CN113899902A - Tyrosine phosphatase substrate identification method - Google Patents

Tyrosine phosphatase substrate identification method Download PDF

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CN113899902A
CN113899902A CN202010573873.7A CN202010573873A CN113899902A CN 113899902 A CN113899902 A CN 113899902A CN 202010573873 A CN202010573873 A CN 202010573873A CN 113899902 A CN113899902 A CN 113899902A
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leu
glu
gly
tyrosine phosphatase
ser
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范高峰
庄敏
张佳丽
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ShanghaiTech University
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention relates to the technical field of biology, in particular to a method for identifying a tyrosine phosphatase substrate. The tyrosine phosphatase substrate identification method provided by the invention comprises the following steps: placing the system comprising the potential substrate for interaction with tyrosine phosphatase in the presence of a tyrosine phosphatase-PafA fusion protein, a pup protein, and labeling the pup protein; enriching potential substrates for interaction with tyrosine phosphatases by the labeled pup protein; the tyrosine phosphatase-PafA fusion protein comprises a tyrosine phosphatase fragment and a PafA fragment, wherein the tyrosine phosphatase fragment has a substrate capture mutant. The method and the system for identifying the tyrosine phosphatase substrate can popularize biological research aiming at the tyrosine phosphatase to any important tyrosine phosphatase family member, and have good industrialization prospect.

Description

Tyrosine phosphatase substrate identification method
Technical Field
The invention relates to the technical field of biology, in particular to a method for identifying a tyrosine phosphatase substrate.
Background
Signal transduction in an organism requires a very complex and precise balance, requiring the involvement of various protein adaptors, protein effectors, proteases and substrates, and other protein complexes to deliver extracellular signals to intracellular targets in a timely and accurate manner. There are many ways of controlling this equilibrium, which can be regulated by phosphorylation and dephosphorylation of important proteins in kinase and phosphatase regulated signal transduction processes. Therefore, the study of the dynamic equilibrium regulation of kinases and phosphatases in organisms is of great importance for the study of signal transduction in cells.
Protein-protein interactions have been a leading hot problem in biochemical and cell biological research. With the progress of research, the transient nature of most intracellular protein interactions has become more and more recognized, including the mode of action between tyrosine phosphatases and catalytic substrates. Conventional protein interaction detection methods, including co-immunoprecipitation combined with mass spectrometry, Fluorescence Resonance Energy Transfer (FRET), and yeast two-hybrid techniques, have failed to meet the need for real-time detection of protein-protein interactions. The proximity labeling based on ligase mediation is a novel technology which is reported in recent years and can convert the interaction between proteins (including weak interaction and transient interaction) into stable covalent linkage under the condition of living cells, and the emergence of the technology undoubtedly provides powerful technical guarantee for detecting the protein interaction. Currently, the widely applied proximity labeling technologies include BioID, APEX, etc., however, these two methods still have limitations: BioID background is too high and APEX requires the addition of hydrogen peroxide, which is damaging to cells.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for identifying a substrate for tyrosine phosphatase, which solves the problems of the prior art.
To achieve the above and other related objects, according to one aspect of the present invention, there is provided a method for identifying a substrate for tyrosine phosphatase, comprising:
1) placing the system comprising the potential substrate for interaction with tyrosine phosphatase in the presence of a tyrosine phosphatase-PafA fusion protein, a pup protein, and labeling the pup protein;
2) enriching potential substrates for interaction with tyrosine phosphatases by the labeled pup protein;
the tyrosine phosphatase-PafA fusion protein comprises a tyrosine phosphatase fragment and a PafA fragment, wherein the tyrosine phosphatase fragment has a substrate capture mutant.
In some embodiments of the invention, the tyrosine phosphatase fragment is selected from the group consisting of a SHP1 fragment, a SHP2 fragment, a PTP1B fragment, a TCPTP fragment, a PTPRK fragment, and a CD45 fragment.
In some embodiments of the invention, in step 1), the system including the potential substrate interacting with the tyrosine phosphatase is the target cell, and the step 1) is specifically: the target cells were cultured in the presence of the tyrosine phosphatase-PafA fusion protein, the pup protein, and labeled with the pup protein.
In some embodiments of the invention, said step 1) further comprises; the cells obtained from the culture are lysed to provide a lysate.
In some embodiments of the invention, the tyrosine phosphatase-PafA fusion protein and/or the pup protein is expressed by a cell of interest.
In some embodiments of the invention, the pup protein comprises a pup fragment and a tag protein fragment, wherein the tag protein is selected from the group consisting of biotin tag proteins.
In some embodiments of the invention, in step 1), the pup protein is labeled with biotin.
In some embodiments of the invention, in step 2), the substrate potentially interacting with the target protein is enriched by the labeled pup protein based on the biotin-avidin system.
In another aspect, the invention provides a tyrosine phosphatase substrate identification system comprising a combination of a tyrosine phosphatase-PafA fusion protein comprising a tyrosine phosphatase fragment and a PafA fragment, and a pup protein, the tyrosine phosphatase fragment having a substrate capture mutation.
In some embodiments of the invention, a system of potential substrates for interaction with tyrosine phosphatases is also included.
In some embodiments of the invention, a marker for labeling the pup protein is also included.
In another aspect, the invention provides an expression system comprising a construct or genome of polynucleotides encoding tyrosine phosphatase-PafA fusion proteins and pup proteins having exogenous polynucleotides encoding tyrosine phosphatase-PafA fusion proteins and pup proteins integrated therein.
Drawings
FIG. 1 shows a schematic diagram of the principle of searching for a phosphatase substrate for PEPSI according to the invention.
FIG. 2 shows an analytical volcano plot of mass spectra results of SHP1-DA VS SHP1-WT of example 1 of the present invention.
FIG. 3 is a graph showing the results of experiments on the interaction of THEMIS and SHP1 in example 2 of the present invention.
FIG. 4 is a graph showing the experimental results of LCK phosphorylation THEMIS in example 3 of the present invention.
FIG. 5 is a graph showing the results of the experiment in which LCK acts on THEMIS Tyr34 site in example 4 of the present invention.
FIG. 6 is a graph showing the results of an experiment for dephosphorylating THEMIS according to example 5 of the present invention by SHP 1.
FIG. 7 is a schematic diagram showing the experimental results of the peptide fragment of THEMIS phosphorylated at the dephosphorylated Tyr34 site of SHP1 in example 6 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.
The inventors of the present invention have made extensive experimental studies, and found that when a tyrosine phosphatase-PafA fusion protein having a substrate trapping mutation is used in combination with a phosphorylation site capable of interacting with a substrate in the surrounding environment, PafA catalyzes a pup protein to modify the pup protein to a lysine site of the substrate interacting with a tyrosine phosphatase fragment in the fusion protein, and can separate and identify the substrate of tyrosine phosphatase by labeling the pup protein, thereby providing a highly efficient method and system for identifying a substrate of tyrosine phosphatase.
In a first aspect, the present invention provides a method for identifying a substrate for tyrosine phosphatase, comprising:
1) placing the system comprising the potential substrate for interaction with tyrosine phosphatase in the presence of a tyrosine phosphatase-PafA fusion protein, a pup protein, and labeling the pup protein;
2) enriching potential substrates for interaction with tyrosine phosphatases by the labeled pup protein;
the tyrosine phosphatase-PafA fusion protein comprises a tyrosine phosphatase fragment and a PafA fragment, wherein the tyrosine phosphatase fragment has a substrate trapping mutation (substrate trapping mutation).
In the method for identifying a tyrosine phosphatase substrate provided by the invention, the tyrosine phosphatase-PafA fusion protein can comprise a tyrosine phosphatase fragment. The tyrosine phosphatase fragment may be a fragment of various enzymes belonging to the tyrosine phosphatase family, for example, the tyrosine phosphatase fragment may be a SHP1(Gene ID:5777) fragment, a SHP2(Gene ID:5781) fragment, a PTP1B (Gene ID:5770) fragment, a TCPTP (Gene ID:5771) fragment, a PTPRK (Gene ID:5796) fragment, a CD45(Gene ID:5788) fragment, and the like. The tyrosine phosphatase fragment can be generally derived from human (homo sapiens) or mouse (mus musculus) and has high homology. As described above, the tyrosine phosphatase fragment in the tyrosine phosphatase-PafA fusion protein usually has a substrate bridging mutation, and the substrate bridging mutation usually means that the mutated tyrosine phosphatase fragment can still be combined with a substrate phosphorylation site, but the tyrosine phosphatase fragment is not easy to separate after being combined with the substrate due to the loss of the activity of the tyrosine phosphatase, and compared with the corresponding wild-type tyrosine phosphatase, the tyrosine phosphatase fragment with the substrate bridging mutation has stronger affinity with the substrate and longer combination time, thereby being used for searching substrates acting with the tyrosine phosphatase. In 1997 Andrew J.Flint et al found that the tyrosinase family all have highly conserved PTP domain comprising 27 invariant residues, which when mutated at one of the positions, change its spatial conformation, for example, D181A mutation of PTP1B makes it have substrate capture properties (DOI: 10.1073/pnas.94.5.1680). The different members of the tyrosine phosphatase family differ in their amino acid sequence and therefore differ in their substrate tracking mutation sites, but are regularly reproducible, typically in highly conserved substrate-binding pockets in the PTP domain, either as aspartic acid, Asp, or cysteine, Cys sites. For example: the mutation sites of different phosphatases are different, such as SHP 1D 419A, PTP1B D181A, SHP 2D 425A, and TCPTP D182A. Specifically, the amino acid sequence of the tyrosine phosphatase fragment can comprise: a) an amino acid sequence as shown in one of SEQ ID NO. 1-4; or b) an amino acid sequence having a sequence similarity of 80% or more to the amino acid sequence represented by any one of SEQ ID nos. 1 to 4, and having the function of the amino acid sequence defined in a). Specifically, the amino acid sequence in b) specifically refers to: the polypeptide fragment which is obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) amino acids to the amino acid sequence shown in any one of SEQ ID NO.1 to 4, or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2 or 3) amino acids to the N-terminal and/or C-terminal and has the functions of the polypeptide fragment shown in any one of SEQ ID NO.1 to 4, for example, a polypeptide fragment which can bind to a substrate phosphorylation site but loses the activity of tyrosine phosphatase, and the bonding of the substrate and the substrate is not easy to separate. The amino acid sequence in b) can have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity with one of SEQ ID No. 1-4.
In the method for identifying a substrate for tyrosine phosphatase provided by the invention, the tyrosine phosphatase-PafA fusion protein can comprise a PafA fragment. The PafA fragment is typically derived from Corynebacterium glutamicum (Corynebacterium glutamicum). The amino acid sequence of the PafA fragment may include: c) an amino acid sequence shown as SEQ ID NO. 4; or d) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.4 and having the function of the amino acid sequence defined in c). Specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown as SEQ ID No.4 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment with the amino acid shown as SEQ ID No.4, for example, can be a lysine site of a substrate capable of catalyzing the modification of the pup protein to interact with a tyrosine phosphatase fragment of the tyrosine phosphatase-PafA fusion protein. The amino acid sequence in d) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 4.
