CN114524871A - Variant SH2 domains with high affinity for tyrosine-containing sulfated-modified peptides - Google Patents

Variant SH2 domains with high affinity for tyrosine-containing sulfated-modified peptides Download PDF

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CN114524871A
CN114524871A CN202210108803.3A CN202210108803A CN114524871A CN 114524871 A CN114524871 A CN 114524871A CN 202210108803 A CN202210108803 A CN 202210108803A CN 114524871 A CN114524871 A CN 114524871A
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李磊
赵东平
周长景
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Qingdao Cancer Research Institute
Qingdao Sanuo Gene Technology Co ltd
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Abstract

The present invention discloses variant SH2 domains with high affinity for peptides containing tyrosine sulfation modifications. The invention obtains a variant SH2 structural domain with enhanced combination affinity to the peptide containing sTyr by introducing single or multiple amino acid residue substitution to a specific region of a parent SH2 structural domain, and the amino acid sequence of the variant SH2 structural domain is shown in any one of SEQ ID NO. 3-7. The variant SH2 structural domain can effectively enrich and detect the peptide containing the sTyr, and can also be used for detecting the concentration of the peptide containing the sTyr in a sample, or detecting the activity of tyrosine sulfotransferase by detecting the sulfation degree of protein in the sample, and the like. In addition, the variant SH2 domain can also be used as a substitute for anti-sTyr antibodies for studies using anti-sTyr antibodies, such as western blotting, immunofluorescence, proteomics, enrichment of sulfate proteins/peptides, and the like.

Description

Variant SH2 domains with high affinity for tyrosine-containing sulfated-modified peptides
Technical Field
The invention belongs to the technical field of protein tyrosine sulfation detection. More specifically, it relates to variant SH2 domains with high affinity for peptides containing tyrosine sulfation modifications.
Background
Tyrosine protein sulfation of proteins catalyzed by Tyrosine Protein Sulfatase (TPST) is a common type of post-translational modification that acts on secreted and membrane proteins. Many proteins are tyrosine sulfated before being recognized and thus involved in protein-protein interactions. TPST and tyrosine sulfated proteins are involved in many physiological processes, such as viral invasion, inflammation, coagulation and infertility, and are closely related to a variety of important biological activities. Tyrosine-containing sulfated proteins can also affect the affinity of cellular receptors for interacting with the corresponding ligands, thereby causing oxidative stress damage or autoimmune disease in the body. Therefore, the detection and enrichment of proteins/peptides containing tyrosine sulfation modifications are of great interest in a number of research efforts.
However, due to the low abundance of protein tyrosine sulfation in organisms and the few kinds of modified amino acid residues, and the lack of tools for effectively enriching Sulfated tyrosine (sTyr), the number of sTyr-containing polypeptides or proteins and sTyr sites reported so far is limited (Panlu et al. The Anti-sTyr antibody has the ability to specifically bind to sTyr, but has a weak affinity for sTyr. Therefore, a product or a method which has high affinity to sTyr and can effectively enrich polypeptide or protein of sTyr is developed, and the method has important significance for research of sTyr.
The Src homology 2 (SH 2) domain is the major element in the transmission of intracellular phosphorylation modification signals, as is the Fyn SH2 domain. The wild-type Fyn SH2 domain is capable of binding phosphotyrosine (pTyr), but not sTyr. Studies have shown that SH2-trm is used as a template and a phage display technology is applied to target polypeptide containing sTyr, and mutants SH2-58.6, SH2-1.8 and SH2-3.1 which can bind to sTyr are developed (Lawrie, et al. ACS Chem biol.2021,16(8): 1508-. However, the affinity of these mutants to the sTyr peptide is still weak, and the Kd value of the strongest mutant SH2-58.6 is only 188nM, which can not effectively enrich the sTyr-containing peptide.
Disclosure of Invention
The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art and to provide a variant SH2 domain with high affinity to tyrosine-containing sulfated-modified peptides.
It is a first object of the present invention to provide a variant SH2 domain with high affinity to tyrosine-containing sulfated-modified peptides.
The second object of the present invention is to provide a DNA sequence encoding the variant SH2 domain.
