prepared in Example 1. The Ndel site (CATATG) sequence used for integration was replaced with CAGATG using Quick Change (Quiagene) to eliminate the Ndel site. Next, the Ncol site (CCATGG) used when Vav (170-375) is incorporated into pProExHTb-Pres is replaced with ATATGG using Quick Change (Quiagene) to create a new Ndel site. This plasmid is hereinafter referred to as pET- Duet SKP1 Crk.

[0087] Next, a synthetic gene composed of the following multicloning sites is phosphorylated and mixed to prepare a double-stranded DNA fragment. No. 33) Number 34)

Insert a double-stranded multicloning site fragment into the vector fragment of pET Duet SKP1 Crk treated with NdelZXhoI.

[0088] (2) Preparation of plasmid pTYR Vav (170-375)

PCR is performed using the Vav cDNA as a saddle with the following primers, and incorporated into the pTYR vector fragment treated with NdelZXhoI.

GCCATATGGGGGACGAGATCTACGAGG (F) (SEQ ID NO: 35)

GCGCTCGAGTCACCTCTTGACCTCGTTCACG (R) (SEQ ID NO: 36)

In the same manner as in Example 2, the chimeric protein (SEQ ID NO: 37) of SKP1 and Crkll N-terminal SH3 domain, 6 X His tag, precision protease cleavage site and Vav (170-375) was placed in E. coli. Co-expressed and purified. The purified chimeric protein was electrophoresed on a 15% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.). A difference in mobility was observed with non-phosphate Crk-Vav (170-375) prepared in Example 4, and it was confirmed that phosphate was added (FIG. 25).

Example 7 [0089] (1) Construction of plasmid pTYR (—)

Example 6 pTYR prepared in (1) was digested with (Notl / Xhol), and the fragment containing the Crkll SH3 gene was incorporated into the vector fragment digested with pET-Duet (Notl / Xhol). pTYR (—) (Fig. 26) was prepared. This plasmid has a structure converted into a multicloning site derived from the kinase gene of pTYR (SKP1) ¾ ET-Duet.

[0090] (2) Construction of plasmid pSH3-NMT

Glover et al. (Glover. J. Human N—myristoyltransferase amino—terminal domain involved in targeting the enzyme to the ribosomal subcellular fraction. "; J. Biol. Chem., 272, 28680-28689, 1997). was prepared _C DNA encoding NMT1 (N- myristoyltransfera s _e) according to the method. the cDNA and铸型, PCR was performed using the following primers, 81 th -496 th amino acids of the catalytic domain (NMT1 of NMT1 A DNA encoding a sequence domain) was obtained as an amplified fragment.

GGTCTAGACATATG AACTCTTTGCCAGCAGAGAGG (F) (SEQ ID NO: 38) CGCCTCGAGTCATTGTAGCACCAGTCCAACC (R) (SEQ ID NO: 39)

The amplified fragment digested with Ndel / Xhol was incorporated into a vector of pTYR (—) that was also digested with Ndel / Xhol to prepare pSH3-NMT (FIG. 27).

[0091] (3) Construction of plasmid pGBl-Myr

PDE¾-532, geneservice, nttp: //www.geneservice.co.uK/) Use the following primers to perform PCR, and use the B1 domain (GB1 domain) of Streptococcal protein G in the plasmid DNA encoding was obtained as an amplified fragment.

GACCCATGGAGTACAAACTGATCC (F) (SEQ ID NO: 40)

GAAGG (R) (SEQ ID NO: 41)

In addition, the following synthetic DNA was mixed to prepare a double-stranded DNA fragment.

CAACGACCGAAAACCTGTATTTTCAG GGGCAGCAGCCTGGAAAAGTTCTTG GGGACCAAAGAAGGCCTAGTTTGGCCATGGCAC (F) (SEQ ID NO: 42) GTGATGGTGATGGTAGTACGACATATGTCTAGA (R) (SEQ ID NO: 43) Both fragments obtained by treating the amplified fragment with Ncol / Xhol and the double-stranded DNA fragment with Ndel / Ncol were incorporated into pET22-b treated with Ndel / Xhol, and plasmid pGBl -Myr (Fig. 28) was created. This plasmid has a 6xHis tag on the N-terminal side of the GB1 domain, a TEV protease digestion site and a myristoylation sequence consisting of 17 amino acid residues (Hantschel 0 et al., Cell, 20 03, 112, No. 6, No. 6, 845-857) and a gene encoding a fusion protein containing a CrkSH3 binding sequence on the C-terminal side.

