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 . 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. 
     
     
         2 . A mutant membrane protein according to  claim 1  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. 
     
     
         3 . A mutant membrane protein according to  claim 2  which is a transmembrane transporter. 
     
     
         4 . A mutant membrane protein according to  claim 3  which is a transmembrane transporter and a member of the neurotransmitter sodium symporter family (NSS). 
     
     
         5 . A mutant membrane protein according to  claim 4  which is the cocaine-sensitive rat serotonin transporter (SERT) protein. 
     
     
         6 . A mutant membrane protein according to  claim 3  which is norepinephrine (NET), dopamine (DAT) or the glycine transporter (GlyT). 
     
     
         7 . A mutant membrane protein according to  claim 5  which comprises one or more amino acid mutations selected from P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A. 
     
     
         8 . A mutant membrane protein according to  claim 7  comprising three mutations selected from G278A, A505L, L99A and P499A. 
     
     
         9 . A mutant membrane protein according to  claim 7  comprising mutations L99A, G278A and A505L. 
     
     
         10 . A mutant membrane protein according to  claim 7  comprising mutations L405A, P499A and A505L. 
     
     
         11 . A mutant membrane protein according to  claim 1  comprising at least one or more mutations which are at the corresponding amino acid positions of P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406A as defined in the amino acid sequence of SERT shown in  FIG. 9 . 
     
     
         12 . A mutant membrane protein according to  claim 1  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. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . A mutant membrane protein according to  claim 1  which is bound to a ligand. 
     
     
         16 .- 24 . (canceled)