The third purpose of the invention is to provide a vector containing the DNA sequence.
The fourth purpose of the invention is to provide a polypeptide containing the variant SH2 domain.
The fifth object of the present invention is to provide the use of said variant SH2 domain for the detection of peptides containing tyrosine sulfation modifications or for the preparation of a product for the detection of peptides containing tyrosine sulfation modifications.
The sixth object of the present invention is to provide the use of said variant SH2 domain for detecting the concentration of a peptide containing a tyrosine sulfation modification or for the preparation of a product for detecting the concentration of a peptide containing a tyrosine sulfation modification.
It is a seventh object of the present invention to provide a method for detecting a peptide containing a tyrosine sulfation modification.
An eighth object of the present invention is to provide a method for isolating a peptide containing a tyrosine sulfation modification.
A ninth object of the present invention is to provide a method for detecting the concentration of a peptide containing a tyrosine sulfation modification.
The above purpose of the invention is realized by the following technical scheme:
the present invention obtains a variant SH2 domain with significantly enhanced binding affinity for peptides containing tyrosine sulfation modifications by introducing single or multiple amino acid residue substitutions to specific regions of the parent SH2 domain.
Specifically, the variant SH2 domain is any one of variant SH2 domains 1-5.
Wherein the amino acid sequence of the SH2 structural domain 1 of the variant is shown as SEQ ID NO. 3; the amino acid sequence of the structural domain 2 of the variant SH2 is shown as SEQ ID NO. 4; the amino acid sequence of the structural domain 3 of the variant SH2 is shown as SEQ ID NO. 5; the amino acid sequence of the SH2 structural domain 4 of the variant is shown as SEQ ID NO. 6; the amino acid sequence of domain 5 of variant SH2 is shown in SEQ ID NO. 7.
The variant SH2 domain of the invention may be prepared by using recombinant DNA techniques known to those skilled in the art. For example, it can be obtained by introducing a suitable expression vector (i.e., a recombinant expression vector) in a suitable manner to induce expression using a DNA sequence encoding the variant SH2 domain of the present invention.
Thus, the present application protects the DNA sequence encoding the variant SH2 domain.
The invention also claims a vector containing a DNA sequence encoding the variant SH2 domain.
The invention also provides a polypeptide comprising a plurality of SH2 domains, said plurality of SH2 domains comprising at least one variant SH2 domain of the invention.
Since the variant SH2 domain of the invention exhibits high affinity to sTyr, the variant SH2 domain of the invention may be tagged with a label to facilitate its detection in a variety of assays. Therefore, the invention also applies to protect the application of the variant SH2 structural domain in detecting tyrosine sulfation modified peptides or preparing products for detecting the tyrosine sulfation modified peptides.
The invention also discloses the application of the variant SH2 structural domain in detecting the concentration of the peptide containing the tyrosine sulfation modification or preparing a product for detecting the concentration of the peptide containing the tyrosine sulfation modification.
The invention also discloses application of the variant SH2 structural domain in binding or detecting sTyr residues in-vitro or in-vivo peptides or proteins.
The invention also discloses application of the variant SH2 structural domain in preparing products for binding or detecting the sTyr residue in the peptide or protein in vitro or in vivo.
The labels of the labeled variant SH2 domain of the invention include, but are not limited to, radioactive labels, cytotoxic labels or fluorescent labels. The SH2 monomer of the invention may also be provided with a carrier, and the peptide may be coupled to the solid phase carrier by covalent or non-covalent coupling. The peptide may be directly or indirectly labeled with a label selected from the group consisting of, but not limited to, biotin, fluorescein, an enzyme, horseradish peroxidase.
In particular, the product comprises a variant SH2 domain bound to an affinity column or bound on a lateral flow strip.
The invention also provides a method for detecting the peptide containing the tyrosine sulfation modification, a sample is contacted with any one of the variant SH2 structural domains, if the peptide containing the tyrosine sulfation modification exists in the sample, a compound containing the peptide containing the tyrosine sulfation modification/the variant SH2 structural domain is formed, and whether the compound is formed or not is detected, namely, whether the peptide containing the tyrosine sulfation modification exists or not is detected.
