title: Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line.
abstract: An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI(-) HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI(-) stable cell line showed it to be Man(5)GlcNAc(2). In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI(-)-inducible cell line at levels comparable with those obtained with WT rhodopsin.
authors.affiliation: Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.forename: Roland
authors.initials: R
authors.lastname: Contreras
authors.forename: H Gobind
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000818:Animals; D019149:Bioreactors; D002417:Cattle; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012276:Ricin / Q000494:pharmacology; D017299:Rod Opsins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D012276:Ricin; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D013752:Tetracycline