Cambio - Excellence in Molecular Biology

Membrane protein work

Membrane protein work: Nanodisc Active GPCR

Nanodiscs provide an exciting alternative for the stabilization of membrane proteins. Cube Biotech offers everything you need to get started with this novel technology. There are two options to reconstitute membrane proteins into nanodiscs:

GPBAR1 (TGR5) protein

GPBAR1 (TGR5) protein

Cambio

Description

Active, purified GPBAR1 (G-protein coupled bile receptor 1)

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 11 nM.

 

 

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Notes

Protein

GPBAR1 (G-protein coupled bile receptor 1)

Alternative names

 TGR5, GPCR19, M-BAR, BG37

UniProt / UniGene

Q8TDU6 / 155918

Protein class

 GPCR, class A (Rhodopsin-like receptors)

Organism

 Human (Homo sapiens)

Sequence
Full-length, wildtype sequence
N-terminal HA tag (underlined), initial methionine in bold, C-terminal 10x His-tag in red,
HRV 3C protease site in blue, spacer in bold grey, Rho1D4 tag in bold green
 
MKTIIALSYIFCLVFA MTPNSTGEVP SPIPKGALGL SLALASLIIT 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 SQSSVDLDLNGGHHHHHHHHHHLEVLFQGPGSSGTETSQVAPA

Affinity tags

 His / Rho1D4 (both C-terminal)
Size (excluding
additional elements)
379 (330) amino acids
40,606 (35,248) Da

Expression system

 Sf9 (baculovirus)

Purified via

 PureCube Rho1D4 Agarose

Buffer

 100 mM NaCl, 20 mM NaH2PO4 pH 7.4, 0.1% Foscholine-12, 0.01% cholesterolhemisuccinate (CHS)

Purity (SDS-PAGE)

 >98%, see Fig. 1

Homogeneity

 Size exclusion chromatography

Activity
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 kD was determined to be 11 nM.

Function
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.
Literature  references:
1. Kawamata, Y. et al. (2003) A G Protein-coupled receptor responsive to bile acids, J. Biol. Chem. 278, 11, pp9435-9440
2. Chen, X. et al. (2011) TGR5: A Novel Target for Weight Maintenance and Glucose Metabolism. Exp. Diabetes Res. Article ID 853501
3. Pols, T.W.H. et al. (2013) The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation. J. Hepatol. 54(6):1263-1272

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

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If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200