Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2.
A biotin-protein ligase recognition site (BRS) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (CB2), thioredoxin A, and a polyhistidine tag at the carboxy terminus. Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were approximately 1mg per liter of bacterial culture. The biotinylated CB2-fusion fully retained its ligand-binding capacity. Introduction of the BRS at the C-terminus of the CB2 fusion protein (construct CB2-109) resulted in its complete in vivo biotinylation; the biotinylated protein was streptavidin-binding competent. Positioning of the BRS near the N-terminus of CB2 (CB2-112) resulted in a very low level of biotinylation in vivo. However, the detergent solubilized and purified CB2-112 fusion protein were successfully biotinylated in vitro by action of a BirA biotin-protein ligase. The biotinylated CB2-112 fusion protein was cleaved by the tobacco etch virus protease at specifically inserted sites, and deposited onto monomeric avidin agarose beads. Biotinylation of the recombinant CB2 receptor enabled not only purification but also immobilization of the GPCR on a solid support in homogeneous orientation which is beneficial for subsequent structural characterization.
Krepkiy
Dmitriy
D
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcoholism and Alcohol Abuse, National Institutes of Health, 5625 Fishers Lane, Bethesda, MD 20892, USA.
Wong
Karen
K
Gawrisch
Klaus
K
Yeliseev
Alexei
A
D001667:Binding, Competitive; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008024:Ligands; D043885:Receptor, Cannabinoid, CB2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D008024:Ligands; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D010450:Endopeptidases; C099488:TEV protease