Method to produce a receptor chip using biotinylated protein

Abstract

The present invention provides a method for detecting modified LDL, abnormal cells or bacteria using an intermolecular interaction analysis method, in which a region involved in ligand recognition by a receptor is expressed, without modification or as a biotinylated protein, in cells or in a test tube, and thereafter, the expressed region or the expressed biotinylated protein is immobilized via avidin or streptavidin to a solid phase while the orientation thereof is maintained, and the immobilized protein is utilized; and a kit for detecting the modified LDL or the like.

What is claimed is:  
     
         1 . A receptor chip, on which a recombinantly expressed biotinylated receptor protein is immobilized via a factor capable of specifically binding to biotin.  
     
     
         2 . A receptor chip according to  claim 1 , wherein the biotinylated receptor protein is expressed in a bacterial host.  
     
     
         3 . A receptor chip according to  claim 1 , wherein the biotinylated receptor protein is expressed in vitro.  
     
     
         4 . A receptor chip according to  claim 2 , wherein the biotinylation of the receptor protein is carried out within a bacterial host.  
     
     
         5 . A receptor chip according to  claim 2 , wherein the biotinylation of the receptor protein is carried out in vitro after expression of the protein.  
     
     
         6 . A receptor chip according to  claim 4 , wherein the immobilized biotinylated receptor protein is obtained by refolding a biotinylated receptor protein expressed as an inclusion body within a bacterium.  
     
     
         7 . A receptor chip according to  claim 6 , wherein the refolding is carried out in a solution containing a cyclic carbohydrate cycloamylose and a polyoxyethylene detergent.  
     
     
         8 . A receptor chip according to  claim 7 , wherein the degree of polymerization of the cyclic carbohydrate cycloamylose is 17 to 50 or 40 to 150.  
     
     
         9 . A receptor chip according to  claim 8 , wherein the degree of polymerization of the cyclic carbohydrate cycloamylose is 40 to 150.  
     
     
         10 . A receptor chip according to  claim 7 , wherein the polyoxyethylene detergent is polyoxyethylenesorbitan ester, polyoxyethylenedodecyl ether, polyoxyethylenehepta methylhexyl ether, polyoxyethyleneisooctylphenyl ether, polyoxyethylenenonylphenyl ether, polyoxyethylene fatty acid ester, or sucrose fatty acid ester.  
     
     
         11 . A receptor chip according to  claim 6 , wherein the refolding is carried out in a solution containing a cyclic carbohydrate cycloamylose and an ionic detergent.  
     
     
         12 . A receptor chip according to  claim 11 , wherein the degree of polymerization of a cyclic carbohydrate cycloamylose is 17 to 50 or 40 to 150.  
     
     
         13 . A receptor chip according to  claim 12 , wherein the degree of polymerization of a cyclic carbohydrate cycloamylose is 40 to 150.  
     
     
         14 . A receptor chip according to  claim 11 , wherein the ionic detergent is cetyltrimethyl ammonium bromide, dodecyl sodium sulfate, sodium deoxycholate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate, hexadecyltrimethyl ammonium bromide, or myristyl sulfo betaine.  
     
     
         15 . A receptor chip according to  claim 1 , wherein the receptor is selected from the group consisting of scavenger receptors, receptors of the insulin receptor family, receptors of the EGF receptor family, receptors of the PDGF receptor family, receptors of the VEGF receptor family, receptors of the FGF receptor family, growth factor receptors of the NGF receptor family, TGF-β super family receptors, Toll-like receptor family, LDL receptor related protein family, and receptors of the G protein coupled receptor family.  
     
     
         16 . A receptor chip according to  claim 15 , wherein the receptor is a scavenger receptor LOX-1.  
     
     
         17 . A receptor chip according to  claim 1 , adapted for detection using surface plasmon resonance, quartz-crystal microbalance, or mass spectrometer.  
     
     
         18 . A method for producing a receptor chip, comprising the steps of: 
 a) recombinantly expressing a biotinylated receptor protein as an inclusion body within a bacterial host;    b) refolding the inclusion body in a solution containing a cyclic carbohydrate cycloamylose and a polyoxyethylene detergent to prepare a soluble biotinylated receptor protein; and    c) immobilizing the refolded soluble biotinylated receptor protein to a solid phase via a factor capable of specifically binding to biotin.    
     