The invention also provides a method for separating the peptide containing tyrosine sulfation modification, wherein the peptide containing tyrosine sulfation modification can be separated by contacting a sample with any one of the variant SH2 structural domains, forming a peptide/variant SH2 structural domain complex containing tyrosine sulfation modification if the peptide containing tyrosine sulfation modification exists in the sample, and releasing the peptide containing tyrosine sulfation modification from the complex.
Further, upon isolation of the tyrosine containing sulfated modified peptides, the concentration of the tyrosine containing sulfated modified peptides in the sample can be determined by measuring the amount of the released tyrosine containing sulfated modified peptides.
In addition, tyrosine sulfatase activity can also be detected by detecting the sulfation level in the sample.
Specifically, the method for detecting the concentration of the peptide containing the tyrosine sulfation modification in the sample comprises the following steps:
s1, fixing the variant SH2 structural domain on resin;
s2. passing the sample through a resin with a bound variant SH2 domain;
s3. releasing any tyrosine containing sulfated modified peptide bound to the resin by adding a solvent that removes the ability of the variant SH2 domain to bind to the tyrosine containing sulfated modified peptide, thereby producing an elution fraction;
s4, determining the concentration of the peptide containing the tyrosine sulfation modification existing in the elution component.
The concentration of the tyrosine-containing sulfated modified peptide can be determined by High Performance Liquid Chromatography (HPLC).
The invention also provides a method for detecting the protein sulfation level in a subject, wherein a tissue sample of the subject is contacted with the variant SH2 structural domain to detect the sulfated protein contained in the sample, and the tyrosine sulfatase transferase activity is detected according to the sulfation level of the sample.
The tissue sample may be a tissue lysate, blood or other body fluid.
In addition to detecting protein sulfation levels in a subject by detecting sulfated proteins in tissue lysates, blood, or other body fluids, the sulfated proteins can also be visualized on tissue sections by using a variant SH2 domain with a fluorescent label; or the variant SH2 domain based on ELISA is combined with a target protein specific antibody to analyze the sulfation in normal or disease tissues/cells, and the like, so as to detect the protein sulfation level of the tissues. Meanwhile, in order to detect the SH2 variant in the sample, it may be labeled with a probe molecule.
The detection of sTyr positive cells can also be carried out by means of a probe comprising at least one polypeptide comprising the domain of variant SH2 and one contrast element.
In particular, the probes can be labeled with a detectable label, which can allow for the detection of the position of sTyr-positive cells, which can allow for following movement and development of sTyr-positive cells.
The contrast component of the probe typically comprises a label which may be those molecules suitable for use in vivo imaging, and methods of labeling are well known to those skilled in the art.
The SH2 monomer probe may be detectably labeled prior to detection, and a detectable label that binds to the hybridization product may be used; these detectable labels include, but are not limited to, any material that has detectable physical or chemical properties and has been well developed in the field of immunoassays.
The label used in the present invention may be any composition detectable by microscopic, photochemical, biochemical, immunochemical, or chemical means.
In addition, the presence or absence of the variant SH2 domain in a sample can also be detected by preparing and using a monoclonal antibody that recognizes any one of the variant SH2 domains described in the present invention.
The SH2 variant with ultra-high affinity for sTyr of the present invention can be used as a substitute for anti-sTyr antibodies, and is used in research fields using anti-sTyr antibodies, such as western blotting, immunofluorescence, proteomics (enrichment of sulfate protein/peptide), etc.
The invention has the following beneficial effects:
the invention obtains the variant SH2 structural domain with obviously enhanced combination affinity to the peptide containing the sTyr by introducing single or multiple amino acid residue substitutions to the specific region of the parent SH2 structural domain, the peptide containing the sTyr can be effectively enriched by utilizing the variant SH2 structural domain, and the variant SH 3556 structural domain can be used for detecting whether the peptide containing the sTyr exists in a sample and detecting the concentration of the peptide containing the sTyr in the sample, or detecting the activity of the tyrosine sulfate transferase by detecting the sulfation degree of protein in the sample, and the like. In addition, the variant SH2 domain can also be used as a substitute for anti-sTyr antibodies for studies using anti-sTyr antibodies, such as western blotting, immunofluorescence, proteomics, enrichment of sulfate proteins/peptides, and the like.
