Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor.
The human peripheral cannabinoid receptor (CB2) was expressed as a fusion with the maltose-binding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag). Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2mg per liter of bacterial culture. The recombinant receptor was ligand binding-competent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. Maltose-binding protein, thioredoxin and a decahistidine tag were removed from the fusion by treatment with Tobacco etch virus (Tev) protease. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy indicated an alpha-helical content of the purified recombinant protein of approximately 54%. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results of reconstitution experiments indicate that the CB2 has retained its ligand-binding properties.
Yeliseev
Alexei
A
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, Bethesda, MD 20892, USA. yeliseeva@mail.nih.gov
Zoubak
Lioudmila
L
Gawrisch
Klaus
K
D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D058619:Maltose-Binding Proteins; D011485:Protein Binding; D017433:Protein Structure, Secondary; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins; C517609:TrxA protein, E coli; D006639:Histidine; D013879:Thioredoxins; D019809:Streptavidin; D010450:Endopeptidases; C099488:TEV protease