Title Yeast secretes high amounts of human calreticulin without cellular stress /
Authors Zinkevičiūtė, Rūta ; Ražanskas, Raimundas ; Kaupinis, Algirdas ; Macijauskaitė, Neringa ; Čiplys, Evaldas ; Houen, Gunnar ; Slibinskas, Rimantas
DOI 10.3390/cimb44050122
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Is Part of Current issues in molecular biology.. Basel : MDPI. 2022, vol. 44, iss. 5, p. 1768-1787.. ISSN 1467-3037. eISSN 1467-3045
Keywords [eng] 2DE ; LC-MSE ; calreticulin ; cellular stress ; proteomics ; recombinant protein ; secretion ; yeast
Abstract [eng] The ER chaperone calreticulin (CALR) also has extracellular functions and can exit the mammalian cell in response to various factors, although the mechanism by which this takes place is unknown. The yeast Saccharomyces cerevisiae efficiently secretes human CALR, and the analysis of this process in yeast could help to clarify how it gets out of eukaryotic cells. We have achieved a secretion titer of about 140 mg/L CALR in our S. cerevisiae system. Here, we present a comparative quantitative whole proteome study in CALR-secreting yeast using non-equilibrium pH gradient electrophoresis (NEPHGE)-based two-dimensional gel electrophoresis (2DE) as well as liquid chromatography mass spectrometry in data-independent analysis mode (LC-MSE). A reconstructed carrier ampholyte (CA) composition of NEPHGE-based first-dimension separation for 2DE could be used instead of formerly commercially available gels. Using LC-MSE, we identified 1574 proteins, 20 of which exhibited differential expression. The largest group of differentially expressed proteins were structural ribosomal proteins involved in translation. Interestingly, we did not find any signs of cellular stress which is usually observed in recombinant protein-producing yeast, and we 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 the cellular secretory machinery. There are only small changes in the cellular proteome of yeast secreting CALR at a high level.
Published Basel : MDPI
Type Journal article
Language English
Publication date 2022
CC license CC license description