Drawings
FIG. 1 is a schematic diagram of the 11 residue positions selected in the SH2 library constructed using SH2-58.6 as a template.
FIG. 2 is a binding curve and Kd value of wild-type and variant SH2 domains to sTyr-containing peptides determined by BLI experiments; wherein, panel a is a binding curve of domain 1 of variant SH 2; panel B is the binding curve for domain 2 of variant SH 2; panel C is a binding curve for domain 3 of variant SH 2; panel D is the binding curve for domain 4 of variant SH 2; panel E is the binding curve for domain 5 of variant SH 2; FIG. F is a binding curve for SH 2-58.6; FIG. G is a binding curve for SH 2-1.8; FIG. H is the binding curve for SH 2-3.1.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The "peptide containing tyrosine sulfation modification", namely "sTyr-containing peptide" or "sTyr-containing peptide" in the invention refers to an amino acid sequence molecule containing sTyr, and comprises polypeptide or protein.
The term "parent SH2 domain" as used herein includes any eukaryotic SH2 domain or polypeptide having at least about 60% sequence identity to the SH2 domain derived from a human protein containing the SH2 domain.
The term "parent SH2 domain" also includes artificially prepared sequences as well as the viral SH2 domain. For example, artificial SH2 domain sequences may be generated or prepared as parent SH2 domains based on one or more mammalian SH2 domain sequences, which would represent a typical SH2 domain sequence, but not identical to any mammalian SH 2.
The term "fragment" refers to any test peptide having a shorter amino acid residue sequence than the peptide having the amino acid residue sequence shown in the present invention.
Optionally, the term "isolated peptide" or "isolated DNA" can be defined as a peptide or DNA molecule that is substantially separated from other cellular components that may naturally accompany the peptide or DNA; the term includes, without limitation, recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized via heterologous systems.
The term "ligand" means a molecule that binds another molecule or target.
The term "peptide" or "polypeptide" is defined as a chain of amino acid residues, typically having a defined sequence; as used herein, "peptide" is mutually inclusive of "polypeptide", "peptide" and "protein".
The terms "variant SH2 domain", "SH2 variant", "SH2 monomer", "SH2 structural variant" are used indifferently to refer to the parent SH2 domain into which substitutions are introduced for the affinity enhancement of the invention.
Example 1 design and characterization of variant SH2 domains of Fyn
The invention identifies a variant SH2 domain capable of binding to an sTyr-containing peptide by randomly replacing amino acid residues in a specific region of a human Fyn SH2 domain with one of 20 natural amino acids.
The number of all amino acid residues of the HUMAN Fyn SH2 domain is based on the full-length isoform of the UniProt database entry (entry) Fyn _ HUMAN. The gene encoding the wild type Fyn SH2 domain between Ala139 and Gly249 (see SEQ ID NO.1 for its amino acid sequence) was subcloned into the pDEST15 vector (Invitrogen Canada Inc.).
Three cysteine residues in SEQ ID No.1 were replaced by serine residues by a QuikChange II site-directed mutagenesis kit (Qiagen Inc.) (avoiding potential screening failures due to disulfide bond formation by cysteine residue interaction during phage screening), and the protein sequence shown in SEQ ID No.2 was generated by mutagenesis.
The gene encoding the protein shown in SEQ ID NO.2 was fused to the gene encoding the coat protein of M13 phage. By mutating the site of the SH2 domain region that binds to sTyr, a diverse library was formed for screening of high affinity mutants.
Simultaneous randomization was performed by the Kunkel Method (see in particular: Sidhu et al, 2000, Method enzymol. vol.328, pp.333) at the 11 amino acid positions detailed below, which can be numbered consecutively from position 1 to position 11, as shown in fig. 1. These 11 sites correspond to residues Arg176 (site 1), Glu177 (site 2), Ser178 (site 3), Glu179 (site 4), Thr180 (site 5), Thr181 (site 6), Tyr185 (site 7, Ser186 (site 8), His202 (site 9), Tyr203 (site 10), Lys204 (site 11) of wild-type full-length human Fyn SH2 in SEQ ID No.1, these 11 sites are Arg38 (site 1), Glu39 (site 2), Ser40 (site 3), Glu41 (site 4), Thr42 (site 5), Thr43 (site 6), Tyr47 (site 7), Ser48 (site 8), His64 (site 9), Tyr65 (site 10), Lys66 (site 11). mutations generate a library of Fyn SH2 domains containing randomly substituted amino acid residues at 11 sites.