In the method for identifying a substrate for tyrosine phosphatase provided by the present invention, the pup protein may include a pup fragment. The Pup protein is usually derived from Corynebacterium glutamicum (Corynebacterium glutamicum). The amino acid sequence of the pup fragment may include: e) an amino acid sequence shown as SEQ ID NO. 5; or f) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.5 and having the function of the amino acid sequence defined in e). Specifically, the amino acid sequence in f) specifically refers to: the amino acid sequence shown as SEQ ID No.5 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids are added to the N-terminal and/or C-terminal of the polypeptide fragment shown as SEQ ID No.5, and the polypeptide fragment having the function of the polypeptide fragment shown as SEQ ID No.5 is, for example, a polypeptide fragment which can be catalyzed by a PafA fragment in a tyrosine phosphatase-PafA fusion protein to be modified at a lysine site of a substrate interacting with a tyrosine phosphatase fragment in the tyrosine phosphatase-PafA fusion protein . The amino acid sequence in f) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 5.
The method for identifying the tyrosine phosphatase substrate provided by the invention can comprise the following steps: the system comprising the potential substrate for interaction with tyrosine phosphatase is placed in the presence of a tyrosine phosphatase-PafA fusion protein, the pup protein, and the pup protein is labeled. The system comprising a substrate potentially interacting with tyrosine phosphatase generally means various systems which may comprise a substrate capable of interacting with tyrosine phosphatase, for example, a target cell or the like. Interacting a system comprising a substrate potentially interacting with tyrosine phosphatase with a tyrosine phosphatase-PafA fusion protein and a pup protein, binding a tyrosine phosphatase fragment of the tyrosine phosphatase-PafA fusion protein to a substrate possibly present in the system and capable of interacting with tyrosine phosphatase, and catalyzing the pup protein by the PafA fragment to modify the pup protein to a lysine site of the substrate capable of interacting with the tyrosine phosphatase fragment of the fusion protein, so that the substrate is labeled with the fusion protein as a whole, and the label on the pup protein is retained after separation of the substrate from the tyrosine phosphatase fragment, so that, by subsequent processing, if the system of the substrate potentially interacting with tyrosine phosphatase does comprise a substrate capable of interacting with tyrosine phosphatase, then the substrate capable of interacting with tyrosine phosphatase can be enriched by the labeled pup protein.
In the method for identifying the tyrosine phosphatase substrate provided by the invention, the system comprising the potential substrate capable of interacting with the tyrosine phosphatase can be the target cell, namely, the target cell can be placed in the presence of the tyrosine phosphatase-PafA fusion protein and the pup protein, so that the substrate capable of interacting with the tyrosine phosphatase, which is possibly arranged in the target cell, can interact with the tyrosine phosphatase-PafA fusion protein and the pup protein, and if the target cell does comprise the substrate capable of interacting with the tyrosine phosphatase, the substrate capable of interacting with the tyrosine phosphatase can be enriched by the labeled pup protein. The target cell may be generally a eukaryotic cell or the like, more specifically a mammalian cell or the like, for example, a human cell, a mouse cell, a rat cell, a hamster cell or the like, and in one embodiment of the invention, the target cell may be Jurkat, HEK293, Hela, HEPG2, CAOV4, K562, MCF7, RAW264.7, C2C12, 3T3L1, CHO-K1 or the like.
In the method for identifying a tyrosine phosphatase substrate provided by the invention, when the system comprising the substrate potentially interacting with the tyrosine phosphatase is a target cell, the target cell can be generally enabled to express the tyrosine phosphatase-PafA fusion protein and/or the pup protein, so that the target cell is placed in the presence of the tyrosine phosphatase-PafA fusion protein and the pup protein. Suitable methods for expressing the tyrosine phosphatase-PafA fusion protein and/or the pup protein in the target cell will be known to those skilled in the art, for example, a construct comprising a polynucleotide encoding the tyrosine phosphatase-PafA fusion protein and the pup protein can be transfected into the target cell, or a foreign polynucleotide encoding the tyrosine phosphatase-PafA fusion protein and the pup protein can be integrated into the genome of the target cell. Furthermore, when the system comprising the potential substrate for interaction with tyrosine phosphatase is a target cell, it is usually necessary to lyse the cells obtained by the culture to provide a lysate, so that the substrate capable of interacting with tyrosine phosphatase can be enriched by the labeled pup protein.
In the method for identifying a tyrosine phosphatase substrate provided by the invention, an appropriate labeling system is generally selected to label the pup protein, so that the potential substrate interacting with the tyrosine phosphatase can be enriched by the labeled pup protein. For example, the pup protein may include a pup fragment as described above, and may further include a tag protein fragment, and the tag protein may be biotin tag protein, tag protein FLAG, tag protein MYC, tag protein HA, and the like, and for example, the amino acid sequence of biotin tag protein may include the sequence shown in SEQ ID No.6, the amino acid sequence of tag protein FLAG may include the sequence shown in SEQ ID No.7, the amino acid sequence of tag protein MYC may include the sequence shown in SEQ ID No.8, and the amino acid sequence of tag protein HA may include the sequence shown in SEQ ID No. 9. When the pup protein includes a tag protein fragment, one skilled in the art can select an appropriate label to label the pup protein, and can select an appropriate method to enrich the substrate for potential interaction with tyrosine phosphatase by the labeled pup protein. For example, when the pup protein is a fusion protein comprising a biotin tag protein fragment, biotin can be enriched by the pup protein, and thus a substrate potentially interacting with a target protein can be enriched based on the biotin-avidin system. For another example, when the pup protein is a fusion protein including fragments of tag proteins such as FLAG, MYC, HA, etc., the substrates potentially interacting with the target protein can be enriched by the corresponding antibodies.
The method for identifying the tyrosine phosphatase substrate provided by the invention can further comprise the following steps: the enriched obtained substrate which interacts with the tyrosine phosphatase is identified to provide a substrate capable of interacting with the tyrosine phosphatase. Suitable methods for identifying the substrate will be known to those skilled in the art and may, for example, be by mass spectrometry or the like.
In a second aspect, the invention provides a tyrosine phosphatase substrate identification system comprising a combination of a tyrosine phosphatase-PafA fusion protein comprising a tyrosine phosphatase fragment and a PafA fragment, and a pup protein, the tyrosine phosphatase fragment having a substrate capture mutation. As described above, when the tyrosine phosphatase-PafA fusion protein with the substrate capture mutation is combined with a phosphorylation site capable of interacting with its substrate in the surrounding environment, PafA catalyzes the pup protein to modify it to a lysine site of the substrate interacting with the tyrosine phosphatase fragment in the fusion protein, and the substrate of tyrosine phosphatase can be isolated and identified by labeling the pup protein.
The tyrosine phosphatase substrate identification system provided by the invention can also comprise a marker for marking the pup protein and/or a system comprising a substrate potentially interacting with tyrosine phosphatase.
In a third aspect, the invention provides an expression system comprising a construct or genome of polynucleotides encoding tyrosine phosphatase-PafA fusion proteins and pup proteins having exogenous polynucleotides encoding tyrosine phosphatase-PafA fusion proteins and pup proteins integrated therein. The expression system may typically be the target cell to be investigated, which may include a substrate potentially interacting with a tyrosine phosphatase. The expression system can be constructed from a cell line corresponding to a target cell to be studied, and the cell can be a eukaryotic cell, more specifically a mammalian cell, such as human cell, mouse cell, rat cell, hamster cell, etc., and in one embodiment of the invention, the target cell can be Jurkat, HEK293, Hela, HEPG2, CAOV4, K562, MCF7, RAW264.7, C2C12, 3T3L1, CHO-K1 cell, etc.
The method and the system for identifying the tyrosine phosphatase substrate can be applied to various tyrosine phosphatases, explore the interaction proteome and the substrate thereof, and capture the transient interaction between the tyrosine phosphatase and the substrate thereof. The human tyrosine phosphatase family has 105 members, each member has important functions, and different cell lines can be selected for researching different functions of the same kind of phosphatase in different organs, so that the tyrosine phosphatase substrate identification method and system provided by the invention can popularize biological research aiming at the tyrosine phosphatase to any important tyrosine phosphatase family member, thereby being beneficial to the fields of cancer, obesity, diabetes, neurodegenerative diseases and other serious diseases and having good industrialization prospects.
The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
Example 1
The cytoplasmic protein SHP1 encoded by PTPN6 gene belongs to non-receptor type tyrosine phosphatase in tyrosine phosphatase superfamily I, mainly comprises an N-SH2 structural domain, a C-SH2 structural domain and a PTP catalytic structural domain, wherein the dephosphorylation of a tyrosine site is mainly performed by a PTP catalytic activity center in signal transduction. The protein tyrosine phosphatase PTPN6/SHP1 is used as a signal regulation molecule and participates in physiological processes of cell proliferation and differentiation, cell apoptosis, intracellular signal transduction and the like. In 1975, studies by Green, M.C. et al found that the mutant phenotype called moteaten (me) in C57BL/6J mice was an autoimmune disease caused by a disturbance in the autoimmune system. The phenotype is caused by that the third exon of the SHP1 gene lacks a cytidine, thereby causing frame shift mutation, so that the transcribed and translated protein almost completely loses the dephosphorylation enzyme activity. In 1984, Shultz, L.D. et al again found a phenotype similar to that of moteaten, with the same symptoms, but the mice with this phenotype survived longer, hence the name moteaten viable (mev). mev is the partial loss of dephosphorylating enzyme activity of a protein transcribed and translated by insertion/deletion in the ninth exon of SHP1 gene. Complete loss of SHP1 function resulted in the development of a moteaten phenotype in mice with disturbed immune system leading to systemic skin ulceration, suggesting that SHP1 is critical for immune system development. While the T cell antigen receptor (TCR) is a receptor which is combined with the Major Histocompatibility Complex (MHC) on the surface of an antigen presenting cell on the surface of a T lymphocyte, various documents report that SHP1 can regulate T cell development, TCR signals and the like, and conditional knockout of SHP1 in a T cell expressing CD4 can enhance the strength of TCR signals, so that the number of apoptotic cells and the number of mature cells are increased in the development selection process, but the development process is not influenced, and the development process of T cells of a germline SHP1 knock-out mouse is damaged, which indicates that SHP1 plays a very important role in the development process of the T cells. However, the molecular mechanism of regulation of SHP1 activity in immune cells is not well understood, and little is known about the substrate of SHP 1.
