Variant SH2 structural domain having high affinity with tyrosine-containing phosphorylation modification peptide

A technology of tyrosine phosphorylation and structural domains, which is applied in the field of variant SH2 domains with high affinity to modified peptides containing tyrosine phosphorylation, which can solve the problems of inability to use and minimize the possibility of immune response Effect

Active Publication Date: 2021-01-22
QINGDAO CANCER INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although phosphotyrosine-specific antibodies have high affinity for pTyr-containing polypeptides, they cannot be used intracellularly

Method used

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  • Variant SH2 structural domain having high affinity with tyrosine-containing phosphorylation modification peptide
  • Variant SH2 structural domain having high affinity with tyrosine-containing phosphorylation modification peptide
  • Variant SH2 structural domain having high affinity with tyrosine-containing phosphorylation modification peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1. Identification of variant SH2 domains by phage display technology

[0072] Amino acid residues at eight positions in the human Fyn SH2 domain were randomly substituted with one of 20 natural amino acids to identify variant SH2 domains that bind pTyr-containing peptides.

[0073] The number of all amino acid residues in the human Fyn SH2 domain is the full-length isoform according to the UniProt database entry (entry) FYN_HUMAN. The gene encoding the wild-type FynSH2 domain between Ala139 and Gly249 (see SEQ ID NO: 1 for its amino acid sequence) was subcloned into pDEST15 vector (Invitrogen Canada Inc.). The three cysteine ​​residues in SEQ ID NO: 1 were replaced with serine residues by QuikChange II Site-Directed Mutagenesis Kit (Qiagen Inc.). This mutation produces the protein sequence shown in SEQ ID NO:2.

[0074] The gene encoding the protein shown in SEQ ID NO: 2 was fused to the gene encoding the major coat protein of M13 bacteriophage. By mutating 8...

Embodiment 2

[0077] Example 2, Enhanced binding of variant Fyn SH2 domains to pTyr-containing peptides in vitro

[0078] Single or multiple substitutions were introduced into the wild-type Fyn SH2 domain, and the degree of affinity enhancement resulting from the introduction of these substitutions was determined. Using this wild-type construct as a template, three variant Fyn SH2 domains were constructed by a site-directed mutagenesis approach.

[0079] In the following description, the residue to be substituted is specified using a position number. For example, T8V indicates a substitution of Thr at position 8 to Val in the wild type construct. T4V / S6A represents 2 substitutions in combination, Thr at position 4 to Val and Ser at position 6 to Ala for the wild type construct. The amino acid sequences between positions 1 and 8 of the SH2 domains of the three variants are listed in the sequence listing. By substituting the wild-type construct (SEQ ID NO:1), the following variant Fyn SH2 ...

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Abstract

The invention belongs to the technical field of application of protein tyrosine kinase (PTKs), and relates to a variant SH2 structural domain having high affinity with tyrosine-containing phosphorylation modification peptide. The invention provides the variant SH2 structural domain having high affinity with the tyrosine-containing phosphorylation modification peptide. The variant SH2 structural domain comprises amino acid sequences selected from SEQ ID NOs: 3, 4, 5 or 6. According to the invention, a residue is used for substituting a pTyr-binding region which is introduced into an SH2 domain,and a favorable substitution for enhanced binding affinity for a pTyr-containing peptide is set forth. Different substitution combinations exhibit different degrees of impact on affinity enhancement.The variant SH2 structural domain shows that the strength of the variant SH2 structural domain is much higher than that of a wild type SH2 structural domain in in-vitro binding analysis of pTyr-containing protein.

Description

technical field [0001] The invention belongs to the technical field of application of protein tyrosine kinases (PTKs), and relates to a variant SH2 domain with high affinity for tyrosine-containing phosphorylated modified peptides, in particular to using the variant for the treatment of protein tyrosine kinases Related disorders, such as immunological and oncological disorders, relate to methods of using the variant for diagnosing protein tyrosine kinase-related disorders, involving the use of the variant for detecting, tracking or monitoring Methods of tyrosine phosphorylation events, methods of using the variants for enrichment or purification of phosphotyrosine-containing peptides or proteins, and pharmaceutical compositions containing the variants. Background technique [0002] Protein tyrosine kinases and their substrates play important roles in a variety of cellular processes such as proliferation, differentiation and apoptosis. Aberrant kinase activation and concomit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12G01N33/68C07K1/22C07K19/00A61K38/17A61P35/00A61P37/02
CPCC07K14/4703C12N9/1205C12Y207/01112G01N33/68G01N33/6812C07K1/22A61P35/00A61P37/02C07K2319/00A61K38/00
Inventor 李磊黄海明李树浩
Owner QINGDAO CANCER INST
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