High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli.
Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.
Yoshikawa
F
F
Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo, Minato-ku, 108-8639, Japan.
Uchiyama
T
T
Iwasaki
H
H
Tomomori-Satoh
C
C
Tanaka
T
T
Furuichi
T
T
Mikoshiba
K
K
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D008024:Ligands; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C081655:inositol-1,3,4,5-tetrakisphosphate; D010833:Phytic Acid; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; C062616:inositol 2,4,5-trisphosphate