Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

Abstract

Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor.

Simplifying G Protein-Coupled Receptor Isolation with a Calcium-Dependent Fragment Complementation Affinity System

“…The autocleavage of the MBP-CB 2 fusion protein prior to or during the purification procedure at the N-terminally located TEV cleavage site is significant, although the strong signal seen on the stained gel is due to both the 47 kDa fragment and the 45 kDa MBP-containing fragment and the associated signal for the CB 2 fragment is lower than that for the full-length protein indicating that some of the fusion protein is still uncleaved. Partial self-cleavage of the MBP-CB 2 fusion constructs prior to TEV protease treatment has been observed for CB 2 -125 and CB 2 -130 constructs previously. , The 106 kDa CB 2 -354 receptor fusion is efficiently enriched to a high level of purity in this single step despite evidence of limited proteolysis evident from the immunostaining at multiple molecular weights. It is important to take into account though that the intensity of bands in the Instant Blue-stained gels do not adequately represent the true purity of the CB 2 preparations, since the bulk of hydrophobic CB 2 protein is screened from interaction with the dye by a large detergent belt.…”

“…Recombinant CB 2 fusions in E. coli BL21 (DE3) cells were expressed in double strength YT broth (16 g of digest peptone, 10 g of yeast extract, and 5 g of NaCl per liter), supplemented with ampicillin (50 mg L –1 ) and glucose (0.2% (w/v)) as described previously …”

Overproduction of Human Zip (SLC39) Zinc Transporters in Saccharomyces cerevisiae for Biophysical Characterization

“…To corroborate this possible effect of the charged tag on production, we included a construct where the His-tag was exchanged to StrepII-tag. StrepII-tag, an octa-amino acid polypeptide, typically does not alter bioactivity of its fusion partner and can be used for affinity purification under mild conditions, maintaining the functionality of produced protein [ 71 ]. Interestingly, the production levels of the StrepII-TEV-hZIP1 variant were marginally decreased compared to the His-TEV-hZIP1 construct, again without any detectable changes in detergent extraction efficacy ( Figure S2B ).…”

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB 2 , a G protein-coupled cannabinoid receptor

“…We previously described the tandem affinity chromatography for purification of CB 2 from detergent-containing solutions, using a combination of the IMAC and the chromatography on a Strep-Tactin resin. (3, 8, 16, 19) . This allows isolation of the full-length CB 2 receptor with high yield and purity.…”

“…The Strep-tag system has been thoroughly described in various articles and reviews (8, 16-18, 22-26) . Currently, two different streptavidin binding tags are being used most commonly: the Strep-tag II (WSHPQFEK) (22) , and its tandem variant– the Twin-Strep-tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) (22) .…”