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J Vis Exp. 2017 Oct 4;(128). doi: 10.3791/56302.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins.

Author information

1
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario.
2
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario; Peter.Stathopulos@schulich.uwo.ca.

Abstract

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

PMID:
29053695
PMCID:
PMC5752362
[Available on 2019-10-04]
DOI:
10.3791/56302
[Indexed for MEDLINE]

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