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 baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter. We investigated the influence of different molecular constructions on receptor expression levels: the receptor was fused either to an amino- or a carboxy-terminal histidine tag (hMOR-N-His and hMOR-C-His respectively), or to the cleavable sequence signal of the baculovirus gp64 glycoprotein (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 culture 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 obtained 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 carboxy-terminal position.
Massotte
D
D
Département des Récepteurs et Protéines membranaires, CNRS UPR 9050, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch-Graffenstaden, France. massotte@esbsl.u-strasbg.fr
Pereira
C A
CA
Pouliquen
Y
Y
Pattus
F
F
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003470:Culture Media; D015870:Gene Expression; D006801:Humans; D007915:Lepidoptera; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera
D003470:Culture Media; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins