Membrane proteins are the most pharmaceutically relevant protein class. At the same time, it is very difficult to obtain them in pure, active form. Cube Biotech's protein experts have worked hard to produce human GPCRs in sufficient quality that we can now offer to the community. Our first available pure, active membrane protein is GPBAR1 / TGR5, a human GPCR involved in bile acid homeostasis, glucose metabolism, and weight maintenance. The protein is available off-the shelf, ready for ligand binding studies, crystallization, and other biochemical/biophysical experiments.
Fig. 1: SDS-PAGE of GPBAR1, purified via Rho1D4 affinity chromatography. L: Lysate: FT: flow through, W: Wash, E1-E3: elution fractions with purified GPCR.
Fig. 2: Surface plasmon resonance measurement of GPBAR-1 / Taurolithocholic acid sulfate salt interaction. Using a 1:1 binding model, the Kd was determined to be 1.1x10e-8 M.
||GPBAR1 (G-protein coupled bile receptor 1)
||TGR5, GPCR19, M-BAR, BG37
|UniProt / UniGene
||Q8TDU6 / 155918
||GPCR, class A (Rhodopsin-like receptors)
||Human (Homo sapiens)
Full-length, wildtype sequence N-terminal HA signal sequence (underlined), 10x His-tag with spacer (red), HRV 3C protease site (blue) spacer (gray), Rho1D4 tag (green)
MKTIIALSYIFCLVFAMTPNSTGEVPSPIPKGALGLSLALASLIIT ANLLLALGIA WDRRLRSPPA GCFFLSLLLA GLLTGLALPT LPGLWNQSRR GYWSCLLVYL APNFSFLSLL ANLLLVHGER YMAVLRPLQP PGSIRLALLL TWAGPLLFAS LPALGWNHWT PGANCSSQAI FPAPYLYLEV YGLLLPAVGA AAFLSVRVLA TAHRQLQDIC RLERAVCRDE PSALARALTW RQARAQAGAM LLFGLCWGPY VATLLLSVLA YEQRPPLGPG TLLSLLSLGS ASAAAVPVAM GLGDQRYTAP WRAAAQRCLQ GLWGRASRDS PGPSIAYHPS SQSSVDLDLNGGHHHHHHHHHHLEVLFQGPGS SGTETSQVAPA
||His / Rho1D4 (both C-terminal)
|Size (excluding additional elements)
||379 (330) amino acids 40,606 (35,248) Da
||PureCube Rho1D4 Agarose
||100 mM NaCl, 20 mM NaH2PO4 pH 7.4, 0.1% Foscholine-12, 0.01% cholesterolhemisuccinate (CHS)
||>98%, see Fig. 1
||Size exclusion chromatography
||Ligand binding measured by SPR using Taurolithocholic Acid Sulfate Disodium Salt (CAS 64936-83-0) at 10°C. Using a 1:1 binding model, the dissociation constant (Kd) was determined to be 1.1x10e-8 M.
||Receptor for bile acid. Bile acid-binding induces its internalization, activation of extracellular signal-regulated kinase and intracellular cAMP production. May be involved in the suppression of macrophage functions by bile acids, and in modulating inflammation.
- Kawamata, Yuji, et al. "AG protein-coupled receptor responsive to bile acids." Journal of Biological Chemistry 278.11 (2003): 9435-9440.