The ER chaperone Calreticulin (CALR) has extracellular functions and can leave the mammalian cell in response to various factors although, the mechanism of how it is carried out is unknown. Yeast Saccharomyces cerevisiae efficiently secretes CALR, and the analysis of this process in yeast could help to clarify how it exits eukaryotic cells. We achieved about 140 mg/L secretion titer of CALR in our S. cerevisiae system. Here we present a comparative proteomics study in CALR secreting yeast using non-equilibrium pH gradient electrophoresis (NEPHGE)-based two-dimensional gel electrophoresis (2DE) and liquid chromatography-mass spectrometry (LC-MS) techniques. Our restored carrier ampholyte (CA) composition of NEPHGE-based first-dimension separation for 2DE proved it can be used instead of formerly commercially available gels. Using LC-MS we identified 1574 proteins, 20 out of which exhibited differential expression. Interestingly, we did not find any signs of cellular stress which is usually observed in recombinant protein producing yeast and did not identify any secretory pathway proteins that exhibited changes in expression. Taken together, high-level secretion of human recombinant CALR protein in S. cerevisiae does not induce cellular stress and does not burden cellular secretory machinery. There are only very small changes in the cellular proteome of yeast secreting CALR at the high level.
[doi:10.25345/C5J678X51]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Calreticulin, secretion, yeast, 2DE, LC-MS, proteomics, recombinant protein, cellular stress
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Algirdas Kaupinis, Proteomics Centre Institute of Biochemistry Vilnius University, Lithuania |
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