The present invention uses a phage display method to display these introduced substituted Fyn SH2 domains on the surface of M13 phage. Carrying out phage screening by using an sTyr peptide with a biotin label, wherein the sequence of the sTyr peptide is shown as follows (SEQ ID NO. 11):
biotin-labeled sTyr peptides: Biotin-a-a-E-P-Q-sTyr-E-E-I-P-I-Y-L
Wherein, a represents 6-amino acetic acid, sTyr represents sulfated tyrosine;
the invention analyzes the change of each variant SH2 binding domain when the sTyr peptide with biotin labels is used for screening libraries through high-throughput sequencing, and identifies the variant SH2 structural domain bound to the sTyr peptide by combining with an experimental verification mode, thereby obtaining 5 Fyn variant SH2 structural domains. The residues of the resulting variant SH2 domain at the 11 amino acid positions in a particular region of the parent SH2 domain are shown in Table 1. In each variant, at least one of the positions contains an amino acid substitution to a wild-type residue.
Table 1 mutant sequences of variant SH2 domains
Figure BDA0003494321110000071
Example 2 variant SH2 Domain sequences
In the following description, the substituted residues are specifically designated using site numbering. For example, E2N indicates a Glu substitution at position 2 to Asn for the wild type construct; E2C/T5R/T6R/S8V/K11L represents 5 substitutions in combination. By substituting the wild-type construct (SEQ ID No.2) of the SH2 domain of Fyn without cysteine, the following 5 variant SH2 domains were obtained, the amino acid sequence between position 1 and position 11 of the 5 variant SH2 domains being as follows:
variant SH2 domain 1: E2N/S3Q/E4P/T5G/T6V/S8V/K11R, and the amino acid sequence thereof is shown as follows (SEQ ID NO. 3): arg Asn Gln Pro Gly Val Lys Gly Ala Tyr Val Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Arg
Variant SH2 domain 2: E2C/T5R/T6V/Y7H/S8V/K11L, the amino acid sequence of which is shown as follows (SEQ ID NO. 4): arg Cys Ser Glu Arg Val Lys Gly Ala His Val Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
Variant SH2 domain 3: E2C/E4A/T5R/T6V/Y7H/S8V/K11L, and the amino acid sequence of the amino acid sequence is shown as follows (SEQ ID NO. 5): arg Cys Ser Ala Arg Val Lys Gly Ala His Val Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
Variant SH2 domain 4: E2Q/E4A/T5R/T6V/Y7F/S8V/K11L, and the amino acid sequence of the amino acid sequence is shown as follows (SEQ ID NO. 6): arg Gln Ser Ala Arg Val Lys Gly Ala Phe Val Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
Variant SH2 domain 5: E2C/T5R/T6R/S8V/K11L, and the amino acid sequence is shown as follows (SEQ ID NO. 7): arg Cys Ser Glu Arg Arg Lys Gly Ala Tyr Val Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
Example 3 affinity of variant SH2 domains for sTyr-containing peptides in vitro
The invention utilizes synthesized sTyr-containing peptide, namely biotin-a-a-E-P-Q-sTyr-E-E-I-P-I-Y-L to carry out binding analysis on a variant SH2 structural domain shown in example 2 and reported variants of SH2-58.6, SH2-1.8 and SH2-3.1 (the sequences are sequentially shown as SEQ ID NO. 8-10).
Coli BL21(DE3) strain was cultured in LB medium (EMD Chemicals) to produce each variant and the wild-type SH2 domain by overexpression. Expression was induced by 0.5mM isopropyl-beta-D-1-thiogalactoside (IPTG) and cell cultures were incubated for 20 hours at 18 ℃. Cells were collected by centrifugation in a buffer solution containing 20mM sodium phosphate, 0.1M NaCl, 20mM imidazole, 1mg/mL lysozyme and 1% Triton-X100 at pH 7.8 and disrupted on ice. Affinity purification was performed with Ni-NTA agarose resin (Qiagen Inc.) following the manufacturer's instructions. The resulting protein was stored in PBT buffer (0.05% Tween 20, 0.5% bovine serum albumin, phosphate buffered saline).
