Glycoprotein hormone receptor mutations

Abstract

A mutant thyroid stimulating hormone receptor (TSHR) or fragment thereof comprises one or more mutations, wherein the mutant TSHR has increased thermostability with respect to the equivalent wild type TSHR or fragment. The one or more mutation is preferably within the extracellular leucine-rich repeat domain (LRD) of the TSHR, or within residues 22 to 260 (TSHR260) of the TSHR, or may be in the transmembrane domain (TMD), A mutant TSHR or fragment thereof of the invention preferably consists of, or consists essentially of, the subdomain TSHR260 of the TSHR receptor. A mutant TSHR or fragment thereof according to the invention has a greater thermostability than the equivalent wild type TSHR or fragment as determined by its half-life at a given temperature, and can be purified whilst retaining activity. A mutant TSHR or fragment thereof according to the invention may also be deglycosylated whilst retaining activity. Methods, kits and uses employing the mutant TSHR or fragment thereof according to the invention are also provided.

1 . A mutant thyroid stimulating hormone receptor (TSHR) which is a mutated form of human wild type TSHR as shown in SEQ ID NO 2 or fragment thereof, which mutant or fragment thereof is able to bind TSHR autoantibodies, wherein the mutant or fragment thereof comprises at least 80% or more of SEQ ID No 2, or wherein the fragment comprises residues 22-260 of the TSHR as shown in SEQ ID NO 4 or a sequence having 80% sequence identity therewith, and wherein the mutant or fragment thereof comprises one, two, three, four, five or six mutation(s) within said residues 22 to 260 of the TSHR selected from: P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D143P, D151E, S166T, I167F, P168Y, V169R, N170W, T179C, I253R and R255Y, wherein the mutant TSHR or fragment thereof has increased thermostability with respect to the equivalent wild type TSHR or fragment thereof, wherein thermostability refers to the half-life of the mutant TSHR or fragment thereof, as compared to the half-life of the equivalent wild type TSHR or fragment thereof, as measured under identical conditions in a binding assay which determines the amount of mutant TSHR or fragment thereof that retains the ability to bind, at the test temperature, an autoantibody selected from the group consisting of: M22, K1-70; and K1-18, wherein the half-life of the mutant or fragment at 42° C. is 1.5 times greater or more than the half-life of the equivalent wild type TSHR or fragment. 
     
     
         2 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant comprises two point mutations, one of which is I253R and the second of which is selected from: P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D143P, D151E, S166T, 1167F, P168Y, V169R, N170W, T179C, and R255Y. 
     
     
         3 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant comprises three point mutations, one of which is I253R, the second of which is D143P, and the third of which is selected from: P28E, L59F, T62V, H63C, L64Y, R112P, P142I, D151E, S166T, P168Y, V169R and N170W. 
     
     
         4 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant comprises four point mutations, one of which is I253R, the second of which is D143P, the third of which is R112P, and a fourth of which is selected from: L59F, H63C, D151E, S166T, V169R and N170W. 
     
     
         5 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant comprises five point mutations, one of which is I253R, the second of which is D143P, the third of which is R112P, a fourth of which is D151E or H63C, and a fifth of which is selected from: L59F, H63C, D151E, S166T and V169R. 
     
     
         6 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant comprises six point mutations, one of which is I253R, the second of which is D143P, the third of which is R112P, a fourth of which is D151E, a fifth of which is H63C, and a sixth of which is either S166T or V169R. 
     
     
         7 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant comprises a set of mutations selected from the group consisting of:
 1) I253R   2) D143P+I253R   3) R112P+D143P+I253R   4) R112P+D143P+D151E+I253R   5) R112P+D143P+D151E+V169R+I253R   6) H63C+R112P+D143P+D151E+V169R+I253R   7) H63C+R112P+D143P+V169R+I253R   8) H63C+R112P+D143P+S166T+I253R.   
     
     
         8 . The mutant TSHR or fragment thereof according to  claim 1 , which binds a TSHR autoantibody selected from the group consisting of: M22, K1-70; and K1-18. 
     
     
         9 . The mutant TSHR or fragment thereof according to  claim 1 , further comprising one or more mutation(s) in the transmembrane domain of the TSHR (TMD). 
     
     
         10 . The mutant TSHR or fragment thereof according to  claim 9  wherein the one or more mutations within the transmembrane domain (TMD) are selected from the group consisting of: E409K, D410K, H443N, L452Y, N455A, M463V, Y466F, L467P, T477I, Q489H, K565L, V595I, C600R, Y601F, I648L, K660D, Y667V, S671A, Y678L, Y678A, which mutations are as shown with reference to the said wild type TSHR in SEQ ID Nos 89-108. 
     
