Arrays of cytosolic accessory proteins immobilized on a surface and pertinent methods
(19)AUSTRALIAN PATENT OFFICE (54) Title Arrays of cytosolic accessory proteinsimmobilized on a surface and pertinent methods (51)6 International Patent Classification(s) C07K 014/705 C07K 001/107 C07K 014/72 G01N 033/68 (21) Application No: 2003212526 (22) Application Date: 2003.03.13 (87) WIPO No: WO03/078464 (30) Priority Data (31) Number (32) Date 0205910.3 2002.03.13 (33) Country GB 200510277 (43) (43) Publication Date : 2003 .09.29 Publication Journal Date : 2003 .11.06 (71) Applicant(s) SENSE PROTEOMIC LIMITED (72) Inventor(s) Blackburn, Jonathan Michael; Hart, Darren James; Kozlowski, Roland; Davies, Andrew; Godber, Benjamin Leslie James(-1-1) Application NoAU2003212526 A8(19)AUSTRALIAN PATENT OFFICE (54) Title Arrays of cytosolic accessory proteinsimmobilized on a surface and pertinent methods (51)6 International Patent Classification(s) C07K 014/705 C07K 001/107 C07K 014/72 G01N 033/68 (21) Application No: 2003212526 (22) Application Date: 2003.03.13 (87) WIPO No: WO03/078464 (30) Priority Data (31) Number (32) Date 0205910.3 2002.03.13 (33) Country GB 200510277 (43) (43) Publication Date : 2003 .09.29 Publication Journal Date : 2003 .11.06 (71) Applicant(s) SENSE PROTEOMIC LIMITED (72) Inventor(s) Blackburn, Jonathan Michael; Hart, Darren James; Kozlowski, Roland; Davies, Andrew; Godber, Benjamin Leslie James-1- Arrays of cytosolic accessory proteins are provided, together with methods of screening, using such arrays. Such arrays comprise a surface having attached thereto at least one cytosolic accessory protein free from its membrane protein components or other subunits with which it is normally complexed. An array comprising a surface having attached thereto at least one cytosolic accessory protein of a membrane protein selected from ion channels, G protein coupled receptors and transmembrane transporter proteins, wherein said cytosolic accessory protein is free from membrane protein components or other subunits of said ion channel, G protein coupled receptor or transmembrane transporter protein complex. An array as claimed in claim 1, comprising a plurality of cytosolic accessory proteins selected from ion-channel subunits, G protein coupled receptor cytosolic accessory proteins, transmembrane transporter cytosolic accessory proteins, K+-channel β-subunits, Ca2+-channel β-subunits, G protein subtypes, Kv channel β-subunits, Calcium channel β-subunits, Gs family, Gt family, Gi family, Gi-0 family, Gq-11 family, Gα-sensory family and βγ family proteins. An array as claimed in claim 1, comprising a plurality of cytosolic accessory proteins which are identical and are selected from ion-channel subunits, G protein coupled receptor cytosolic accessory proteins, transmembrane transporter cytosolic accessory proteins, K+-channel β-subunits, Ca2+-channel β-subunits, G protein subtypes, Kv channel β-subunits, Calcium channel β-subunits, Gs family, Gt family, Gi family, Gi-0 family, Gq-1 1 family, Gα-sensory family and βγ family proteins. An array as claimed in claim 2 or 3, wherein the array comprises at least one K+-channel β-subunit selected from: β1.1, β1.2, β1.3, β2.1, β2.2, β3.1, β3.2 and β4. An array as claimed in claim 2 or 3, wherein the array comprises at least one calcium channel β-subunit selected from: β1a, β1b, β1c, β2a, β2b, β2c, β3a, β3b and β4. An array as claimed in any preceding claim, wherein the cytosolic accessory protein is an ion channel subunit domain. An array as claimed in any preceding claim, wherein cytosolic accessory protein subunits are provided as tagged protein constructs. An array as claimed in claim 7, wherein the tagged protein construct comprises an affinity tag. An array as claimed in claim 7 or claim 8, wherein the tag is a His, biotin, FLAG, myc, or VSV tag. An array as claimed in any preceding claim, wherein the protein moieties are attached to the surface via a common marker moiety. An array as claimed in any preceding claim, wherein each position in the array contains one or more copies of a single protein type in the form of a monomer, dimer, trimer, tetramer or higher multimer. A method for determining which cytosolic proteins interact with a given membrane protein, or vice versa, said method comprising the steps of :
(i) providing an array of cytosolic accessory proteins as claimed in any preceding claim;(ii) contacting the array with cytosolic fragments of said membrane protein and/or cytosolic fragments of other related membrane protein family members; and(iii) detecting and identifying the interacting partners. A method for screening compounds or peptides or proteins for the ability to interact selectively with a cytosolic protein, said method comprising the steps of :
(i) providing an array as claimed in any of claims 1-11;(ii) contacting the array with candidate compounds or peptides or proteins; and(iii) identifying the interacting partners. A method as claimed in claim 13, further comprising the step of quantitating the interaction of the interacting partners by measuring the binding or catalytic constants KD or KM, or by normalising amount bound against protein quantity. A method for screening compounds or peptides or proteins for the ability to selectively modulate the interaction between a cytosolic protein and a membrane protein, said method comprising the steps of:
(i) providing an array as claimed in any of claims 1-11;(ii) contacting the array with candidate compounds or peptides or proteins and with one or more membrane proteins or cytosolic fragments thereof, either simultaneously or in sequence; and(iii) determining whether said interaction is modulated by the presence of said compounds or peptides or proteins. A method according to claim 15 wherein the cytosolic fragment of a membrane protein is a soluble functional domain. A method as claimed in claim 15 or claim 16, further comprising the step of quantitating the degree of modulation of the interaction by measuring the binding or catalytic constants KD or KM, or by normalising amount bound against protein quantity. A method as claimed in any one of claims 15 to 16 wherein the concentration of a compound required to inhibit a given interaction is measured by determination of the IC50 value. The use of an array of cytosolic accessory proteins as claimed in any of claims 1-11 to measure the relative catalytic activity of different members of a family of said cytosolic accessory proteins. The use of an array of cytosolic accessory proteins as claimed in any of claims 1-11 as an affinity surface on which to select antibodies from a library of phenotype-genotype-linked antibodies (such as phage displayed antibodies). The use of an array of cytosolic accessory proteins as claimed in any of claims 1-11 for determining the effect of post-translational modifications on the interactions of said cytosolic accessory proteins with membrane proteins.