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

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

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.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • Cysteine / metabolism*
  • Glycosylation
  • Humans
  • Magnetic Resonance Spectroscopy / methods*
  • Mass Spectrometry / methods*
  • Recombinant Proteins / metabolism*
  • Sulfhydryl Compounds / metabolism*

Substances

  • Recombinant Proteins
  • Sulfhydryl Compounds
  • Cysteine