High efficient expression of the functional ligand binding site of the inositol 1,4,5-trisphosphate receptor in Escherichia coli

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1), an inositol 1,4,5-t risphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-termi nal ligand-binding region. Here we report an improved Escherichia coli expr ession system in which large amounts of the IP3 binding sites could be effi ciently produced as soluble active proteins. We have found that the structu res of IP3 binding constructs expressed in E. coli significantly affect the ir production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fra ction. As a result, soluble active T604 would be 19 mg per liter of culture . The affinity for IP3 of T604 (K-d = 45 nM) is comparable to that of the n ative IP(3)R1, whereas that of an R441Q mutant is much higher (8.1 nM). Thi s system should provide an invaluable and powerful means to unveil the mole cular recognition of IP(3)R1 for IP3. (C) 1999 Academic Press.