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

Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13419-24. doi: 10.1073/pnas.212519299. Epub 2002 Oct 7.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bioreactors
  • Cattle
  • Cell Line
  • Gene Expression / drug effects
  • Glycosylation
  • Humans
  • In Vitro Techniques
  • Mutation
  • N-Acetylglucosaminyltransferases / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Rhodopsin / chemistry*
  • Rhodopsin / genetics
  • Rhodopsin / metabolism*
  • Ricin / pharmacology
  • Rod Opsins / genetics
  • Tetracycline / pharmacology
  • Transfection

Substances

  • Recombinant Proteins
  • Rod Opsins
  • Rhodopsin
  • Ricin
  • N-Acetylglucosaminyltransferases
  • alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
  • Tetracycline