Parameters influencing human mu opioid receptor over-expression in baculovirus-infected insect cells

The cDNA encoding the human mu opioid receptor (hMOR) was cloned in the bac ulovirus Autographa californica (AcMNPV) under the control of the polyhedri n promoter. We investigated the influence of different molecular constructi ons on receptor expression levels: the receptor was fused either to an amin o- or a carboxy-terminal histidine tag (hMOR-N-His and hMOR-C-His respectiv ely), or to the cleavable sequence signal of the baculovirus gp64 glycoprot ein (gp-hMOR and gp-hMOR-C-His). Two cell lines, Spodoptera frugiperda (Sf9 ) and Trichoplusia ni (BTI-TN-5B1-4), in combination with three different c ulture media were also tested for their ability to produce maximal protein expression. Molecular constructions and culture conditions were both shown to influence substantially protein production. The best results were obtain ed using cells adapted to serum-free medium combined with constructions in fusion with the endogenous signal sequence of the baculovirus gp64 protein. Those conditions led to maximal expression and shortened the time required for receptor production. We also showed that an amino-terminal location of a hexahistidine tag was more detrimental to the expression level than a ca rboxy-terminal position. (C) 1999 Elsevier Science B.V. All rights reserved .