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Protein for promoting erythrocyte growth factor activity and its use

A protein and red blood cell technology, applied in peptide/protein components, medical preparations containing active ingredients, extracellular fluid diseases, etc., can solve problems such as insufficient amino acids, achieve the effect of less protein consumption and accurate determination of binding constants

Inactive Publication Date: 2007-05-02
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0026]We use the protein function grafting calculation strategy developed by our laboratory (Biopolymers, 54: 515-523, 2000) for the calculation of EPO surface functional region grafting. The key residues of the interaction interface between EPO (Protein Data Bank code leer) and its receptor EPOR: 48 PHE, 147 ASN, and 150 ARG are the key residues for grafting. We used a vector matching-based grafting strategy to graft the key binding site of the protein to another non-homologous protein, and successfully found a candidate protein backbone PH domain for grafting EPO function (Protein Data Bank code is 1mai) . Through a computer program we wrote according to this strategy, we calculated that the complementarity score of this skeleton was 561, and the mean square deviation of key residues was 0.843 angstroms. The three key residues that need to be mutated correspond to the following:

[0027] 63 bit GLU→PHE; 47 bit ASP→...

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 (residues 1-140; Protein Data Bank code is 1mai) of the rat (Rat) phospholipase C delta1 (Phospholipase C Delta 1) is pGST4 through primer pair 1 (primer sequence shown in Table 1) / PLCdelta1 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). The amplified product was purified and then digested with NdeI and EcoRI. The digested product was ligated into the pET-28a vector (purchased from Novagen) that was digested with NdeI and EcoRI by T4 DNA ligase to obtain the expression vector pETPHD. The sequence was verified by DNA sequencing to be correct.

[0033] Primer pair

Primer sequence (5’-3’)

Sequence number (SEQ ID NO)

1

Forward primer

CACTCTA C...

Embodiment 3

[0066] Example 3: In vitro receptor binding ability detection

[0067] The binding ability of wtNH-PHD and its mutant protein to EPOR (EPO receptor) was tested using surface plasmon resonance (SPR) technology. The experimental equipment was Biacore 3000 (purchased from Uppsala, Sweden), and the recombinant human EPO soluble receptor (rhEPO sR) was purchased from R&D Systems. The buffer used in the experiment is HBS-EP (10 mM Hepes, 150 mM NaCl, 3.7 mM EDTA, pH 7.4, 0.005% P20), and the chip is a CM5 chip.

[0068]The rhEPO sR was dissolved in a 100 mM sodium acetate solution of pH 3.1 to obtain a solution of pH 4 for immobilization. During the immobilization process, the flow rate is maintained at 5μL·min -1 . The second channel of the CM5 chip was activated with 35μL N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide / N-hydroxysuccinimide (EDC / NHS, 1:1), then 45μL rhEPO sR was injected, and finally 35μL 1M ethanolamine- HCl, pH 8.5 closes the channel. The fixed rhEPO sR is 1200RU. Use...

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PUM

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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 factor activity, in particular to a pleckstrin homology domain that is widely present in organisms and a mutant with erythrocyte growth factor activity. Background technique [0002] Protein is the main substance that completes various life functions in organisms. The study of protein structure and function law is one of the central topics of life sciences. It not only includes the analysis and research of natural protein structure and function law, but also includes the use of existing knowledge for protein design. The ultimate goal of protein design is to design a protein with any specified structure and function from scratch. Whether it is to make an existing protein have a new function or to introduce a function into a newly designed protein, it has very important theoretical significance and potential application value for studying the relationship bet...

Claims

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Application Information

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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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