Oct RED96 System (ForteBio) was used in a biofilm interference experiment to analyze the strength of affinity of the variant SH2 domain to sTyr-containing peptides, as measured using a streptavidin biosensor (cat # 18-5019). Synthetic sTyr-containing peptides were immobilized on biosensors. The concentration gradient of the variant SH2 domain was 300nM, 150nM and 0nM in this order for the measurement.
The binding curves and Kd values of wild-type and variant SH2 domains to sTyr-containing peptides determined by BLI experiments are shown in fig. 2, and the Kd values are summarized in table 2. Wherein, FIG. 2A is the combination curve of the structural domain 1 (abbreviated as variant 1 in the figure) of the variant SH2 and the sTyr-containing peptide; fig. 2B is a graph of the binding of variant SH2 domain 2 to an sTyr-containing peptide; fig. 2C is a graph of the binding of variant SH2 domain 3 to an sTyr-containing peptide; fig. 2D is a graph of the binding of domain 4 of variant SH2 to an sTyr-containing peptide; fig. 2E is a graph of the binding of domain 5 of variant SH2 to an sTyr-containing peptide; FIG. 2F is the binding curve of SH2-58.6 to sTyr-containing peptides; FIG. 2G is a graph showing the binding curves of SH2-1.8 to sTyr-containing peptides; FIG. 2H is the binding curve of SH2-3.1 to sTyr-containing peptides; since wild-type Fyn SH2 did not bind to the sTyr-containing peptide, there was no binding curve.
The smaller the Kd value represents the higher the affinity, and the results shown in FIG. 2 and Table 2 show that the affinity of the variant SH2 domain (variant SH2 domain 1-5) and the sTyr-containing peptide is obviously stronger than that of the reported SH2 mutant (SH2-58.6, SH2-1.8 and SH2-3.1), compared with the SH2-58.6 with the highest relative affinity in three controls, the affinity of the SH variant 2 domain 1-5 is improved by 22% -48%, and the sTyr-containing peptide can be effectively enriched, and can be used for detecting and separating the sTyr-containing peptide or preparing products for detecting and separating the sTyr-containing peptide.
TABLE 2 Kd values of wild-type and variant SH2 domains and sTyr-containing peptides
Figure BDA0003494321110000091
Figure BDA0003494321110000101
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Qingdao Sainuo Gene science and technology Co., Ltd
Qingdao Cancer Research Institute
<120> variant SH2 domain with high affinity to tyrosine-containing sulfated-modified peptides
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 111
<212> PRT
<213> Homo sapiens
<400> 1
Ala Pro Val Asp Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu
1 5 10 15
Gly Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg
20 25 30
Gly Thr Phe Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Ser
35 40 45
Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His
50 55 60
Tyr Lys Ile Arg Lys Leu Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg
65 70 75 80
Ala Gln Phe Glu Thr Leu Gln Gln Leu Val Gln His Tyr Ser Glu Arg
85 90 95
Ala Ala Gly Leu Cys Cys Arg Leu Val Val Pro Cys His Lys Gly
100 105 110
<210> 2
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ala Pro Val Asp Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu
1 5 10 15
Gly Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg
20 25 30
Gly Thr Phe Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Ser
35 40 45
Leu Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His
50 55 60
Tyr Lys Ile Arg Lys Leu Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg
65 70 75 80
Ala Gln Phe Glu Thr Leu Gln Gln Leu Val Gln His Tyr Ser Glu Arg
85 90 95
Ala Ala Gly Leu Ser Ser Arg Leu Val Val Pro Ser His Lys Gly
100 105 110
<210> 3
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Arg Asn Gln Pro Gly Val Lys Gly Ala Tyr Val Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Arg
20 25
<210> 4
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Arg Cys Ser Glu Arg Val Lys Gly Ala His Val Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 5
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Arg Cys Ser Ala Arg Val Lys Gly Ala His Val Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 6
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Arg Gln Ser Ala Arg Val Lys Gly Ala Phe Val Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 7
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Arg Cys Ser Glu Arg Arg Lys Gly Ala Tyr Val Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 8
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Arg Gln Leu Ala Arg Val Lys Gly Ala Tyr Ala Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 9
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Arg Glu Ala Glu Arg Val Lys Gly Ala Tyr Ala Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 10
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Arg Glu His Pro Phe Val Lys Gly Ala Tyr Ala Leu Ser Ile Arg Asp
1 5 10 15
Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr Leu
20 25
<210> 11
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Glu Pro Gln Tyr Glu Glu Ile Pro Ile Tyr Leu
1 5 10

Claims (10)

1. A variant SH2 domain having high affinity with a peptide containing tyrosine sulfation modification is characterized in that the amino acid sequence of the variant is shown in any one of SEQ ID NO. 3-7.
