Solving the Glycosylation Problem in Glycoprotein Structural Genomics
Author Information
Author(s): Chang Veronica T., Crispin Max, Aricescu A. Radu, Harvey David J., Nettleship Joanne E., Fennelly Janet A., Yu Chao, Boles Kent S., Evans Edward J., Stuart David I., Dwek Raymond A., Jones E. Yvonne, Owens Raymond J., Davis Simon J.
Primary Institution: University of Oxford
Hypothesis
Can transient expression of glycoproteins in mammalian cells with N-glycosylation inhibitors enhance crystallization for structural analysis?
Conclusion
The study demonstrates that using kifunensine or swainsonine during transient expression of glycoproteins in HEK293T cells can improve their crystallization properties.
Supporting Evidence
- Transient expression in mammalian cells can yield glycoproteins suitable for crystallization.
- Kifunensine and swainsonine enhance the sensitivity of glycoproteins to endo H.
- The methods developed can potentially be applied to other mammalian expression systems.
Takeaway
This research shows that adding special chemicals when making proteins in cells can help scientists see their shapes better.
Methodology
Glycoproteins were expressed in HEK293T cells with N-glycosylation inhibitors and analyzed for endo H sensitivity and crystallization.
Limitations
The expression levels in GnTI-deficient cells were lower than in HEK293T cells, which may limit high-throughput applications.
Digital Object Identifier (DOI)
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