[0092] (4) E. coli BL-21 (DE3) is transformed with pSH3-NMT prepared in (2), and a fusion protein (SEQ ID NO: 44, NMT1 catalytic domain and Crkll SH3 and 6 X His-tagging force) is also obtained. Hereinafter, SH3-NMT) was expressed in E. coli, and SH3-NMT was purified by the same method as in Example 2 to prepare a glycerol solution of SH3-NMT. In addition, pGBl-Myr prepared in (3) is used to transform E. coli BL-21 (DE3), which also includes 6 X His tag, TEV protease digestion site, myristylation sequence, GB1 domain, and Crkll SH3 binding sequence. The fraction containing the fusion protein expressed in E. coli as a protein (SEQ ID NO: 45) and TEV-digested, the fusion protein in which the modified residue Gly is exposed at the N-terminus (hereinafter referred to as GBl-Myr) Was prepared.

[0093] 20 mM Tris-HCl / lmM EDTA / 150 mM NaCl (pH 7.5) 10 0 1 containing 10 mg / ml GB1-Myr was mixed with lOxAbl buffer solution 100 1 used in Comparative Example 1, and this was mixed with myristoyl-CoA. (Sigma) is added to a final concentration of O.lmM. Add 10 μl or 1 μl of SH3-NMT / glycerol solution or 1 μl of ζ / ζ and the volume of the solution to 1 ml. Was prepared with water and incubated at 25 ° C for 2 hours. The sample after the reaction was run on a 18.8% polyacrylamide gel using an electrophoresis apparatus (Nippon Aid Co., Ltd.) (FIG. 29). As a result, the change in the band associated with myristoyl was observed in the sample with SH3-NMT of 101, while the sample with SH3-NMT of 1 μ1 had not reacted with myristoylated GBl-Myr. Two bands originating from GBl-Myr were observed. In addition, two types of sampnore, one with SH3-NMT and one without SH3-NMT, were subjected to mass spectrometry using the MAS spectrometer Voeager from Applied Biosystem. The increase in the molecular weight of 210 was observed with the addition of SH3-NMT. It was confirmed that GBl-Myr (theoretical molecular weight 9036.7, measured molecular weight 9038.1625) was force myristoylated (theoretical molecular weight 9246.7, measured molecular weight 9248.4779).

Example 8

[0094] Chernofife method (Chernoff J., Schievella AR, Jost CA, Erikson R 丄., Neel BG; 〃 Cloning of a cDNA for a major human protein- tyrosine-phosphatase .; roc. Natl. Acad. Sci. USA 87: 2735-2739 (1990)), cDNA encoding human PTB-IB (human protein-tyrosine-phosphatase) was prepared. Using this cDNA as a saddle, PCR was performed using the following primers, and DNA encoding the phosphatase domain of PTP-1B was obtained as an amplified fragment.

GCGCCATGGAGATGGAAAAGGAGTTCGAGCAGATC (F) (SEQ ID NO: 46) (R) (SEQ ID NO: 47)

The obtained amplified fragment was treated with Ncol / Xhol and incorporated into pET21-d that was also treated with Ncol / Xhol to prepare plasmid ρΡΤΡΙΒ-PxxP (FIG. 30). This plasmid is a fusion protein containing the Crkll SH3 binding sequence and 6 X His tag on the C-terminal side of the catalytic domain of human PTP-1B ty rosine phosphatase (domain consisting of the 1st to 300th amino acid sequences of PTB-1B). (SEQ ID NO: 48, hereinafter referred to as PTP-1B-PxxP) is expressed.