2. A DNA sequence encoding the variant SH2 domain of claim 1.
3. A vector comprising the DNA sequence of claim 2.
4. A polypeptide comprising a plurality of SH2 domains, at least one of said plurality of SH2 domains being the variant SH2 domain of claim 1.
5. Use of the variant SH2 domain of claim 1 for the detection of a tyrosine containing sulfated modified peptide or for the preparation of a product for the detection of a tyrosine containing sulfated modified peptide.
6. Use of the variant SH2 domain of claim 1 for detecting the concentration of a peptide containing tyrosine sulfation modifications or for preparing a product for detecting the concentration of a peptide containing tyrosine sulfation modifications.
7. The use of claim 5 or 6, characterized in that the product comprises a variant SH2 domain bound to an affinity column or bound on a lateral flow strip.
8. A method for detecting a peptide containing a tyrosine sulfation modification, which comprises contacting a sample with the variant SH2 domain of claim 1, wherein if a peptide containing a tyrosine sulfation modification is present in the sample, a complex containing the peptide containing the tyrosine sulfation modification/the variant SH2 domain is formed, and detecting the formation of the complex, thereby detecting the presence of the peptide containing the tyrosine sulfation modification.
9. A method for isolating a peptide containing a tyrosine sulphation modification, comprising contacting a sample with the variant SH2 domain of claim 1, wherein if a peptide containing a tyrosine sulphation modification is present in the sample, a complex containing the tyrosine sulphation modification peptide/variant SH2 domain is formed, and wherein the peptide containing the tyrosine sulphation modification is released from the complex, thereby isolating the peptide containing the tyrosine sulphation modification.
10. A method for determining the concentration of a peptide containing a tyrosine sulfation modification, which is characterized in that the concentration of the peptide containing a tyrosine sulfation modification in a sample is determined by determining the amount of the peptide containing a tyrosine sulfation modification released on the basis of the method of claim 9.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115947862A (en) * 2022-08-29 2023-04-11 嘉华药锐生物科技(昆山)有限公司 SH2 super-parent protein and conjugate formed by conjugation of SH2 super-parent protein and solid phase

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002171977A (en) * 1999-07-29 2002-06-18 Herikkusu Kenkyusho:Kk New human sh2 protein
CN105026422A (en) * 2012-03-27 2015-11-04 西安大略大学 Sh2 domain variants
CN112250750A (en) * 2020-10-21 2021-01-22 青岛肿瘤研究院 Variant SH2 domain with high affinity to tyrosine-containing phosphorylated modified peptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002171977A (en) * 1999-07-29 2002-06-18 Herikkusu Kenkyusho:Kk New human sh2 protein
CN105026422A (en) * 2012-03-27 2015-11-04 西安大略大学 Sh2 domain variants
CN112250750A (en) * 2020-10-21 2021-01-22 青岛肿瘤研究院 Variant SH2 domain with high affinity to tyrosine-containing phosphorylated modified peptides

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115947862A (en) * 2022-08-29 2023-04-11 嘉华药锐生物科技(昆山)有限公司 SH2 super-parent protein and conjugate formed by conjugation of SH2 super-parent protein and solid phase
CN115947862B (en) * 2022-08-29 2024-01-26 嘉华药锐生物科技(昆山)有限公司 SH2 super parent protein and conjugate formed by conjugation of SH2 super parent protein and solid phase

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