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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
Open accessAbstract
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.
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Measurement of protein using bicinchoninic acid
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., & Klenk, D. C. (1985) · Analytical Biochemistry · doi:10.1016/0003-2697(85)90442-7 · Cited by 18,482
Nathans, J. (1990) · Biochemistry · doi:10.1021/bi00493a034 · Cited by 323
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Scally, S. W., Petersen, J., Law, S. C., Dudek, N. L., Nel, H. J., Loh, K. L., Wijeyewickrema, L. C., Eckle, S. B. G., van Heemst, J., Pike, R. N., et al. (2013) · Journal of Experimental Medicine · doi:10.1084/jem.20131241 · Cited by 429
Glycans on influenza hemagglutinin affect receptor binding and immune response
Wang, C.-C., Chen, J.-R., Tseng, Y.-C., Hsu, C.-H., Hung, Y.-F., Chen, S.-W., Chen, C.-M., Khoo, K.-H., Cheng, T.-J., Cheng, Y.-S. E., et al. (2009) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0909696106 · Cited by 316
Metadata
- Authors
- Reeves, P. J., Callewaert, N., Contreras, R., & Khorana, H. G.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 2002-10-07
- Volume
- 99
- Issue
- 21
- Pages
- 13419-13424
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W2052745304
- Cited by
- 640
- References
- 2
- FWCI
- 2.05
- Type
- journal article
- Language
- en
- Open access
- green
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