     
         19 . A method according to  claim 18 , wherein the degree of polymerization of the cyclic carbohydrate cycloamylose is 17 to 50 or 40 to 150.  
     
     
         20 . A method according to  claim 19 , wherein the degree of polymerization of the cyclic carbohydrate cycloamylose is 40 to 150.  
     
     
         21 . A method according to  claim 18 , wherein the polyoxyethylene detergent is polyoxyethylenesorbitan ester, polyoxyethylenedodecyl ether, polyoxyethyleneheptamethylhexyl ether, polyoxyethyleneisooctylphenyl ether, polyoxyethylenenonylphenyl ether, polyoxyethylene fatty acid ester, or sucrose fatty acid ester.  
     
     
         22 . A method according to  claim 18 , wherein the receptor is selected from the group consisting of scavenger receptors, receptors of the insulin receptor family, receptors of the EGF receptor family, receptors of the PDGF receptor family, receptors of the VEGF receptor family, receptors of the FGF receptor family, growth factor receptors of the NGF receptor family, TGF-β super family receptors, Toll-like receptor family, LDL receptor related protein family, and receptors of the G protein coupled receptor family.  
     
     
         23 . A method according to  claim 22 , wherein the receptor is a scavenger receptor LOX-1.  
     
     
         24 . A method according to  claim 18 , wherein the solid phase is adapted for detection using surface plasmon resonance, quartz-crystal microbalance, or mass spectrometer.  
     
     
         25 . A method for producing a receptor chip, comprising the steps of: 
 a) recombinantly expressing a biotinylated receptor protein as an inclusion body within a bacterial host;    b) refolding the inclusion body in a solution containing a cyclic carbohydrate cycloamylose and an ionic detergent to prepare a soluble biotinylated receptor protein; and    c) immobilizing the refolded soluble biotinylated receptor protein to a solid phase via a factor capable of specifically binding to biotin.    
     
     
         26 . A method according to  claim 25 , wherein the degree of polymerization of the cyclic carbohydrate cycloamylose is 17 to 50 or 40 to 150.  
     
     
         27 . A method according to  claim 26 , wherein the degree of polymerization of the cyclic carbohydrate cycloamylose is 40 to 150.  
     
     
         28 . A method according to  claim 25 , wherein the ionic detergent is cetyltrimethyl ammonium bromide, dodecyl sodium sulfate, sodium deoxycholate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate, hexadecyltrimethyl ammonium bromide, or myristyl sulfo betaine.  
     
     
         29 . A method according to  claim 25 , wherein the receptor is selected from the group consisting of scavenger receptors, receptors of the insulin receptor family, receptors of the EGF receptor family, receptors of the PDGF receptor family, receptors of the VEGF receptor family, receptors of the FGF receptor family, growth factor receptors of the NGF receptor family, TGF-β super family receptors, Toll-like receptor family, LDL receptors related protein family, and receptors of the G protein coupled receptor family.  
     
     
         30 . A method according to  claim 29 , wherein the receptor is a scavenger receptor LOX-1.  
     
     
         31 . A method according to  claim 25 , wherein the solid phase is adapted for detection using surface plasmon resonance, quartz-crystal microbalance, or mass spectrometer.  
     
     
         32 . A receptor chip produced by a method according to  claim 18  or  25 .  
     
     
         33 . A method for detecting modified LDL, an abnormal cell, or a bacterium, using a receptor chip according to  claim 16 .  
     
     
         34 . A receptor chip produced by a method according to  claim 23  or  30 .  
     
     
         35 . A method for detecting modified LDL, an abnormal cell, or a bacterium, using a receptor chip according to  claim 34 .  
     
     
         36 . A detection kit, comprising a receptor chip produced by a method according to  claim 18  or  25 .  
     
     
         37 . A detection kit, comprising a receptor chip according to  claim 16 .  
     
     
         38 . A detection kit, comprising a receptor chip produced by a method according to  claim 23  or  30 .  
     
     
         39 . A detection kit, comprising a receptor chip according to  claim 34.