Biosensor with covalently attached membrane-spanning proteins

Abstract

The invention provides a product comprising: a membrane-spanning protein; a lipid membrane formed from amphiphilic molecules and membrane-spanning protein molecules; and a substrate: characterised in that the membrane protein is directly coupled to the substrate. The invention also provides a method for producing such a product which i) comprises treating a substrate with a hydrophilic coating agent; ii) providing at least one membrane-spanning protein; iii) bringing the protein into contact with the treated substrate under conditions for the coupling of the protein directly to the treated substrate; iv) adding amphiphilic molecules to the protein-coupled substrate to form a lipid membrane. The product is useful for biosensors, protein arrays and the like.

1 . A product comprising: a membrane-spanning protein; a lipid membrane formed from amphiphilic molecules and membrane-spanning protein molecules; and a substrate: characterised in that the membrane protein is coupled to the substrate by a direct covalent coupling.  
     
     
         2 . A product according to  claim 1  wherein at least some of the amphiphilic molecules are coupled to the substrate by a direct covalent coupling.  
     
     
         3 . A product according to  claim 1  or  2  wherein the protein is coupled to the substrate via at least one exposed cysteine residue located in the periplasmic side of the protein.  
     
     
         4 . A product according to any preceding claim wherein the product is a biosensor.  
     
     
         5 . A product according to any preceding claim wherein the product is a protein array.  
     
     
         6 . A product according to any preceding claim wherein the protein comprises a polypeptide of more than 20 L-amino acid residues purified from a cell extract.  
     
     
         7 . A product according to any preceding claim wherein the protein comprises a D-barrel structure.  
     
     
         8 . A product according to any of claims  1 - 6  wherein the protein comprises a α-helix structure.  
     
     
         9 . A product according to any preceding claim wherein the protein is an integral membrane protein in which secondary structural units span the membrane at least once and join regions exposed to an aqueous phase.  
     
     
         10 . A product according to any preceding claim wherein the protein is an ion channel forming protein (ionophore).  
     
     
         11 . A product according to any of claims  1 - 9  wherein the protein is a receptor for a ligand or family of ligands.  
     
     
         12 . A product according to any of claims  1 - 9  wherein the protein is an enzyme.  
     
     
         13 . A product according to any of claims  1 - 12  wherein the protein is an engineered (recombinant) protein comprising a membrane-spanning protein and a synthetic polypeptide.  
     
     
         14 . A product according to any of claims  1 - 12  wherein the protein is an engineered (recombinant) protein comprising a membrane-spanning protein and all or part of a soluble protein of more than 20 L-amino acid residues purified from a cell extract.  
     
     
         15 . A product according to any of claims  1 - 12  wherein the protein is an engineered (recombinant) protein into which has been introduced, by insertion or mutation, at least one exposed cysteine residue located in the periplasmic side of the protein.  
     
     
         16 . A product according to  claim 10  wherein the protein is a porin.  
     
     
         17 . A product according to  claim 16  wherein the porin is selected from Table 1.  
     
     
         18 . A product according to any preceding claim wherein the amphiphilic molecules comprise or consist of thiolipid.  
     
     
         19 . A product according to  claim 18  wherein the thiolipid is selected from the group consisting of: 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]; 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]; N-(14′-mercapto-1′,11′-dioxo-3′,6′,9′-trioxa-12′-azatetradecyl)-2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine; (8′mercapto-3′,6′-dioxa-octyl)-1,2-dipalymitoyl-sn-glycero-3-phosphatidic acid.  
     
     
         20 . A product according to any of claims  1 - 19  wherein the substrate is a metal.  
     
     
         21 . A product according to  claim 20  wherein the metal is selected from: gold; chromium; platinum; silver.  
     
     
         22 . A product according to any of claims  1 - 21  wherein the substrate has a surface modified to react with thiol groups.  
     
     
         23 . A product according to any of claims  1 - 19  wherein the surface is selected from: glass; plastic; silicon; hydrogen terminated silicon.  
     
     
         24 . A method for the preparation of a product according to any of claims  1 - 23  comprising: 
 i. treating a substrate with a hydrophilic coating agent;  
 ii. providing at least one membrane-spanning protein;  
 iii. bringing the protein into contact with the treated substrate under conditions for the covalent coupling of the protein directly to the treated substrate;  
 iv. adding amphiphilic molecules to the protein coupled substrate to form a lipid membrane.  
 
     
     
         25 . A method according to  claim 24  wherein the hydrophilic agent is a thiol.  
     
     
         26 . A method according to  claim 25  wherein the thiol is of general formula OH—(CH 2 ) n —SH, wherein n is an integer and the alkyl chain is straight or branched.  
     
     
         27 . A method according to  claim 24  wherein the substrate is treated with a hydrophilic agent selected from: 2-mercaptoethanol; mercaptopropionic acid; 1-mercapto-2-propanol; 2,3 dimercapto-1-propanol; 2-mercapto-3-butanol; erythro-1,4-dimercapto-2,3-butanediol, (DTE); threo-1,4-dimercapto-2,3-butanediol, (DTT); mixtures of DTE and DTT; thiol glycerol.  
     
     
         28 . A method according to  claim 27  wherein the hydrophilic agent is 2-mercaptoethanol.  
     
     
         29 . A method according to any of claims  24 - 28  wherein the substrate is gold.  
     
     
         30 . A method according to any of claims  24 - 29  wherein the protein is an ionophore.  
     
     
         31 . A method according to  claim 24  wherein the amphiphilic molecules are covalently coupled to the substrate.  
     
     
         32 . A method according to  claim 31  wherein the protein is a porin.  
     
     
         33 . A method according to any of claims  24 - 32  wherein at least one exposed cysteine residue is provided in the periplasmic side of the protein, whereby coupling of the protein to the substrate is via coupling of at least one cysteine to at least one thiol group on the substrate.  
     
     
         34 . A method according to  claim 33  wherein the porin is modified OmpA, formed by N-terminal addition of MHHHHHHSS-Cys- to the mature OmpA protein.  
     
     
         35 . A method according to  claim 33  wherein the porin is modified OmpF, formed by mutation of glutamate 183 to cysteine.  
     
     
         36 . A product obtainable by the method according to any of claims  24 - 35 .  
     
     
         37 . Use of the product according to any of claims  1 - 23  to interact with at least one of following molecules: polypeptides; antigenic polypeptides; antibodies or fragments of antibodies; receptors; ligands; antibiotics; drugs; pesticides; sugars; amino acids; fatty acids; peptides; hormones; steroids; nucleic acids (DNA, RNA, cDNA); peptide nucleic acids; metals; inorganic ions.