Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete Mucor circinelloides
2003
Houghton-Larsen, J. | Pedersen, P.A.
We have produced the rat adenosine A1 receptor in Mucor circinelloides using a translational fusion to the endogenous glucoamylase (GlaM) gene. The fusion protein produced from an episomal plasmid was correctly processed as judged by western blotting, since only a 33 kDa band was detected in membrane preparations from M. circinelloides expressing the receptor. This corresponds to the mass of the full-length receptor released from the fused GlaM protein. The presence of a high affinity binding site with a Kd value of 0.5 nM for the receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in membrane preparations suggests that the receptor was correctly folded and inserted into the membranes. A receptor expression level of 100-300 fmol/mg total membrane protein was achieved as judged by binding of the antagonist [3H]-DPCPX.
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