In this example, SHP1, a protein tyrosine phosphatase, was used as a subject, and its substrate was searched in jurkat.t cell line by the PEPSI method (i.e., the method for identifying a substrate for tyrosine phosphatase provided by the present invention). As shown in FIG. 1, a schematic diagram of a substrate search using PEPSI is shown for SHP1 mapping mutant (SHP 1-D419A).
The Jurkat cell line for inducing and expressing the fusion protein of the biotin tag protein and the small protein pup is constructed, a plasmid (pHR-Tet3G rtTA; pHR-Tet3G Bio-PupE-IRES-BFP) of a Tet-On system containing the sequence (SEQ ID NO.10) of the fusion protein and a lentivirus packaging plasmid are transfected into HEK293T cells through liposome, the obtained lentivirus is infected with the Jurkat cells to obtain the Jurkat cell line for inducing and expressing the fusion protein of the pup and the biotin tag protein by Doxycycline, and the BFP is expressed while the pup is expressed, so that the positive cell line can be screened through BFP fluorescence. Then, a plasmid containing SHP1-WT + PafA-Myc-6His fusion protein and a plasmid containing SHP1-DA + PafA-Myc-6His fusion protein are respectively transfected into HEK293T cells together with a lentivirus packaging plasmid through liposome, and the obtained lentivirus is infected with the Jurkat cell line for inducing expression of pup as described above, so that the Jurkat cell line stably expressing the SHP1-WT + PafA-Myc-6His fusion protein or the SHP1-DA + PafA-Myc-6His fusion protein is obtained for mass spectrometry.
The amino acid sequence of the SHP1-WT + PafA-Myc-6His fusion protein is as follows:
whereas the SHP1-DA + PafA-Myc-6His fusion protein differs from SHP1-WT + PafA-Myc-6His in that the bold and underlined D becomes A.
Figure BDA0002550334050000091
After adjusting the cell concentration to 0.5 million/ml and starting cells of 10-20million per sample, the above cell line was treated with 2. mu.g/ml Doxycyline (SELLECK S4163) and 4. mu.m Biotin (Hu test 67000260), and the cells after drug addition were placed in an incubator at 37 ℃ and 5% carbon dioxide for 28-32h, so that pup could complete the process of labeling potential substrates, while the Biotin-tagged protein fused to pup could enrich Biotin, and thus could enrich potential substrates with streptavidin-conjugated magnetic beads. After labeling was completed, the cells were taken out from the incubator, collected by centrifugation, horizontally rotated at room temperature at 500g for 3min, and the supernatant was removed. Cells were lysed for 30-60min on ice using 900. mu.l of lysis buffer (50mM Tris 7.5; 200mM NaCl; 2% Triton; 0.1% SDS) supplemented with the protease inhibitor Cocktail (APExBIO K1007) per sample. 13000g was then centrifuged at 4 ℃ for 15min to remove the DNA from the lysate, and 800ul of the supernatant was added urea to a final concentration of 8M (0.384g) and DTT to a final concentration of 10mM (MDBio D023-5g), 56 ℃ (Thermomixer) for 1 h. IAM (iodoacetamide, currently available ABCONE I53892-25G) was added to a final concentration of 25mM, protected from light, and left at room temperature for 45 min. DTT was added to a final concentration of 25mM and allowed to stand at room temperature for 0.5 h. The pup-labeled protein was enriched by adding 50ul of beads to streptavidin beads (NEB S1420S), and then bound on a homogenizer at room temperature for 1 h. The beads were washed to remove non-specifically bound proteins, using 4 buffers in sequence, each time on a homogenizer and spun at room temperature for 5 min.
Buffer 1: 2 times (8M Urea; 50mM Tris 8.0; 200mM NaCl; 0.2% SDS)
Buffer 2: 2 times (8M Urea; 50mM Tris 8.0; 200mM NaCl)
Buffer 3: 2 times (50mM Tris 8.0; 0.5mM EDTA; 1mM DTT)
Buffer 4: 2 times (50mM ammonium bicarbonate)
The protein was digested to peptides by adding 6ug of pancreatin (promega V5113) and shaking overnight on a 37 ℃ Thermomixer instrument with shaking overnight on a shaker at 37 ℃ with 15ul of pancreatin +110ul of 50mM ammonium bicarbonate.
The overnight cleaved peptide fragments were desalted using ZipTip (Merck/Millipore ZTC18S096), the 37 ℃ overnight cleaved tube was removed, and the supernatant was transferred to a new EP tube with the aid of a magnet. 10% formic acid was added to a final concentration of 1% in order to inactivate the enzyme while precipitating the protein. After the addition, the pH is detected by using a pH test paper, and the pH is ensured to be less than 3. 100% acetonitrile soaks Ziptip, 200ul rifle head inhales acetonitrile and sheathes Ziptip, beats off acetonitrile slowly, makes ZipTip soak. Ziptip was equilibrated with 0.1% TFA (trifluoroacetic acid) and directly aspirated, then added to waste stream 2 times. The sample was then combined and blown 4-5 times, taking care that no air bubbles could be generated. The sample was then desalted by washing with 0.1% TFA, and the sample was removed after each inhalation and repeated 2-3 times. Finally, 50ul of 70% ACN-0.1% TFA was taken alone with a new 100ul pipette tip, followed by sheathing with Ziptip containing the sample, eluting the sample into a new EP tube, pipetting 2-3 times, and finally vacuum-drying the sample. The identification of the protein component was performed on the samples by mass spectrometry and the mass spectrometry data was analyzed using the perseus platform. unique peptides represent the specificity of peptide fragments, and the higher the value of the peptides is in a certain sense, the higher the content of the protein in a sample is, and the protein can be used as a reference index. In this case, the candidate genes were ranked according to unique peptides, THEMIS was selected as the candidate gene for further study, and the specific results are shown in fig. 2.
Example 2
The mass spectrum result is verified, and THEMIS-FLAG and SHP1-WT or SHP1-DA are cloned into pEF6/myc-His A vector by using a homologous recombination method to construct an overexpression plasmid.
The base sequence of SHP-WT is as follows:
atggtgaggtggtttcaccgagacctcagtgggctggatgcagagaccctgctcaagggccgaggtgtccacggtagcttcctggctcggcccagtcgcaagaaccagggtgacttctcgctctccgtcagggtgggggatcaggtgacccatattcggatccagaactcaggggatttctatgacctgtatggaggggagaagtttgcgactctgacagagctggtggagtactacactcagcagcagggtgtcctgcaggaccgcgacggcaccatcatccacctcaagtacccgctgaactgctccgatcccactagtgagaggtggtaccatggccacatgtctggcgggcaggcagagacgctgctgcaggccaagggcgagccctggacgtttcttgtgcgtgagagcctcagccagcctggagacttcgtgctttctgtgctcagtgaccagcccaaggctggcccaggctccccgctcagggtcacccacatcaaggtcatgtgcgagggtggacgctacacagtgggtggtttggagaccttcgacagcctcacggacctggtggagcatttcaagaagacggggattgaggaggcctcaggcgcctttgtctacctgcggcagccgtactatgccacgagggtgaatgcggctgacattgagaaccgagtgttggaactgaacaagaagcaggagtccgaggatacagccaaggctggcttctgggaggagtttgagagtttgcagaagcaggaggtgaagaacttgcaccagcgtctggaagggcagcggccagagaacaagggcaagaaccgctacaagaacattctcccctttgaccacagccgagtgatcctgcagggacgggacagtaacatccccgggtccgactacatcaatgccaactacatcaagaaccagctgctaggccctgatgagaacgctaagacctacatcgccagccagggctgtctggaggccacggtcaatgacttctggcagatggcgtggcaggagaacagccgtgtcatcgtcatgaccacccgagaggtggagaaaggccggaacaaatgcgtcccatactggcccgaggtgggcatgcagcgtgcttatgggccctactctgtgaccaactgcggggagcatgacacaaccgaatacaaactccgtaccttacaggtctccccgctggacaatggagacctgattcgggagatctggcattaccagtacctgagctggcccgaccatggggtccccagtgagcctgggggtgtcctcagcttcctggaccagatcaaccagcggcaggaaagtctgcctcacgcagggcccatcatcgtgcactgcagcgccggcatcggccgcacaggcaccatcattgtcatcgacatgctcatggagaacatctccaccaagggcctggactgtgacattgacatccagaagaccatccagatggtgcgggcgcagcgctcgggcatggtgcagacggaggcgcagtacaagttcatctacgtggccatcgcccagttcattgaaaccactaagaagaagctggaggtcctgcagtcgcagaagggccaggagtcggagtacgggaacatcacctatcccccagccatgaagaatgcccatgccaaggcctcccgcacctcgtccaaacacaaggaggatgtgtatgagaacctgcacactaagaacaagagggaggagaaagtgaagaagcagcggtcagcagacaaggagaagagcaagggttccctcaagaggaag(SEQ ID NO.12)
the base sequence of SHP1-DA is as follows:
atggtgaggtggtttcaccgagacctcagtgggctggatgcagagaccctgctcaagggccgaggtgtccacggtagcttcctggctcggcccagtcgcaagaaccagggtgacttctcgctctccgtcagggtgggggatcaggtgacccatattcggatccagaactcaggggatttctatgacctgtatggaggggagaagtttgcgactctgacagagctggtggagtactacactcagcagcagggtgtcctgcaggaccgcgacggcaccatcatccacctcaagtacccgctgaactgctccgatcccactagtgagaggtggtaccatggccacatgtctggcgggcaggcagagacgctgctgcaggccaagggcgagccctggacgtttcttgtgcgtgagagcctcagccagcctggagacttcgtgctttctgtgctcagtgaccagcccaaggctggcccaggctccccgctcagggtcacccacatcaaggtcatgtgcgagggtggacgctacacagtgggtggtttggagaccttcgacagcctcacggacctggtggagcatttcaagaagacggggattgaggaggcctcaggcgcctttgtctacctgcggcagccgtactatgccacgagggtgaatgcggctgacattgagaaccgagtgttggaactgaacaagaagcaggagtccgaggatacagccaaggctggcttctgggaggagtttgagagtttgcagaagcaggaggtgaagaacttgcaccagcgtctggaagggcagcggccagagaacaagggcaagaaccgctacaagaacattctcccctttgaccacagccgagtgatcctgcagggacgggacagtaacatccccgggtccgactacatcaatgccaactacatcaagaaccagctgctaggccctgatgagaacgctaagacctacatcgccagccagggctgtctggaggccacggtcaatgacttctggcagatggcgtggcaggagaacagccgtgtcatcgtcatgaccacccgagaggtggagaaaggccggaacaaatgcgtcccatactggcccgaggtgggcatgcagcgtgcttatgggccctactctgtgaccaactgcggggagcatgacacaaccgaatacaaactccgtaccttacaggtctccccgctggacaatggagacctgattcgggagatctggcattaccagtacctgagctggcccgcccatggggtccccagtgagcctgggggtgtcctcagcttcctggaccagatcaaccagcggcaggaaagtctgcctcacgcagggcccatcatcgtgcactgcagcgccggcatcggccgcacaggcaccatcattgtcatcgacatgctcatggagaacatctccaccaagggcctggactgtgacattgacatccagaagaccatccagatggtgcgggcgcagcgctcgggcatggtgcagacggaggcgcagtacaagttcatctacgtggccatcgcccagttcattgaaaccactaagaagaagctggaggtcctgcagtcgcagaagggccaggagtcggagtacgggaacatcacctatcccccagccatgaagaatgcccatgccaaggcctcccgcacctcgtccaaacacaaggaggatgtgtatgagaacctgcacactaagaacaagagggaggagaaagtgaagaagcagcggtcagcagacaaggagaagagcaagggttccctcaagaggaag(SEQ ID NO.13)
overexpression of the protein was achieved by integration of the plasmids of THEMIS-FLAG and SHP1-WT or SHP1-DA into Jurkat cells using electrical breakdown (i.e., electroporation) methods. Before electrotransfer, DNA of each sample (30 ug of plasmid in total per sample) is mixed well, centrifuged for 5min at 800g to collect cells, supernatant is discarded, the cells are resuspended to a final concentration of 40M/ml with OPti-MEM (Gibco,31985070), 300ul (12M) of resuspended cells are added to each mixed plasmid, the tube wall is flicked to mix them evenly, the mixed solution of the mixed plasmid and cells is allowed to stand at room temperature for 15min, and then transferred to an electric shock cup (Bio-rad, 1652088), the cell is allowed to stand for 15min in the electric shock cup by using a preset program of Jurkat cells in an apparatus of Bio-rad, parameters are adjusted to 250V and 0.4cm, the cells are allowed to stand for 15min after electric shock, the cells after electrotransfer are placed in 10ml of preheated culture medium, and then an incubator with a carbon dioxide concentration of 5% at 37 ℃ for 24-32 h.
