Mutant proteins and methods for their production

Abstract

The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallisation studies and also in screening to identify compounds for use in drug discovery and therapy.

1 .- 24 . (canceled) 
     
     
         25 . A mutant membrane protein which has increased conformational stability compared to its parent membrane protein, wherein the one or more mutations are located at the interfaces between transmembrane alpha-helices, or in a kinked region or in an alpha-helix adjacent to a kink. 
     
     
         26 . A mutant membrane protein according to  claim 25  which is a T-cell receptor complex, a growth factor receptor, a ligand-gated transmembrane ion channel, a voltage-gated transmembrane ion channel, a transmembrane transporter, an enzyme, a carrier protein, or an ion pump. 
     
     
         27 . A mutant membrane protein according to  claim 26  which is a transmembrane transporter; optionally wherein the transmembrane transporter is a member of the solute carrier proteins (SLC) family. 
     
     
         28 . A mutant membrane protein according to  claim 27  which is a transmembrane transporter and a member of the neurotransmitter sodium symporter family (NSS). 
     
     
         29 . A mutant membrane protein according to  claim 28  which is a member of the SLC6 subclass of the NSS family; optionally a mammalian SLC6 protein. 
     
     
         30 . A mutant membrane protein according to  claim 29  wherein the mutant membrane protein is a norepinephrine transporter (NET) protein, a dopamine transporter (DAT) protein, or the a glycine transporter (GlyT) protein, or a serotonin transporter (SERT) protein. 
     
     
         31 . A mutant membrane protein according to  claim 29  which is a monoamine transporter protein. 
     
     
         32 . A mutant membrane protein according to  claim 31  which is a norepinephrine transporter (NET) protein, a dopamine transporter (DAT) protein, or a serotonin transporter (SERT) protein. 
     
     
         33 . A mutant membrane protein according to  claim 25 , wherein the mutant membrane protein is a serotonin transporter (SERT) protein; optionally a mammalian SERT protein . 
     
     
         34 . A mutant membrane protein according to  claim 25 , wherein the one or more mutations comprises three or more mutations. 
     
     
         35 . A mutant membrane protein according to  claim 25 , wherein each of the one or more mutations is i) an amino acid residue replaced with alanine or ii) if the amino acid residue was already alanine, then it is replaced with leucine. 
     
     
         36 . A mutant membrane protein according to  claim 25 , wherein the mutant membrane protein comprises at least one or more mutations which are within a window of amino acids either side of the position which corresponds to the amino acid positions of one or more of P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A as defined in the amino acid sequence of the serotonin transporter (SERT) shown in  FIG.  9   ; wherein the window of amino acids refers to a window of i plus or minus four amino acid residues where i is defined as the stabilising mutation. 
     
     
         37 . A mutant membrane protein according to  claim 25  which has increased conformational stability when compared to its parent to one or more of heat, a detergent, a chaotrope or an extreme of pH. 
     
     
         38 . A mutant membrane protein according to  claim 25  which is bound to a ligand.