E. coli BL-21 (DE3) was transformed with pPTP-lB-PxxP, and PTP-lB-PxxP was purified as a glycerol solution by the same method as in Example 2. 20 μM Tris-HCl / lmM EDTA / 150 mM NaCl (pH 7.5) 100 μl containing 10 mg / ml of phosphorylated Crkll prepared by the method of Example 2 and lO XAbl buffer solution 100 / zl used in Comparative Example 1 Mix, add PTP-1B-PxxP / glycerol solution to 10 1 or 1 μ 1 or 01, adjust the volume to 1 ml with water, and incubate at 25 ° C for 2 hours . The sample after the reaction was run on a 15% polyacrylamide gel using an electrophoresis apparatus (Nihon Aid Co., Ltd.) (FIG. 31). As a result, a change in the band accompanying dephosphorylation was observed in 101 PPT-lB-PxxP, and dephosphorylated phosphorous was observed in 1 P1 of PTP-1 B-PxxP. Two bands derived from oxidized Crkll and unreacted phosphorylated Crk II were observed. In addition, phosphorylation was confirmed by immunostaining similar to Example 2 (FIG. 32). As a result, phosphorous was not obtained with 10 / zl of PTP-1B-PxxP. The disappearance of the oxidized Crkll band was confirmed. When 1 μl of PTP-1B-PxxP was added, it was confirmed that the band was thin, and dephosphorylation was confirmed.

Industrial applicability

The present invention is an epoch-making technique for efficiently performing a protein modification reaction by using a common phenomenon called protein-protein interaction. For example, even in the case where the normal method is not fully phosphorylated, the affinity between the two is increased by generating a protein-protein interaction between the substrate protein and the kinase, thereby reducing the amount of the enzyme. The substrate protein can be phosphorylated efficiently. Phosphorylation is an important process in intracellular and intercellular signal transduction, and the use of the phosphorylated protein or the induction of a biological reaction is controlled by the method of the present invention capable of efficiently producing phosphorylated protein. be able to.

In other words, gene therapy that enables artificial control of signal transduction via phosphates by introducing kinases or substrate proteins into cells and efficiently performing phosphorylation in cells. Application to screening of phosphorylation regulators, efficient production of phosphoproteins useful in medicine and industry is also expected.

Claims

The scope of the claims

[1] A method for producing a modified protein comprising modifying a substrate protein with an enzyme protein, wherein the substrate protein and Z or the enzyme protein can increase the affinity between the substrate protein and the enzyme protein. The method for producing a modified protein, which is a chimeric protein to which a binding domain is added.

[2] The substrate protein or enzyme protein that binds to a chimeric protein to which a protein binding domain has been added is a chimeric protein to which an amino acid sequence to which the protein binding domain has affinity has been added. Production method.

[3] Protein-binding domain and Z, or an amino acid sequence in which the protein-binding domain shows affinity. Force Attached to substrate protein and Z or enzyme protein via an amino acid sequence that can be cleaved enzymatically or chemically. Item 3. The manufacturing method according to Item 1 or 2.

[4] The method for producing a modified protein according to any one of claims 1 to 3, wherein the enzyme protein is a kinase.

[5] The method for producing a modified protein according to any one of claims 1 to 4, wherein the protein binding domain is an SH3 domain.

[6] The SH3 domain is the following peptide (a) or (b):

(a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1

(b) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having affinity for an SH3 domain binding sequence, wherein the SH3 The domain binding sequence is PXXPXZ! /, ZXXPXXP or PXXXRXXK (wherein X represents an arbitrary amino acid, Z represents K or R), and has a proline-rich sequence represented by claim 5. The manufacturing method as described.

[7] The SH3 domain is a peptide of (c) or (d) below:

(c) Peptide having the amino acid sequence of SEQ ID NO: 26

(d) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 26, and having affinity for an SH3 domain binding sequence, wherein the SH3 6. The production according to claim 5, wherein the domain binding sequence has a sequence represented by PX [V or I] [D or N] RXXKP (wherein X represents any amino acid). Method.

8. The production method according to claim 6, wherein the substrate protein has an SH3 domain, and the kinase has an SH3 domain binding sequence.

[9] The production method according to any one of [6] and [7], wherein the substrate protein has an SH3 domain binding sequence, and the kinase has an SH3 domain.

[10] The production method according to claim 3, comprising a step of cleaving an amino acid sequence that can be cleaved enzymatically or chemically after the reaction between the substrate protein and the enzyme protein to recover the modified protein.

[11] The host protein is transformed using a vector that expresses the substrate protein and Z or enzyme protein that is a chimeric protein and the substrate protein or enzyme protein to which the chimeric protein binds, and the substrate protein and enzyme protein are transformed into the host. 4. The production method according to any one of claims 1 to 3, which is also capable of co-expression in cells.

[12] The method for producing a modified protein according to any one of claims 1 to 3, wherein the enzyme protein is myristoyltransferase.

[13] The method for producing a modified protein according to any one of claims 1 to 3, wherein the enzyme protein is a protein dephosphorylating enzyme.