Wherein the amino acid sequence of the fusion protein of THEMIS-FLAG is as follows: MALSLEEFVHSLDLRTLPRVLEIQAGIYLEGSIYEMFGNECCFSTGEVIKITGLKVKKIIAEICEQIEGCESLQPFELPMNFPGLFKIVADKTPYLTMEEITRTIHIGPSRLGHPCFYHQKDIKLENLIIKQGEQIMLNSVEEIDGEIMVSCAVARNHQTHSFNLPLSQEGEFYECEDERIYTLKEIVEWKIPKNRTRTVNLTDFSNKWDSTNPFPKDFYGTLILKPVYEIQGVMKFRKDIIRILPSLDVEVKDITDSYDANWFLQLLSTEDLFEMTSKEFPIVTEVIEAPEGNHLPQSILQPGKTIVIHKKYQASRILASEIRSNFPKRHFLIPTSYKGKFKRRPREFPTAYDLEIAKSEKEPLHVVATKAFHSPHDKLSSVSVGDQFLVHQSETTEVLCEGIKKVVNVLACEKILKKSYEAALLPLYMEGGFVEVIHDKKQYPISELCKQFRLPFNVKVSVRDLSIEEDVLAATPGLQLEEDITDSYLLISDFANPTECWEIPVGRLNMTVQLVSNFSRDAEPFLVRTLVEEITEEQYYMMRRYESSASHPPPRPPKHPSVEETKLTLLTLAEERTVDLPKSPKRHHVDITKKLHPNQAGLDSKVLIGSQNDLVDEEKERSNRGATAIAETFKNEKHQKPGLEPWKLMDYKDDDDKDI (SEQ ID NO.14)
The horizontal rotor was rotated at room temperature 300g, centrifuged for 5min to collect cells, added with 800ul of lysis buffer (20mM Hepes pH 7.5; 150mM NaCl; 1% NP40) to lyse cells, lysed on ice for 10min, centrifuged at 4 ℃ 15000rpm for 10min to remove DNA, collected as supernatant, 200ul of which was taken as INPUT sample to indicate transfection, the remaining 550ul was added with 8ul of FLAG beads (SigmaA2220-1ML) to enrich THEMIS protein by immunoprecipitation, and WB was used to detect whether proteins of SHP1-WT or SHP1-DA were also precipitated. The results are shown in FIG. 3, where LANE1 was the control cell transfected with 2.5ug of PEF6/myc-His A empty plasmid, LANE2 was the cell transfected with 1.25ug of THEMIS-FLAG over-expression plasmid and 1.25ug of PEF6/myc-HisA empty plasmid, LANE3 was the cell transfected with 1.25ug of SHP1-WT over-expression plasmid and 1.25ug of PEF6/myc-His A empty plasmid, LANE4 was the cell transfected with 1.25ug of SHP1-DA over-expression plasmid and 1.25ug of PEF6/myc-His A empty plasmid, LANE5 was the cell transfected with 1.25ug of SHP1-WT and 1.25ug of SHP1-DA over-expression plasmid, and LANE5 was the cell transfected with 1.25ug of THEMIS-FLAG 1 and 1.25ug of SHP1-DA over-empty plasmid. As can be seen from FIG. 3, THEMIS does interact with SHP1 protein, and SHP1-DA binds THEMIS more strongly than SHP 1-WT.
Example 3
SHP1 as phosphatase dephosphorylates substrate, firstly detecting THEMIS can not be phosphorylated, and LCK can phosphorylate THEMIS according to the prior literature report. Thus, LCK as well as THEMIS-WT-FLAG were expressed by lipofection in HEK293T cells. The liposome (Mirus Trans-IT 2020) and the plasmid were mixed in Opti-MEM, left to stand at room temperature for 20min, and then slowly added dropwise to the cell culture medium, and then placed in an incubator at 37 ℃ and 5% carbon dioxide concentration for about 18-24 h. The liposome is added with the volume which is twice of the mass of the plasmid (namely 1ug of the plasmid is added into 2ul of the liposome), the liposome can wrap DNA molecules into the liposome and phosphate radicals of nucleic acid through electrostatic interaction to form a DNA-lipid complex, and can also be adsorbed by cell membranes with negative charges on the surface, and then through fusion of the membranes or endocytosis of the cells, the DNA is transferred into the cells to form inclusion bodies or enter lysosomes, wherein a small part of the DNA can be released from the inclusion bodies, enters cytoplasm, and further enters nucleus for transcription and expression. 24h after transfection, medium supernatant was discarded, 800ul of lysate (20mM Hepes pH 7.5; 150mM NaCl; 1% NP40) was added to lyse the cells, the cells were lysed on ice for 10min, DNA was removed by centrifugation at 4 ℃ and 15000rpm for 10min, the supernatant was collected, 200ul of the supernatant was taken as an INPUT sample to indicate transfection, the remaining 550ul was added with 8ul of FLAG beads (Sigma A2220-1ML) to enrich the THEMIS protein by immunoprecipitation, and phosphorylation level of tyrosine site of the THEMIS protein was detected by WB. Experiments show that LCK can indeed phosphorylate THEMIS, the specific results are shown in FIG. 4, wherein LANE1 is a control cell transfected with 2.5ug PEF6/myc-His A unloaded plasmid, LANE2 is a cell transfected with 1.25ug THEMIS-WT-FLAG over-expression plasmid and 1.25ug PEF6/myc-His A unloaded plasmid, LANE3 is a cell transfected with 1.25ug LCK over-expression plasmid and 1.25ug PEF6/myc-His A unloaded plasmid, and LANE4 is a cell transfected with 1.25ug LCK and 1.25ug THEMIS-WT-FLAG over-expression plasmid simultaneously.
Wherein the amino acid sequence of the LCK protein is as follows: MGCGCSSHPE DDWMENIDVC ENCHYPIVPL DGKGTLLIRN GSEVRDPLVT YEGSNPPASP LQDNLVIALH SYEPSHDGDL GFEKGEQLRI LEQSGEWWKA QSLTTGQEGF IPFNFVAKAN SLEPEPWFFK NLSRKDAERQ LLAPGNTHGS FLIRESESTA GSFSLSVRDF DQNQGEVVKH YKIRNLDNGG FYISPRITFP GLHELVRHYT NASDGLCTRL SRPCQTQKPQ KPWWEDEWEV PRETLKLVER LGAGQFGEVW MGYYNGHTKV AVKSLKQGSM SPDAFLAEAN LMKQLQHQRL VRLYAVVTQE PIYIITEYME NGSLVDFLKT PSGIKLTINK LLDMAAQIAE GMAFIEERNY IHRDLRAANI LVSDTLSCKI ADFGLARLIE DNEYTAREGA KFPIKWTAPE AINYGTFTIK SDVWSFGILL TEIVTHGRIP YPGMTNPEVI QNLERGYRMV RPDNCPEELY QLMRLCWKER PEDRPTFDYL RSVLEDFFTA TEGQYQPQP (SEQ ID NO.15)
Example 4
The THEMIS protein has 19 tyrosine sites in total, 19 tyrosine (Y) sites are respectively mutated into a plasmid with single point mutation of phenylalanine (F) by a point mutation method, and the 19 different chemis YF mutants are detected to change the phosphorylation degree. LCK (SEQ ID NO.15), THEMIS-WT-FLAG (SEQ ID NO.14) and THEMIS 19 YF mutants (Y in the sequence of SEQ ID NO.14 is changed to F, respectively) were also expressed in HEK293T cells by lipofection, liposomes (Mirus Trans-IT 2020) and plasmids were mixed in Opti-MEM (Gibco31985070), left to stand at room temperature for 20min and then slowly dropped into the cell culture medium, cultured in an incubator at 37 ℃ and 5% carbon dioxide concentration for 24h, then the medium supernatant was discarded, 800ul was added to 800ul of lysate (20mM Hepes pH 7.5; 150mM NaCl; 1% NP40) to lyse the cells, lysed on ice for 10min, centrifuged at 4 ℃ and 15000rpm for 10min to remove DNA, the supernatant was collected, 200ul of which was taken as an INPUT sample to indicate transfection, the remaining 200ul of FLAads (Sigma A0-1) was added to the sample to indicate transfection, and the remaining 550ul was enriched by the IMIS protein immunoprecipitation method, the tyrosine phosphorylation level of the THEMIS protein is detected by WB to evaluate whether the phosphorylation degree of the THEMIS protein is influenced by mutating one site, thereby finding out the site where the THEMIS is phosphorylated by LCK. The specific results are shown in FIG. 5, in which EV is the control transfected with the empty plasmid, THEMIS WT only is the cell transfected with the THEMIS-WT overexpression plasmid and the empty plasmid equivalent to LCK, and the rest is the cell transfected with both LCK and the corresponding single point mutant THEMIS overexpression plasmid. As can be seen from FIG. 5, THEMIS Tyr34 is critical for its own phosphorylation, and once Tyr at position 34 is mutated, THEMIS is hardly phosphorylated any more by LCK.
