Stable proteins

A fusion protein comprising, from N-terminus to C-terminus:

• a. a first portion of a Family B G-protein coupled receptor (GPCR) that comprises transmembrane helix (TM)-1, TM2 and TM3 of the GPCR, wherein the TM1 and the TM2 are joined by intracellular loop 1 (ICL1), and the TM2 and TM3 are joined by extracellular loop 1 (ECL1);
• b. a stable protein domain;
• and
• c. a second portion of the GPCR comprising TM4, TM5, TM6 and TM7 of the GPCR wherein the TM4 and the TM5 are joined by extracellular loop 2 (ECL2), the TM5 and the TM6 are joined by intracellular loop 3 (ICL3), and the TM6 and the TM7 are joined by extracellular loop 3 (ECL3);
• wherein the stable protein domain comprises a soluble, well-folded polypeptide that provides N- and C-termini, the distance between which approximates the distance between helices 3 and 4 in the Family B GPCR, and that provides a hydrophilic surface for crystal lattice contacts, thereby facilitating crystallisation,
• wherein the stable protein domain is inserted into the intracellular loop 2 (ICL2) region of the GPCR which loop joins the TM3 in the first portion of the GPCR and the TM4 in the second portion of the GPCR, and
• wherein the fusion protein displays reduced aggregation in the presence of a detergent solution, as compared to a GPCR without insertion of the stable protein domain into the ICL2 region of the GPCR.