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Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13419-24. Epub 2002 Oct 7.

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.

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Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.


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.

[Indexed for MEDLINE]
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