Example 5
7.5ug of THEMIS plasmid and 7.5ug of LCK plasmid were simultaneously expressed in 293T cells (10cm dish) by lipofection, liposomes (Mirus Trans-IT 2020) and plasmids were mixed in Opti-MEM, left to stand at room temperature for 20min and slowly added dropwise to the cell culture medium, incubated at 37 ℃ in an incubator with 5% carbon dioxide concentration for 24h, then the medium supernatant was discarded, 1000ul of lysate (20mM Hepes pH 7.5; 150mM NaCl; 1% NP40) was added to lyse the cells, lysed on ice for 10min, centrifuged at 4 ℃ 15000rpm for 10min to remove DNA, the supernatant was collected, and 20ul of FLAG beads (Sigma A2220-1ML) were added to enrich the THEMIS protein by immunoprecipitation. SHP1 has two states of off-state and on-state, and in general, N-SH2 domain of SHP1 binds to PTP domain, thereby inhibiting its phosphatase activity. In addition, the results of in vitro enzyme activity experiments showed that the phosphatase activity of SHP1-EA (E74A, i.e., the amino acid at position 74 in the aforementioned sequence of SHP-WT was changed from E to A) in the activated state was the highest as compared with WT. Therefore, in the in vitro assay, the dephosphorylation test was carried out using SHP1-EA and inactivated SHP1-CS (C453S, i.e., the 453 th amino acid in the sequence of SHP-WT mentioned above is changed from C to S), FALG beads enriched in phosphorylated THEMIS protein were divided into 12 equal portions, reacted with purified SHP1-EA or SHP1-CS protein at 30 ℃ for 1h, and the amounts of added proteins were, in order from left to right: 0.25 ug; 0.5 ug; 1 ug; 2ug, SHP1-EA corresponds to the amount of protein added from left to right in SHP 1-CS. The experiment was stopped by adding protein loading buffer (250mM Tris-HCl (pH6.8); 10% (W/V) SDS; 0.5% (W/V) bromophenol blue; 50% (V/V) glycerol; 5% (W/V) beta-mercaptoethanol) and the protein was denatured by boiling at 95 ℃ for 5min, and then the phosphorylation level of THEMIS was measured by WB. The specific results are shown in FIG. 6. As can be seen from FIG. 6, SHP1 did dephosphorylate THEMIS, and the degree of phosphorylation of THEMIS decreased continuously with increasing concentration of SHP 1. This also verifies the mass spectrum results obtained by the PEPSI system, THEMIS indeed a novel substrate for SHP 1.
Example 6
In vitro experiments confirmed that SHP1 could indeed dephosphorylate THEMIS, and further confirmed whether its specific site of action was Tyr 34. Artificially synthesizing a Tyr34 site phosphorylation modified peptide segment: EGSI (p-Y) EMFGNECCFS, which was reacted after solubilization (20ng/ul) with purified SHP1-EA or SHP1-CS proteins at 30 ℃ for 1h, with an amount of 360ng peptide fragment added per sample, the amounts of protein added being, in order from left to right: 0.5 ug; 1 ug; 2ug, SHP1-EA corresponds to the amount of protein added from left to right in SHP 1-CS. The experiment was stopped by adding a protein loading buffer containing no bromophenol blue, and the protein was denatured by boiling at 95 ℃ for 5min, the reaction was dropped onto a nitrocellulose membrane, air-dried, and then blocked with a 2.5% BSA solution, and the degree of phosphorylation of the peptide fragment of THEMIS was detected using 4G10(pTyr) antibody. The specific results are shown in FIG. 7. As can be seen from FIG. 7, SHP1-EA was able to dephosphorylate pY34 the thesis peptide.
Taken together, LCK is able to phosphorylate THEMIS and its primary site of action is THEMIS Tyr34, whereas the phosphorylation of THEMIS Tyr34 is able to be abolished by SHP 1.
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> Shanghai science and technology university
<120> a method for identifying a substrate for tyrosine phosphatase
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 595
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr
1 5 10 15
Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser
20 25 30
Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln
35 40 45
Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr
50 55 60
Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr
65 70 75 80
Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu
85 90 95
Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His
100 105 110
Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly
115 120 125
Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp
130 135 140
Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser
145 150 155 160
Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr
165 170 175
Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu
180 185 190
His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr
195 200 205
Leu Arg Gln Pro Tyr Tyr Ala Thr Arg Val Asn Ala Ala Asp Ile Glu
210 215 220
Asn Arg Val Leu Glu Leu Asn Lys Lys Gln Glu Ser Glu Asp Thr Ala
225 230 235 240
Lys Ala Gly Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu Val
245 250 255
Lys Asn Leu His Gln Arg Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly
260 265 270
Lys Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Ser Arg Val Ile
275 280 285
Leu Gln Gly Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala
290 295 300
Asn Tyr Ile Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr
305 310 315 320
Tyr Ile Ala Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp
325 330 335
Gln Met Ala Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg
340 345 350
Glu Val Glu Lys Gly Arg Asn Lys Cys Val Pro Tyr Trp Pro Glu Val
355 360 365
Gly Met Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu
370 375 380
His Asp Thr Thr Glu Tyr Lys Leu Arg Thr Leu Gln Val Ser Pro Leu
385 390 395 400
Asp Asn Gly Asp Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser
405 410 415
Trp Pro Asp His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe
420 425 430
Leu Asp Gln Ile Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly Pro
435 440 445
Ile Ile Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile
450 455 460
Val Ile Asp Met Leu Met Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys
465 470 475 480
Asp Ile Asp Ile Gln Lys Thr Ile Gln Met Val Arg Ala Gln Arg Ser
485 490 495
Gly Met Val Gln Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile
500 505 510
Ala Gln Phe Ile Glu Thr Thr Lys Lys Lys Leu Glu Val Leu Gln Ser
515 520 525
Gln Lys Gly Gln Glu Ser Glu Tyr Gly Asn Ile Thr Tyr Pro Pro Ala
530 535 540
Met Lys Asn Ala His Ala Lys Ala Ser Arg Thr Ser Ser Lys His Lys
545 550 555 560
Glu Asp Val Tyr Glu Asn Leu His Thr Lys Asn Lys Arg Glu Glu Lys
565 570 575
Val Lys Lys Gln Arg Ser Ala Asp Lys Glu Lys Ser Lys Gly Ser Leu
580 585 590
Lys Arg Lys
595
<210> 2
<211> 593
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Thr Ser Arg Arg Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala
1 5 10 15
Glu Asn Leu Leu Leu Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg
20 25 30
Pro Ser Lys Ser Asn Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn
35 40 45
Gly Ala Val Thr His Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp
50 55 60
Leu Tyr Gly Gly Glu Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr
65 70 75 80
Tyr Met Glu His His Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile
85 90 95
Glu Leu Lys Tyr Pro Leu Asn Cys Ala Asp Pro Thr Ser Glu Arg Trp
100 105 110
Phe His Gly His Leu Ser Gly Lys Glu Ala Glu Lys Leu Leu Thr Glu
115 120 125
Lys Gly Lys His Gly Ser Phe Leu Val Arg Glu Ser Gln Ser His Pro
130 135 140
Gly Asp Phe Val Leu Ser Val Arg Thr Gly Asp Asp Lys Gly Glu Ser
145 150 155 160
Asn Asp Gly Lys Ser Lys Val Thr His Val Met Ile Arg Cys Gln Glu
165 170 175
Leu Lys Tyr Asp Val Gly Gly Gly Glu Arg Phe Asp Ser Leu Thr Asp
180 185 190
Leu Val Glu His Tyr Lys Lys Asn Pro Met Val Glu Thr Leu Gly Thr
195 200 205
Val Leu Gln Leu Lys Gln Pro Leu Asn Thr Thr Arg Ile Asn Ala Ala
210 215 220
Glu Ile Glu Ser Arg Val Arg Glu Leu Ser Lys Leu Ala Glu Thr Thr
225 230 235 240
Asp Lys Val Lys Gln Gly Phe Trp Glu Glu Phe Glu Thr Leu Gln Gln
245 250 255
Gln Glu Cys Lys Leu Leu Tyr Ser Arg Lys Glu Gly Gln Arg Gln Glu
260 265 270
Asn Lys Asn Lys Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Thr
275 280 285
Arg Val Val Leu His Asp Gly Asp Pro Asn Glu Pro Val Ser Asp Tyr
290 295 300
Ile Asn Ala Asn Ile Ile Met Pro Glu Phe Glu Thr Lys Cys Asn Asn
305 310 315 320
Ser Lys Pro Lys Lys Ser Tyr Ile Ala Thr Gln Gly Cys Leu Gln Asn
325 330 335
Thr Val Asn Asp Phe Trp Arg Met Val Phe Gln Glu Asn Ser Arg Val
340 345 350
Ile Val Met Thr Thr Lys Glu Val Glu Arg Gly Lys Ser Lys Cys Val
355 360 365
