Abstract
The cDNAs encoding human delta (hDOR), kappa (hKOR) and micro (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.
Highlights
The cDNAs encoding human ␦, and opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag
The three opioid receptor subtypes were best expressed when infection was performed at a cell density of 1 ϫ 106 cells/ml with m.o.i. ϭ 2 for hMOR and m.o.i. ϭ 1 for human ␦ opioid receptor (hDOR) and hKOR (Fig. 1)
The Kd value for [3H]diprenorphine (0.8 Ϯ 0.4 nM) was comparable to that obtained for hMOR expressed in Sf9 (1.2 Ϯ 0.4 nM) and COS cells (0.23 nM) (Fig. 3). hMOR expressed in High Five cells retained its selectivity for the antagonist naloxonazine, whereas Ki values for the agonists DAMGO and dermorphin suggested that the receptor was in a low affinity binding state as inferred in Sf9 cells (Table V)
Summary
The cDNA encoding the human opioid receptor (hMOR) was isolated from human brain [12] and subcloned under the control of the polyhedrin promoter in the EagI and SmaI sites of pVL1392 (Pharmingen). The cells were washed three times with PBS, 0.1% Tween 20 and incubated for 4 h at room temperature in PBS, 0.1% Tween 20, 1% bovine serum albumin with a secondary anti-mouse antibody coupled to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories, Inc.) at a 1:200 dilution. After another wash with PBS, 0.1% Tween 20, nuclei were stained with 4,6-diamidino-2-phenylindole (Sigma), and cells were washed again.
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