Methods and compositions for obtaining and using biologically active multi-protein complexes

Abstract

Methods for isolating and using multi-protein complexes that are biologically active are provided. The complexes contain one or more proteins of interest (e.g. a receptor, ion channel, etc.) and associated scaffolding proteins such as phosphatases, kinases and post synaptic density components. Buffers that do not contain denaturing agents and which may be used to isolate the multi-protein complexes are also provided, as are protein arrays containing the biologically active multi-protein complexes. The protein arrays may be used, for example, for high throughput screening assays.

1 . An isolated multiprotein complex in vitro in a form which retains biological activity, comprising
 one or more proteins of interest and   one or more scaffolding proteins.   
   
   
       2 . The isolated multiprotein complex of  claim 1 , wherein said one or more proteins of interest is multimeric. 
   
   
       3 . The isolated multiprotein complex of  claim 1 , wherein said one or more proteins of interest is selected from the group consisting of receptors and ion channels. 
   
   
       4 . The isolated multiprotein complex of  claim 1 , wherein said one or more scaffolding proteins are selected from the group consisting of phosphatases, kinases, and post synaptic density components. 
   
   
       5 . The isolated multiprotein complex of  claim 1 , wherein said one or more proteins of interest is N-methyl-D-aspartate receptor. 
   
   
       6 . The isolated multiprotein complex of  claim 1 , wherein said one or more proteins of interest is gamma-amino butyric acid receptor. 
   
   
       7 . The isolated multiprotein complex of  claim 1 , wherein said one or more proteins of interest is α-7 nicotinic acetylcholine receptor. 
   
   
       8 . The isolated multiprotein complex of  claim 1 , wherein said one or more proteins of interest is cyclic nucleotide-gated ion channel-HCN4. 
   
   
       9 . An array comprising
 a substrate; and   one or more isolated multiprotein complexes associated with said substrate, wherein said one or more isolated multiprotein complexes comprise one or more proteins of interest and one or more scaffolding proteins, and wherein said one or more isolated multiprotein complexes are biologically active.   
   
   
       10 . The array of  claim 9 , wherein said one or more isolated multiprotein complexes are immobilized on said substrate via an antibody that is attached to said substrate, said antibody being specific for said one or more proteins of interest. 
   
   
       11 . The array of  claim 9 , wherein said one or more proteins of interest is multimeric. 
   
   
       12 . The array of  claim 9 , wherein said one or more proteins of interest is selected from the group consisting of receptors and ion channels. 
   
   
       13 . The array of  claim 9 , wherein said one or more scaffolding proteins is selected from the group consisting of phosphatases, kinases, and post synaptic density components. 
   
   
       14 . The array of  claim 9 , wherein said one or more proteins of interest is N-methyl-D-aspartate receptor. 
   
   
       15 . The array of  claim 9 , wherein said one or more proteins of interest is gamma-amino butyric acid receptor. 
   
   
       16 . The array of  claim 9 , wherein said one or more proteins of interest is α-7 nicotinic acetylcholine receptor. 
   
   
       17 . The array of  claim 9 , wherein said one or more proteins of interest is cyclic nucleotide-gated ion channel-HCN4. 
   
   
       18 . The array of  claim 9 , wherein said substrate is a multiwell plate. 
   
   
       19 . The array of  claim 9 , wherein said isolated multiprotein complexes are associated with said substrate in a manner suitable for high throughput screening. 
   
   
       20 . A method of assaying for biological activity or chemical reactivity, comprising the steps of:
 providing an isolated multiprotein complex in vitro in a form which retains biological activity, said multiprotein complex comprising one or more proteins of interest and one or more scaffolding proteins;   exposing said isolated multiprotein complex to one or more substances; and   detecting a presence or absence of a biological activity or chemical reactivity of said multiprotein complex to said one or more substances.   
   
   
       21 . The method of  20  wherein said providing step is performed with said isolated multiprotein complex in a liquid solution or suspension. 
   
   
       22 . The method of  claim 20  wherein said providing step is performed with said isolated multiprotein complex associated with a substrate. 
   
   
       23 . The method of  claim 22 , wherein said isolated multiprotein complex is associated with said substrate in a manner that is suitable for high throughput screening. 
   
   
       24 . The method of  claim 20  wherein said detecting step detects changes in at least one of the following: chemiluminescence, surface plasmon resonance, phosphorescence, fluorescence, and UV/Vis properties 
   
   
       25 . The method of  claim 20 , wherein said biological activity is selected from the group consisting of a change in phosphorylation and a change in protein-protein interaction. 
   
   
       26 . A method of preparing an assay for biological activity or chemical reactivity, comprising the step of
 isolating a multiprotein complex from a biological sample so that the multiprotein complex is present in vitro in a form which retains biological activity, said multiprotein complex comprising one or more proteins of interest and one or more scaffolding proteins.   
   
   
       27 . The method of  claim 26 , wherein said step of isolating employs a composition which comprises
 one or more non-denaturing detergents,   one or more reducing agents,   one or more buffering agents,   one or more chelating agents, and   one or more protease inhibitors.   
   
   
       28 . The method of  27 , wherein said composition comprises 0.1% Triton X-100, 0.2% β-mercaptoethanol, 50 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride, and has a pH of approximately 7.5. 
   
   
       29 . A kit comprising,
 an isolated multiprotein complex in vitro in a form which retains biological activity, said isolated multiprotein complex comprising one or more proteins of interest and one or more scaffolding proteins, wherein said isolated multiprotein complex is present in a non-denaturing buffer;   reaction buffer;   blocking buffer; and   instructions for use.   
   
   
       30 . The kit of  claim 29 , wherein said isolated multiprotein complex is immobilized on a substrate. 
   
   
       31 . The kit of  claim 29 , wherein said isolated multiprotein complex is in solution. 
   
   
       32 . The kit of  claim 31 , wherein said kit further comprises antibodies that selectively bind said isolated multiprotein complex. 
   
   
       33 . The kit of  claim 32 , wherein said antibodies are conjugated to a detectable label. 
   
   
       34 . The kit of  claim 33 , wherein said detectable label is a fluorescent dye. 
   
   
       35 . The kit of  claim 32 , further comprising a detectable label for conjugation to said antibodies.