Lys Tyr Trp Pro Asp Glu Tyr Ala Leu Lys Glu Tyr Gly Val Met Arg
370 375 380
Val Arg Asn Val Lys Glu Ser Ala Ala His Asp Tyr Thr Leu Arg Glu
385 390 395 400
Leu Lys Leu Ser Lys Val Gly Gln Gly Asn Thr Glu Arg Thr Val Trp
405 410 415
Gln Tyr His Phe Arg Thr Trp Pro Asp His Gly Val Pro Ser Asp Pro
420 425 430
Gly Gly Val Leu Asp Phe Leu Glu Glu Val His His Lys Gln Glu Ser
435 440 445
Ile Met Asp Ala Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly
450 455 460
Arg Thr Gly Thr Phe Ile Val Ile Asp Ile Leu Ile Asp Ile Ile Arg
465 470 475 480
Glu Lys Gly Val Asp Cys Asp Ile Asp Val Pro Lys Thr Ile Gln Met
485 490 495
Val Arg Ser Gln Arg Ser Gly Met Val Gln Thr Glu Ala Gln Tyr Arg
500 505 510
Phe Ile Tyr Met Ala Val Gln His Tyr Ile Glu Thr Leu Gln Arg Arg
515 520 525
Ile Glu Glu Glu Gln Lys Ser Lys Arg Lys Gly His Glu Tyr Thr Asn
530 535 540
Ile Lys Tyr Ser Leu Ala Asp Gln Thr Ser Gly Asp Gln Ser Pro Leu
545 550 555 560
Pro Pro Cys Thr Pro Thr Pro Pro Cys Ala Glu Met Arg Glu Asp Ser
565 570 575
Ala Arg Val Tyr Glu Asn Val Gly Leu Met Gln Gln Gln Lys Ser Phe
580 585 590
Arg
<210> 3
<211> 438
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser Gly Ser Trp
1 5 10 15
Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala Ser Asp Phe Pro Cys
20 25 30
Arg Val Ala Lys Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp
35 40 45
Val Ser Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn
50 55 60
Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser
65 70 75 80
Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp
85 90 95
Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg
100 105 110
Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys
115 120 125
Glu Glu Lys Glu Met Ile Phe Glu Asp Thr Asn Leu Lys Leu Thr Leu
130 135 140
Ile Ser Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu
145 150 155 160
Glu Asn Leu Thr Thr Gln Glu Thr Arg Glu Ile Leu His Phe His Tyr
165 170 175
Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu
180 185 190
Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His
195 200 205
Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr
210 215 220
Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp
225 230 235 240
Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Lys Phe
245 250 255
Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr Leu
260 265 270
Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln
275 280 285
Asp Gln Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu
290 295 300
His Ile Pro Pro Pro Pro Arg Pro Pro Lys Arg Ile Leu Glu Pro His
305 310 315 320
Asn Gly Lys Cys Arg Glu Phe Phe Pro Asn His Gln Trp Val Lys Glu
325 330 335
Glu Thr Gln Glu Asp Lys Asp Cys Pro Ile Lys Glu Glu Lys Gly Ser
340 345 350
Pro Leu Asn Ala Ala Pro Tyr Gly Ile Glu Ser Met Ser Gln Asp Thr
355 360 365
Glu Val Arg Ser Arg Val Val Gly Gly Ser Leu Arg Gly Ala Gln Ala
370 375 380
Ala Ser Pro Ala Lys Gly Glu Pro Ser Leu Pro Glu Lys Asp Glu Asp
385 390 395 400
His Ala Leu Ser Tyr Trp Lys Pro Phe Leu Val Asn Met Cys Val Ala
405 410 415
Thr Val Leu Thr Ala Gly Ala Tyr Leu Cys Tyr Arg Phe Leu Phe Asn
420 425 430
Ser Asn Thr Arg Trp Ala
435
<210> 4
<211> 482
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ser Thr Val Glu Ser Ala Leu Thr Arg Arg Ile Met Gly Ile Glu
1 5 10 15
Thr Glu Tyr Gly Leu Thr Phe Val Asp Gly Asp Ser Lys Lys Leu Arg
20 25 30
Pro Asp Glu Ile Ala Arg Arg Met Phe Arg Pro Ile Val Glu Lys Tyr
35 40 45
Ser Ser Ser Asn Ile Phe Ile Pro Asn Gly Ser Arg Leu Tyr Leu Asp
50 55 60
Val Gly Ser His Pro Glu Tyr Ala Thr Ala Glu Cys Asp Asn Leu Thr
65 70 75 80
Gln Leu Ile Asn Phe Glu Lys Ala Gly Asp Val Ile Ala Asp Arg Met
85 90 95
Ala Val Asp Ala Glu Glu Ser Leu Ala Lys Glu Asp Ile Ala Gly Gln
100 105 110
Val Tyr Leu Phe Lys Asn Asn Val Asp Ser Val Gly Asn Ser Tyr Gly
115 120 125
Cys His Glu Asn Tyr Leu Val Gly Arg Ser Met Pro Leu Lys Ala Leu
130 135 140
Gly Lys Arg Leu Met Pro Phe Leu Ile Thr Arg Gln Leu Ile Cys Gly
145 150 155 160
Ala Gly Arg Ile His His Pro Asn Pro Leu Asp Lys Gly Glu Ser Phe
165 170 175
Pro Leu Gly Tyr Cys Ile Ser Gln Arg Ser Asp His Val Trp Glu Gly
180 185 190
Val Ser Ser Ala Thr Thr Arg Ser Arg Pro Ile Ile Asn Thr Arg Asp
195 200 205
Glu Pro His Ala Asp Ser His Ser Tyr Arg Arg Leu His Val Ile Val
210 215 220
Gly Asp Ala Asn Met Ala Glu Pro Ser Ile Ala Leu Lys Val Gly Ser
225 230 235 240
Thr Leu Leu Val Leu Glu Met Ile Glu Ala Asp Phe Gly Leu Pro Ser
245 250 255
Leu Glu Leu Ala Asn Asp Ile Ala Ser Ile Arg Glu Ile Ser Arg Asp
260 265 270
Ala Thr Gly Ser Thr Leu Leu Ser Leu Lys Asp Gly Thr Thr Met Thr
275 280 285
Ala Leu Gln Ile Gln Gln Val Val Phe Glu His Ala Ser Lys Trp Leu
290 295 300
Glu Gln Arg Pro Glu Pro Glu Phe Ser Gly Thr Ser Asn Thr Glu Met
305 310 315 320
Ala Arg Val Leu Asp Leu Trp Gly Arg Met Leu Lys Ala Ile Glu Ser
325 330 335
Gly Asp Phe Ser Glu Val Asp Thr Glu Ile Asp Trp Val Ile Lys Lys
340 345 350
Lys Leu Ile Asp Arg Phe Ile Gln Arg Gly Asn Leu Gly Leu Asp Asp
355 360 365
Pro Lys Leu Ala Gln Val Asp Leu Thr Tyr His Asp Ile Arg Pro Gly
370 375 380
Arg Gly Leu Phe Ser Val Leu Gln Ser Arg Gly Met Ile Lys Arg Trp
385 390 395 400
Thr Thr Asp Glu Ala Ile Leu Ala Ala Val Asp Thr Ala Pro Asp Thr
405 410 415
Thr Arg Ala His Leu Arg Gly Arg Ile Leu Lys Ala Ala Asp Thr Leu
420 425 430
Gly Val Pro Val Thr Val Asp Trp Met Arg His Lys Val Asn Arg Pro
435 440 445
Glu Pro Gln Ser Val Glu Leu Gly Asp Pro Phe Ser Ala Val Asn Ser
450 455 460
Glu Val Asp Gln Leu Ile Glu Tyr Met Thr Val His Ala Glu Ser Tyr
465 470 475 480
Arg Ser
<210> 5
<211> 64
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Asn Ala Lys Gln Thr Gln Ile Met Gly Gly Gly Gly Arg Asp Glu
1 5 10 15
Asp Asn Ala Glu Asp Ser Ala Gln Ala Ser Gly Gln Val Gln Ile Asn
20 25 30
Thr Glu Gly Val Asp Ser Leu Leu Asp Glu Ile Asp Gly Leu Leu Glu
35 40 45
Asn Asn Ala Glu Glu Phe Val Arg Ser Tyr Val Gln Lys Gly Gly Glu
50 55 60
<210> 6
<211> 84
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ala Gly Lys Ala Gly Glu Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr
1 5 10 15
Val Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln
20 25 30
Thr Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala
35 40 45
Pro Thr Asp Gly Lys Val Glu Lys Val Leu Val Lys Glu Arg Asp Ala
50 55 60
Val Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly Asp Tyr Asp Ile Pro
65 70 75 80
Thr Thr Ala Ser
<210> 7
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 8
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 9
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 10
<211> 450
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggccggga aggcaggcga gggagagatc cccgcaccct tggccggcac ggtcagcaaa 60
atcctggtca aggaaggcga caccgtgaag gctggacaga cggtgttggt actggaggcg 120
atgaagatgg agacagagat caatgccccg accgatggga aggtggagaa ggtgttggtt 180
aaggaaaggg acgccgtgca gggcggtcag ggactgatca agatcggcga ctacgacatc 240
ccgacaaccg ccagcatgaa cgcgaaacag acccagatca tgggtggcgg tggtcgtgac 300
gaagacaatg cggaagactc tgctcaggcg tctggtcagg ttcagatcaa taccgaaggt 360
gttgactctc tgctggacga aatcgacggc ctgctcgaaa acaacgcgga ggaattcgtt 420
cgttcttacg ttcagaaagg tggtgaataa 450
<210> 11
<211> 1112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr
1 5 10 15
Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser
20 25 30
Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln
35 40 45
Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr
50 55 60
Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr
65 70 75 80
Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu
85 90 95
Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp Tyr His
100 105 110
Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly
115 120 125
Glu Pro Trp Thr Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp
130 135 140
Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser
145 150 155 160
Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr
165 170 175
Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu
180 185 190
His Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr
195 200 205
Leu Arg Gln Pro Tyr Tyr Ala Thr Arg Val Asn Ala Ala Asp Ile Glu
210 215 220
Asn Arg Val Leu Glu Leu Asn Lys Lys Gln Glu Ser Glu Asp Thr Ala
225 230 235 240
Lys Ala Gly Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu Val
245 250 255
Lys Asn Leu His Gln Arg Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly
260 265 270
Lys Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Ser Arg Val Ile
275 280 285
Leu Gln Gly Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala
290 295 300
Asn Tyr Ile Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr
