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ERK2 combination polypeptide and preparation thereof

A technology that combines polypeptides and polynucleotides, applied in the field of polypeptides, can solve the problems of not finding FRS2, etc., and achieve the effect of enhancing FGF pathway, regulating proliferation, and inhibiting activation

Inactive Publication Date: 2008-04-09
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, previous work did not find the exact binding site of ERK2 on FRS2

Method used

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  • ERK2 combination polypeptide and preparation thereof
  • ERK2 combination polypeptide and preparation thereof
  • ERK2 combination polypeptide and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 The region that binds to ERK2 in FRS2 is 248-272 amino acids

[0086] From the human cDNA library, use QuikChange Site-Directed Mutagenesis Kits (Stratagene) or obtain two required gene reading frames and clone them into the vector pGEX5X-3 (Amersham Pharmacia Biotech) to construct pGEX5X-3-FRS2 wild type and various pGEX5X-3-FRS2 mutants, the structure is as follows figure 1 shown. The primers and enzymes used in the construction of recombinant plasmids are listed in the following table:

[0087] Recombinant plasmid corresponding

fusion protein

5' end primer

3' end primer

restriction endonuclease

WT-GST

GGAATTCCATGGGTAGCTGTTGTAGC

(SEQ ID NO: 5)

ATAAGAATGCGGCCGCCATGGGCAGATC

AGTACTA (SEQ ID NO: 6)

5'-EcoRI

3'-NotI

NT-GST

GGAATTCCATGGGTAGCTGTTGTAGC

(SEQ ID NO: 5)

CCGCTCGAGTTCAGGTTCAAAGAAGAATC

T (SEQ ID NO: 16)

5'-EcoRI

3'-XhoI

CT-GST

...

Embodiment 2

[0092] Example 2 Mutation of amino acids 248-272 in FRS2 will destroy the combination of FRS2 and ERK2 Using the human pGEX5X-3-FRS2 plasmid as a template, the designed primer sequences are as follows:

[0093] FRS2-FPA

[0094] 5'GAAGGAGTCAATTTTGTTTTTGGGCCAACCCCTGTTCAAAATCAGTTTATGGAAAAAG3' (SEQ ID NO: 22)

[0095] 5'CTTTTTCCATAAACTGATTTTGAACAGGGGTTGGCCCAAAAACAAAATTGACTCCTTC3' (SEQ ID NO: 23)

[0096] FRS2-FPB5'CAACCCCTGTTCAAAATCAGTTTATGGAAAAAGAGAATGTGGAGCAACTTGGAAG3' (SEQ ID NO: 24)

[0097] 5'CTTCCAAGTTGCTCCACATTCTCTTTTTCCATAAACTGATTTTGAACAGGGGTTG3' (SEQ ID NO: 25)

[0098] QuikChange Site-Directed Mutagenesis Kits (products of Stratagene) were selected, and mutants were constructed according to the instructions. FRS2 mutant GST fusion proteins FPA (SEQ ID NO: 26) and FPB (SEQ ID NO: 3) were obtained by the same method as in Example 1. Using the GST-FRS2-FPA plasmid as a template, the GST-FRS2-3kl (SEQ IN NO: 4) plasmid was constructed in the same manner as in Example 1 ...

Embodiment 3

[0101] Example 3 Overexpression of FRS2-wt and FRS2-3kl leads to different performances of aFGF-induced MAPK / ERK and Akt / PKB signaling pathways

[0102]Using pGEX5X-3-FRS2 as a template, the primer sequences were designed as follows: 5'-CCGCTCGAGATGGGTAGCTGTTGTAGC-3' and 5'-ATAAGAATGCGGCCGCCATGGGCAGATCAGTACTA-3', using the same method as in Example 1, after XhoI and NotI double digestion, T4DNA ligase Under the action, it was directional inserted into the protein expression vector pMSCV (product of Clontech Company) that had been cut with the same double restriction enzymes. Infect NIH3T3 cells with pMSCV plasmid or pMSCV-FRS2-wt or pMSCV-FRS2-3kl retrovirus (add 100 μl virus and Polybrene with a final concentration of 4 mg / ml in 900 μl culture medium) NIH3T3 cells, after two days, culture the cells overnight without serum , 100ng / ml aFGF stimulated the cells for different lengths of time, and after SDS-PAGE of the total product of cell lysate, the immunoblotting method used a...

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PUM

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Abstract

The present invention relates to a polypeptide which has the function of regulating the cell proliferation and differentiation. The present invention provides a separated ERK2 combined polypeptide, which is the polypeptide or the conservative variation polypeptide which has an amino acid sequence of SEQIDNO: 1. the present invention further provides a preparation method of the polypeptide. The polypeptide can be combined with ERK2 (Extracellular signal-regulated kinase2) to regulate the cell proliferation and differentiation, and the disclosure of the polypeptide site provides a new targeted inhibition site for an FGFR inhibitor.

Description

technical field [0001] The invention relates to a polypeptide, in particular to a polypeptide with the function of regulating cell proliferation and differentiation and its preparation. Background technique [0002] Adapter proteins are a group of molecular regulators that can recruit different proteins to form complexes and pass on the information of upstream signaling molecules (Pawson T, et al.Signaling through scaffold, anchoring, and adapter proteins.Science, 19, 2075-80 , 1997; Ravichandran KS, et al. Signaling via Shc family adapterproteins. Oncogene, 20(44), 6322-30, 2001). When the signal transduction complex forms and completes its biological function, the adapter protein often undergoes tyrosine phosphorylation, structural changes and subcellular localization changes (Cheng Y, et al. ERK negatively regulates the epidermal growth factor-mediated interaction of Gabl and the phosphatidylinositol 3-kinase. J. Biol Chem, 277, 19382-19388, 2002; Guri Tzivion, et al.14-...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/435C12N15/12C12N15/63C12N1/21C12P21/02
Inventor 陈正军周文超
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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