Protein for promoting erythrocyte growth factor activity and its use

A protein and red blood cell technology, applied in the fields of peptide/protein components, medical preparations containing active ingredients, extracellular fluid diseases, etc., can solve the problem of insufficient amino acids, and achieve the effect of less protein dosage and accurate binding constant measurement.

Inactive Publication Date: 2008-11-12
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Site-directed mutagenesis has shown that only a few residues have a significant impact on binding (usually 3-4 key residues are provided by each side of the binding), but it is clearly not enough to only consider amino acids that play a significant role

Method used

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  • Protein for promoting erythrocyte growth factor activity and its use
  • Protein for promoting erythrocyte growth factor activity and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Selection of PH domain mutation sites

[0026]We used the calculation strategy of protein function grafting developed in our laboratory (Biopolymers, 54:515-523, 2000) for the calculation of functional region grafting on the surface of EPO. The key residues at the interaction interface between EPO (Protein Data Bank code leer) and its receptor EPOR: PHE at position 48, ASN at position 147, and ARG at position 150 are used as key residues for grafting. We used a grafting strategy based on vector matching to transfer the key binding site of the protein to another non-homologous protein, and successfully found the candidate protein backbone PH domain for grafting EPO function (Protein Data Bank code is lmai) . Calculated by a computer program we wrote according to this strategy, the complementarity score of this backbone is 561, and the mean square deviation of key residues is 0.843 angstroms. The three key residues that need to be mutated correspond to the fo...

Embodiment 2

[0030] Example 2: Construction, expression and purification of PH domain mutants

[0031] 1. Gene amplification and expression vector construction

[0032] The gene of the PH domain (residue 1-140; ProteinData Bank code is 1mai) of rat (Rat) phospholipase Cδ1 (Phospholipase C Delta 1) was expressed in pGST4 / PLCδ1 by primer pair 1 (the primer sequence is shown in Table 1) The plasmid (gifted and authorized by Professor Hitoshi Yagisawa; Eur. J. Biochem. 265: 481-490, 1999) was used as a template and amplified by Polymerase Chain Reaction (PCR). After purification, the amplified product was double-digested with NdeI and EcoRI, and the digested product was ligated into the pET-28a vector (purchased from Novagen) that had been double-digested with NdeI and EcoRI by T4 DNA ligase to obtain the expression vector pETPHD. The correct sequence was verified by DNA sequencing.

[0033] Table 1. Primer Design

[0034]

[0035]

[0036] Note: ____ mark is the endonuclease site. C...

Embodiment 3

[0068] Example 3: Detection of receptor binding ability in vitro

[0069]The binding ability of wtNH-PHD and its mutant proteins to EPOR (EPO receptor) was tested by surface plasmon resonance (SPR). The experimental equipment was Biacore 3000 (purchased from Uppsala Company, Sweden), and the recombinant human EPO soluble receptor (rhEPO sR) was purchased from R&D Systems. The buffer used in the experiment was HBS-EP (10 mM Hepes, 150 mM NaCl, 3.7 mM EDTA, pH 7.4, 0.005% P20), and the chip was a CM5 chip.

[0070] rhEPO sR was dissolved in 100 mM sodium acetate solution at pH 3.1 to obtain a solution at pH 4 for immobilization. During the immobilization process, the flow rate was maintained at 5 μL min -1 . Activate the second channel of the CM5 chip with 35 μL N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC / NHS, 1:1), then inject 45 μL rhEPO sR, and finally inject 35 μL 1M ethanolamine- HCl, pH 8.5 blocked the channels. The fixed rhEPO sR was 1200...

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Abstract

This invention relates to a kind of protein that possesses erythropoietin activity and its preparation. This protein is derivatized through its amino acid sequence is substituted by one or more amino acid residue, absence or addition ,in the condition that remaining tertiary structure of pleckstrin homology structural domain to be invariant, it possesses erythropoietin activity. Technology project of this invention is that function of haemopoietin EPO is grafted on backbone of pleckstrin homology structural domain by using protein functional grafting method, progress mutant design, expression and depuration, and determine the binding capability of mutant and EPO acceptor and EPO activity by experiment. This invented protein can be used as EPO succedaneum, used for curing anaemia, chronic renal failure(CRF),HIV infected / ZDU curing patient, arthritis deformans, carcino-anaemia and any other symptoms that can be cured by using EPO.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a protein with erythrocyte growth-stimulating factor activity, in particular to a pleckstrin homology domain widely present in organisms and a mutant with erythrocyte growth-stimulating factor activity. Background technique [0002] Protein is the main substance that completes various life functions in organisms. The study of protein structure and function rules is one of the central topics of life sciences, which includes not only the analysis of natural protein structure and function rules, but also the use of existing knowledge for protein design. The ultimate goal of protein design is to design proteins with arbitrary specified structures and functions de novo. Whether making existing proteins have new functions or introducing functions into newly designed proteins, it has very important theoretical significance and potential application value for studying the relationship between ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/79A61K38/18A61P29/00A61P19/02A61P13/12A61P7/06A61P31/18A61P35/00
Inventor 来鲁华曹傲能刘森刘士勇常智杰王银银程龙
Owner PEKING UNIV
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