305 310 315 320
Tyr Ile Ala Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp
325 330 335
Gln Met Ala Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg
340 345 350
Glu Val Glu Lys Gly Arg Asn Lys Cys Val Pro Tyr Trp Pro Glu Val
355 360 365
Gly Met Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu
370 375 380
His Asp Thr Thr Glu Tyr Lys Leu Arg Thr Leu Gln Val Ser Pro Leu
385 390 395 400
Asp Asn Gly Asp Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser
405 410 415
Trp Pro Asp His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe
420 425 430
Leu Asp Gln Ile Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly Pro
435 440 445
Ile Ile Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile
450 455 460
Val Ile Asp Met Leu Met Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys
465 470 475 480
Asp Ile Asp Ile Gln Lys Thr Ile Gln Met Val Arg Ala Gln Arg Ser
485 490 495
Gly Met Val Gln Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Ala Ile
500 505 510
Ala Gln Phe Ile Glu Thr Thr Lys Lys Lys Leu Glu Val Leu Gln Ser
515 520 525
Gln Lys Gly Gln Glu Ser Glu Tyr Gly Asn Ile Thr Tyr Pro Pro Ala
530 535 540
Met Lys Asn Ala His Ala Lys Ala Ser Arg Thr Ser Ser Lys His Lys
545 550 555 560
Glu Asp Val Tyr Glu Asn Leu His Thr Lys Asn Lys Arg Glu Glu Lys
565 570 575
Val Lys Lys Gln Arg Ser Ala Asp Lys Glu Lys Ser Lys Gly Ser Leu
580 585 590
Lys Arg Lys Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
595 600 605
Ser Gly Gly Gly Ser Gly Met Ser Thr Val Glu Ser Ala Leu Thr Arg
610 615 620
Arg Ile Met Gly Ile Glu Thr Glu Tyr Gly Leu Thr Phe Val Asp Gly
625 630 635 640
Asp Ser Lys Lys Leu Arg Pro Asp Glu Ile Ala Arg Arg Met Phe Arg
645 650 655
Pro Ile Val Glu Lys Tyr Ser Ser Ser Asn Ile Phe Ile Pro Asn Gly
660 665 670
Ser Arg Leu Tyr Leu Asp Val Gly Ser His Pro Glu Tyr Ala Thr Ala
675 680 685
Glu Cys Asp Asn Leu Thr Gln Leu Ile Asn Phe Glu Lys Ala Gly Asp
690 695 700
Val Ile Ala Asp Arg Met Ala Val Asp Ala Glu Glu Ser Leu Ala Lys
705 710 715 720
Glu Asp Ile Ala Gly Gln Val Tyr Leu Phe Lys Asn Asn Val Asp Ser
725 730 735
Val Gly Asn Ser Tyr Gly Cys His Glu Asn Tyr Leu Val Gly Arg Ser
740 745 750
Met Pro Leu Lys Ala Leu Gly Lys Arg Leu Met Pro Phe Leu Ile Thr
755 760 765
Arg Gln Leu Ile Cys Gly Ala Gly Arg Ile His His Pro Asn Pro Leu
770 775 780
Asp Lys Gly Glu Ser Phe Pro Leu Gly Tyr Cys Ile Ser Gln Arg Ser
785 790 795 800
Asp His Val Trp Glu Gly Val Ser Ser Ala Thr Thr Arg Ser Arg Pro
805 810 815
Ile Ile Asn Thr Arg Asp Glu Pro His Ala Asp Ser His Ser Tyr Arg
820 825 830
Arg Leu His Val Ile Val Gly Asp Ala Asn Met Ala Glu Pro Ser Ile
835 840 845
Ala Leu Lys Val Gly Ser Thr Leu Leu Val Leu Glu Met Ile Glu Ala
850 855 860
Asp Phe Gly Leu Pro Ser Leu Glu Leu Ala Asn Asp Ile Ala Ser Ile
865 870 875 880
Arg Glu Ile Ser Arg Asp Ala Thr Gly Ser Thr Leu Leu Ser Leu Lys
885 890 895
Asp Gly Thr Thr Met Thr Ala Leu Gln Ile Gln Gln Val Val Phe Glu
900 905 910
His Ala Ser Lys Trp Leu Glu Gln Arg Pro Glu Pro Glu Phe Ser Gly
915 920 925
Thr Ser Asn Thr Glu Met Ala Arg Val Leu Asp Leu Trp Gly Arg Met
930 935 940
Leu Lys Ala Ile Glu Ser Gly Asp Phe Ser Glu Val Asp Thr Glu Ile
945 950 955 960
Asp Trp Val Ile Lys Lys Lys Leu Ile Asp Arg Phe Ile Gln Arg Gly
965 970 975
Asn Leu Gly Leu Asp Asp Pro Lys Leu Ala Gln Val Asp Leu Thr Tyr
980 985 990
His Asp Ile Arg Pro Gly Arg Gly Leu Phe Ser Val Leu Gln Ser Arg
995 1000 1005
Gly Met Ile Lys Arg Trp Thr Thr Asp Glu Ala Ile Leu Ala Ala Val
1010 1015 1020
Asp Thr Ala Pro Asp Thr Thr Arg Ala His Leu Arg Gly Arg Ile Leu
1025 1030 1035 1040
Lys Ala Ala Asp Thr Leu Gly Val Pro Val Thr Val Asp Trp Met Arg
1045 1050 1055
His Lys Val Asn Arg Pro Glu Pro Gln Ser Val Glu Leu Gly Asp Pro
1060 1065 1070
Phe Ser Ala Val Asn Ser Glu Val Asp Gln Leu Ile Glu Tyr Met Thr
1075 1080 1085
Val His Ala Glu Ser Tyr Arg Ser Glu Gln Lys Leu Ile Ser Glu Glu
1090 1095 1100
Asp Leu His His His His His His
1105 1110
<210> 12
<211> 1785
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atggtgaggt ggtttcaccg agacctcagt gggctggatg cagagaccct gctcaagggc 60
cgaggtgtcc acggtagctt cctggctcgg cccagtcgca agaaccaggg tgacttctcg 120
ctctccgtca gggtggggga tcaggtgacc catattcgga tccagaactc aggggatttc 180
tatgacctgt atggagggga gaagtttgcg actctgacag agctggtgga gtactacact 240
cagcagcagg gtgtcctgca ggaccgcgac ggcaccatca tccacctcaa gtacccgctg 300
aactgctccg atcccactag tgagaggtgg taccatggcc acatgtctgg cgggcaggca 360
gagacgctgc tgcaggccaa gggcgagccc tggacgtttc ttgtgcgtga gagcctcagc 420
cagcctggag acttcgtgct ttctgtgctc agtgaccagc ccaaggctgg cccaggctcc 480
ccgctcaggg tcacccacat caaggtcatg tgcgagggtg gacgctacac agtgggtggt 540
ttggagacct tcgacagcct cacggacctg gtggagcatt tcaagaagac ggggattgag 600
gaggcctcag gcgcctttgt ctacctgcgg cagccgtact atgccacgag ggtgaatgcg 660
gctgacattg agaaccgagt gttggaactg aacaagaagc aggagtccga ggatacagcc 720
aaggctggct tctgggagga gtttgagagt ttgcagaagc aggaggtgaa gaacttgcac 780
cagcgtctgg aagggcagcg gccagagaac aagggcaaga accgctacaa gaacattctc 840
ccctttgacc acagccgagt gatcctgcag ggacgggaca gtaacatccc cgggtccgac 900
tacatcaatg ccaactacat caagaaccag ctgctaggcc ctgatgagaa cgctaagacc 960
tacatcgcca gccagggctg tctggaggcc acggtcaatg acttctggca gatggcgtgg 1020
caggagaaca gccgtgtcat cgtcatgacc acccgagagg tggagaaagg ccggaacaaa 1080
tgcgtcccat actggcccga ggtgggcatg cagcgtgctt atgggcccta ctctgtgacc 1140
aactgcgggg agcatgacac aaccgaatac aaactccgta ccttacaggt ctccccgctg 1200
gacaatggag acctgattcg ggagatctgg cattaccagt acctgagctg gcccgaccat 1260
ggggtcccca gtgagcctgg gggtgtcctc agcttcctgg accagatcaa ccagcggcag 1320
gaaagtctgc ctcacgcagg gcccatcatc gtgcactgca gcgccggcat cggccgcaca 1380
ggcaccatca ttgtcatcga catgctcatg gagaacatct ccaccaaggg cctggactgt 1440
gacattgaca tccagaagac catccagatg gtgcgggcgc agcgctcggg catggtgcag 1500
acggaggcgc agtacaagtt catctacgtg gccatcgccc agttcattga aaccactaag 1560
aagaagctgg aggtcctgca gtcgcagaag ggccaggagt cggagtacgg gaacatcacc 1620
tatcccccag ccatgaagaa tgcccatgcc aaggcctccc gcacctcgtc caaacacaag 1680
gaggatgtgt atgagaacct gcacactaag aacaagaggg aggagaaagt gaagaagcag 1740
cggtcagcag acaaggagaa gagcaagggt tccctcaaga ggaag 1785
<210> 13
<211> 1785
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atggtgaggt ggtttcaccg agacctcagt gggctggatg cagagaccct gctcaagggc 60
cgaggtgtcc acggtagctt cctggctcgg cccagtcgca agaaccaggg tgacttctcg 120
ctctccgtca gggtggggga tcaggtgacc catattcgga tccagaactc aggggatttc 180
tatgacctgt atggagggga gaagtttgcg actctgacag agctggtgga gtactacact 240
cagcagcagg gtgtcctgca ggaccgcgac ggcaccatca tccacctcaa gtacccgctg 300
aactgctccg atcccactag tgagaggtgg taccatggcc acatgtctgg cgggcaggca 360
gagacgctgc tgcaggccaa gggcgagccc tggacgtttc ttgtgcgtga gagcctcagc 420
cagcctggag acttcgtgct ttctgtgctc agtgaccagc ccaaggctgg cccaggctcc 480
ccgctcaggg tcacccacat caaggtcatg tgcgagggtg gacgctacac agtgggtggt 540
ttggagacct tcgacagcct cacggacctg gtggagcatt tcaagaagac ggggattgag 600
gaggcctcag gcgcctttgt ctacctgcgg cagccgtact atgccacgag ggtgaatgcg 660
gctgacattg agaaccgagt gttggaactg aacaagaagc aggagtccga ggatacagcc 720
aaggctggct tctgggagga gtttgagagt ttgcagaagc aggaggtgaa gaacttgcac 780
cagcgtctgg aagggcagcg gccagagaac aagggcaaga accgctacaa gaacattctc 840
ccctttgacc acagccgagt gatcctgcag ggacgggaca gtaacatccc cgggtccgac 900
tacatcaatg ccaactacat caagaaccag ctgctaggcc ctgatgagaa cgctaagacc 960
tacatcgcca gccagggctg tctggaggcc acggtcaatg acttctggca gatggcgtgg 1020
caggagaaca gccgtgtcat cgtcatgacc acccgagagg tggagaaagg ccggaacaaa 1080
tgcgtcccat actggcccga ggtgggcatg cagcgtgctt atgggcccta ctctgtgacc 1140
aactgcgggg agcatgacac aaccgaatac aaactccgta ccttacaggt ctccccgctg 1200
gacaatggag acctgattcg ggagatctgg cattaccagt acctgagctg gcccgcccat 1260
ggggtcccca gtgagcctgg gggtgtcctc agcttcctgg accagatcaa ccagcggcag 1320
gaaagtctgc ctcacgcagg gcccatcatc gtgcactgca gcgccggcat cggccgcaca 1380
ggcaccatca ttgtcatcga catgctcatg gagaacatct ccaccaaggg cctggactgt 1440
gacattgaca tccagaagac catccagatg gtgcgggcgc agcgctcggg catggtgcag 1500
acggaggcgc agtacaagtt catctacgtg gccatcgccc agttcattga aaccactaag 1560
aagaagctgg aggtcctgca gtcgcagaag ggccaggagt cggagtacgg gaacatcacc 1620
tatcccccag ccatgaagaa tgcccatgcc aaggcctccc gcacctcgtc caaacacaag 1680
gaggatgtgt atgagaacct gcacactaag aacaagaggg aggagaaagt gaagaagcag 1740
cggtcagcag acaaggagaa gagcaagggt tccctcaaga ggaag 1785
<210> 14
<211> 660
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Met Ala Leu Ser Leu Glu Glu Phe Val His Ser Leu Asp Leu Arg Thr
1 5 10 15
Leu Pro Arg Val Leu Glu Ile Gln Ala Gly Ile Tyr Leu Glu Gly Ser
20 25 30
Ile Tyr Glu Met Phe Gly Asn Glu Cys Cys Phe Ser Thr Gly Glu Val
35 40 45