     
         11 . The mutant TSHR or fragment thereof according to  claim 9 , wherein the one or more mutations within the transmembrane domain (TMD) contains two point mutations, one of which is T477I or V595I or I648L, and the second of which is a different mutation selected from the group consisting of: E409K, D410K, H443N, L452Y, N455A, M463V, Y466F, L467P, T477I, Q489H, K565L, V595I, C600R, Y601F, I648L, K660D, Y667V, S671A, Y678L, Y678A. 
     
     
         12 . The mutant TSHR or fragment thereof according to  claim 9 , wherein the one or more mutation(s) within the transmembrane domain (TMD) contains three point mutations, one of which is V595I, the second of which is Y678L or K565L, and the third of which is selected from the group consisting of: E409K, D410K, H443N, L452Y, N455A, M463V, Y466F, L467P, T477I, Q489H, K565L, C600R, Y601F, I648L, K660D, Y667V, S671A, Y678L, Y678A. 
     
     
         13 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant further comprises a detectable label selected from the group consisting of: enzymatic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes, alkaline phosphatase (AP) labels and biotin labels. 
     
     
         14 . The mutant TSHR or fragment thereof according to  claim 13 , wherein the mutant further comprises an alkaline phosphatase (AP) label. 
     
     
         15 . The mutant TSHR or fragment thereof according to  claim 14 , selected from the group consisting of:
 TSHR260-AP-I253R=TSHR260-AP+I253R   TSHR260-AP-JMG22=TSHR260-AP+D143P+I253R   TSHR260-AP-JMG37=TSHR260-AP+R112P+D143P+I253R   TSHR260-AP-JMG45=TSHR260-AP+R112P+D143P+D151E+I253R   TSHR260-AP-JMG52=TSHR260-AP+R112P+D143P+D151E+V169R+I253R   TSHR260-AP-JMG55=TSHR260-AP+H63C+R112P+D143P+D151E+V169R+I253R   TSHR260-AP-JMG57=TSHR260-AP+H63C+R112P+D143P+V169R+I253R   TSHR260-AP-JMG58=TSHR260-AP+H63C+R112P+D143P+S166T+I253R,   wherein TSHR260 consists of residues 22-260 of the TSHR as shown in SEQ ID NO 4, and AP indicates an alkaline phosphatase label.   
     
     
         16 . A method of purifying a mutant TSHR or fragment thereof as defined in  claim 1 , which method comprises:
 i) purifying a composition, which composition comprises the mutant TSHR or fragment thereof and optionally culture supernatant, wherein the purifying is by column chromatography using cation or anion exchange chromatography; optionally wherein the method further comprises, either before or after step (i), purifying the said composition by affinity chromatography, optionally wherein the affinity chromatography comprises antibody affinity chromatography and/or metal ion affinity chromatography; and   ii) collecting the purified mutant TSHR or fragment thereof.   
     
     
         17 . The method according to  claim 16 , wherein the method comprises:
 i) purifying a composition comprising the mutant or fragment thereof and optionally culture supernatant, wherein the purifying is by cation-exchange or anion-exchange column chromatography ii) further purifying the mutant or fragment thereof by antibody affinity chromatography; optionally wherein the antibody is a monoclonal antibody which binds a conformational epitope within the TSHR extracellular domain;   iii) optionally further purifying the mutant or fragment thereof by metal-ion affinity chromatography, which optionally comprises nickel-affinity chromatography;   iv) collecting the purified mutant TSHR or fragment thereof.   
     
     
         18 . The method according to  claim 16 , wherein the mutant consists of, or consists essentially of, the subdomain TSHR260 of the TSHR as shown in SEQ ID NO 4 and wherein the mutant consists of one of the following set of mutations:
 1) I253R   2) D143P+I253R   3) R112P+D143P+I253R   4) R112P+D143P+D151E+I253R   5) R112P+D143P+D151E+V169R+I253R   6) H63C+R112P+D143P+D151E+V169R+I253R   7) H63C+R112P+D143P+V169R+I253R   8) H63C+R112P+D143P+S166T+I253R.   
     
     
         19 . Purified mutant TSHR or fragment thereof as defined in  claim 1 , which purified mutant or fragment is produced by a method comprising:
 i) purifying a composition, which composition comprises the mutant TSHR or fragment thereof and optionally culture supernatant, wherein the purifying is by column chromatography using cation or anion exchange chromatography; optionally wherein the method further comprises, either before or after step (i), purifying the said composition by affinity chromatography, optionally wherein the affinity chromatography comprises antibody affinity chromatography and/or metal ion affinity chromatography; and   ii) collecting the purified mutant TSHR or fragment thereof.   
     
     
         20 . The mutant TSHR or fragment thereof according to  claim 1 , wherein the mutant TSHR or fragment thereof is deglycosylated.