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Membrane protein; Heterologous expression; Mouse 5-HT5A receptor: Receptor affinity; Immunogold electron microscopy
Abstract:
The methylotrophic yeast Pichia pastoris was tested for heterologous expression of the mouse 5-HT5A receptor. Three different expression plasmids were constructed where the cDNA of the receptor was cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gen. The expression plasmids differed with respect to the signal sequences used for N-terminal fusion. In two cases the coding region was additionally fused to the c-myc tag to permit immunological detection of the receptor. Expression of functional receptor after transformation of strain GS115 was detected by radioligand binding using [3H]LSD. The construct with the best expression levels in strain GS115 was used for transformation of the protease deficient strain SMD1163. Here, the expression level was 2-8 times higher. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of [3H]LSD with a Kd of approximately 1.9 nM. Receptor concentrations of approximately 22 pmol/mg membrane protein revealed the potential of the P. pastoris expression system for high level expression of membrane proteins. The pharmacological properties were comparable to those reported for the receptor expressed in mammalian systems.