Ile Lys Ile Thr Gly Leu Lys Val Lys Lys Ile Ile Ala Glu Ile Cys
50 55 60
Glu Gln Ile Glu Gly Cys Glu Ser Leu Gln Pro Phe Glu Leu Pro Met
65 70 75 80
Asn Phe Pro Gly Leu Phe Lys Ile Val Ala Asp Lys Thr Pro Tyr Leu
85 90 95
Thr Met Glu Glu Ile Thr Arg Thr Ile His Ile Gly Pro Ser Arg Leu
100 105 110
Gly His Pro Cys Phe Tyr His Gln Lys Asp Ile Lys Leu Glu Asn Leu
115 120 125
Ile Ile Lys Gln Gly Glu Gln Ile Met Leu Asn Ser Val Glu Glu Ile
130 135 140
Asp Gly Glu Ile Met Val Ser Cys Ala Val Ala Arg Asn His Gln Thr
145 150 155 160
His Ser Phe Asn Leu Pro Leu Ser Gln Glu Gly Glu Phe Tyr Glu Cys
165 170 175
Glu Asp Glu Arg Ile Tyr Thr Leu Lys Glu Ile Val Glu Trp Lys Ile
180 185 190
Pro Lys Asn Arg Thr Arg Thr Val Asn Leu Thr Asp Phe Ser Asn Lys
195 200 205
Trp Asp Ser Thr Asn Pro Phe Pro Lys Asp Phe Tyr Gly Thr Leu Ile
210 215 220
Leu Lys Pro Val Tyr Glu Ile Gln Gly Val Met Lys Phe Arg Lys Asp
225 230 235 240
Ile Ile Arg Ile Leu Pro Ser Leu Asp Val Glu Val Lys Asp Ile Thr
245 250 255
Asp Ser Tyr Asp Ala Asn Trp Phe Leu Gln Leu Leu Ser Thr Glu Asp
260 265 270
Leu Phe Glu Met Thr Ser Lys Glu Phe Pro Ile Val Thr Glu Val Ile
275 280 285
Glu Ala Pro Glu Gly Asn His Leu Pro Gln Ser Ile Leu Gln Pro Gly
290 295 300
Lys Thr Ile Val Ile His Lys Lys Tyr Gln Ala Ser Arg Ile Leu Ala
305 310 315 320
Ser Glu Ile Arg Ser Asn Phe Pro Lys Arg His Phe Leu Ile Pro Thr
325 330 335
Ser Tyr Lys Gly Lys Phe Lys Arg Arg Pro Arg Glu Phe Pro Thr Ala
340 345 350
Tyr Asp Leu Glu Ile Ala Lys Ser Glu Lys Glu Pro Leu His Val Val
355 360 365
Ala Thr Lys Ala Phe His Ser Pro His Asp Lys Leu Ser Ser Val Ser
370 375 380
Val Gly Asp Gln Phe Leu Val His Gln Ser Glu Thr Thr Glu Val Leu
385 390 395 400
Cys Glu Gly Ile Lys Lys Val Val Asn Val Leu Ala Cys Glu Lys Ile
405 410 415
Leu Lys Lys Ser Tyr Glu Ala Ala Leu Leu Pro Leu Tyr Met Glu Gly
420 425 430
Gly Phe Val Glu Val Ile His Asp Lys Lys Gln Tyr Pro Ile Ser Glu
435 440 445
Leu Cys Lys Gln Phe Arg Leu Pro Phe Asn Val Lys Val Ser Val Arg
450 455 460
Asp Leu Ser Ile Glu Glu Asp Val Leu Ala Ala Thr Pro Gly Leu Gln
465 470 475 480
Leu Glu Glu Asp Ile Thr Asp Ser Tyr Leu Leu Ile Ser Asp Phe Ala
485 490 495
Asn Pro Thr Glu Cys Trp Glu Ile Pro Val Gly Arg Leu Asn Met Thr
500 505 510
Val Gln Leu Val Ser Asn Phe Ser Arg Asp Ala Glu Pro Phe Leu Val
515 520 525
Arg Thr Leu Val Glu Glu Ile Thr Glu Glu Gln Tyr Tyr Met Met Arg
530 535 540
Arg Tyr Glu Ser Ser Ala Ser His Pro Pro Pro Arg Pro Pro Lys His
545 550 555 560
Pro Ser Val Glu Glu Thr Lys Leu Thr Leu Leu Thr Leu Ala Glu Glu
565 570 575
Arg Thr Val Asp Leu Pro Lys Ser Pro Lys Arg His His Val Asp Ile
580 585 590
Thr Lys Lys Leu His Pro Asn Gln Ala Gly Leu Asp Ser Lys Val Leu
595 600 605
Ile Gly Ser Gln Asn Asp Leu Val Asp Glu Glu Lys Glu Arg Ser Asn
610 615 620
Arg Gly Ala Thr Ala Ile Ala Glu Thr Phe Lys Asn Glu Lys His Gln
625 630 635 640
Lys Pro Gly Leu Glu Pro Trp Lys Leu Met Asp Tyr Lys Asp Asp Asp
645 650 655
Asp Lys Asp Ile
660
<210> 15
<211> 509
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Gly Cys Gly Cys Ser Ser His Pro Glu Asp Asp Trp Met Glu Asn
1 5 10 15
Ile Asp Val Cys Glu Asn Cys His Tyr Pro Ile Val Pro Leu Asp Gly
20 25 30
Lys Gly Thr Leu Leu Ile Arg Asn Gly Ser Glu Val Arg Asp Pro Leu
35 40 45
Val Thr Tyr Glu Gly Ser Asn Pro Pro Ala Ser Pro Leu Gln Asp Asn
50 55 60
Leu Val Ile Ala Leu His Ser Tyr Glu Pro Ser His Asp Gly Asp Leu
65 70 75 80
Gly Phe Glu Lys Gly Glu Gln Leu Arg Ile Leu Glu Gln Ser Gly Glu
85 90 95
Trp Trp Lys Ala Gln Ser Leu Thr Thr Gly Gln Glu Gly Phe Ile Pro
100 105 110
Phe Asn Phe Val Ala Lys Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe
115 120 125
Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro
130 135 140
Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala
145 150 155 160
Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu
165 170 175
Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr
180 185 190
Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His
195 200 205
Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys
210 215 220
Gln Thr Gln Lys Pro Gln Lys Pro Trp Trp Glu Asp Glu Trp Glu Val
225 230 235 240
Pro Arg Glu Thr Leu Lys Leu Val Glu Arg Leu Gly Ala Gly Gln Phe
245 250 255
Gly Glu Val Trp Met Gly Tyr Tyr Asn Gly His Thr Lys Val Ala Val
260 265 270
Lys Ser Leu Lys Gln Gly Ser Met Ser Pro Asp Ala Phe Leu Ala Glu
275 280 285
Ala Asn Leu Met Lys Gln Leu Gln His Gln Arg Leu Val Arg Leu Tyr
290 295 300
Ala Val Val Thr Gln Glu Pro Ile Tyr Ile Ile Thr Glu Tyr Met Glu
305 310 315 320
Asn Gly Ser Leu Val Asp Phe Leu Lys Thr Pro Ser Gly Ile Lys Leu
325 330 335
Thr Ile Asn Lys Leu Leu Asp Met Ala Ala Gln Ile Ala Glu Gly Met
340 345 350
Ala Phe Ile Glu Glu Arg Asn Tyr Ile His Arg Asp Leu Arg Ala Ala
355 360 365
Asn Ile Leu Val Ser Asp Thr Leu Ser Cys Lys Ile Ala Asp Phe Gly
370 375 380
Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Glu Gly Ala
385 390 395 400
Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ile Asn Tyr Gly Thr
405 410 415
Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu
420 425 430
Ile Val Thr His Gly Arg Ile Pro Tyr Pro Gly Met Thr Asn Pro Glu
435 440 445
Val Ile Gln Asn Leu Glu Arg Gly Tyr Arg Met Val Arg Pro Asp Asn
450 455 460
Cys Pro Glu Glu Leu Tyr Gln Leu Met Arg Leu Cys Trp Lys Glu Arg
465 470 475 480
Pro Glu Asp Arg Pro Thr Phe Asp Tyr Leu Arg Ser Val Leu Glu Asp
485 490 495
Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro Gln Pro
500 505

Claims (10)

1. A method of identifying a tyrosine phosphatase substrate comprising:
1) placing the system comprising the potential substrate for interaction with tyrosine phosphatase in the presence of a tyrosine phosphatase-PafA fusion protein, a pup protein, and labeling the pup protein;
2) enriching potential substrates for interaction with tyrosine phosphatases by the labeled pup protein;
the tyrosine phosphatase-PafA fusion protein comprises a tyrosine phosphatase fragment and a PafA fragment, wherein the tyrosine phosphatase fragment has a substrate capture mutant.
2. The method for identifying a tyrosine phosphatase substrate according to claim 1, wherein the tyrosine phosphatase fragment is selected from the group consisting of a SHP1 fragment, a SHP2 fragment, a PTP1B fragment, a TCPTP fragment, a PTPRK fragment, and a CD45 fragment.
3. The method for identifying a substrate for tyrosine phosphatase as claimed in claim 1, wherein in step 1), the system comprising the substrate potentially interacting with tyrosine phosphatase is the target cell, and the step 1) comprises: the target cells were cultured in the presence of the tyrosine phosphatase-PafA fusion protein, the pup protein, and labeled with the pup protein.
4. A method of identifying a substrate for tyrosine phosphatase as claimed in claim 3, wherein said step 1) further comprises; the cells obtained from the culture are lysed to provide a lysate.
5. A method of identifying a tyrosine phosphatase substrate according to claim 3, wherein the tyrosine phosphatase-PafA fusion protein and/or the pup protein is expressed by the target cell.
6. The method for identifying a substrate for tyrosine phosphatase according to claim 1, wherein in the step 1), the pup protein comprises a pup fragment and a tag protein fragment, and the tag protein is selected from the group consisting of a biotin tag protein;
and/or, labeling the pup protein by biotin;
and/or, in the step 2), the substrates potentially interacting with the target protein are enriched by the labeled pup protein based on the biotin-avidin system.
7. A tyrosine phosphatase substrate identification system comprising a combination of a tyrosine phosphatase-PafA fusion protein comprising a tyrosine phosphatase fragment and a PafA fragment, said tyrosine phosphatase fragment having a substrate capture mutation, and a pup protein.
8. A tyrosine phosphatase substrate identification system according to claim 7 further comprising a system of potential substrates for interaction with tyrosine phosphatases.
9. The substrate identification system for tyrosine phosphatase according to claim 7, further comprising a marker for labeling the pup protein.
10. An expression system comprising a construct or genome of polynucleotides encoding tyrosine phosphatase-PafA fusion protein and pup protein having exogenous polynucleotides encoding tyrosine phosphatase-PafA fusion protein and pup protein integrated therein.
CN202010573873.7A 2020-06-22 2020-06-22 Tyrosine phosphatase substrate identification method Pending CN113899902A (en)

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