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Title: Comprehensive analysis and identification of the human STIM1 domains for structural and functional studies
Authors: Eshaghi, Said
How, Jonathan
Zhang, Ai
Phillips, Margaret
Reynaud, Aline
Lu, Si Yan
Pan, Lucy Xin
Ho, Hai Ting
Yau, Yin Hoe
Guskov, Albert
Pervushin, Konstantin
Shochat, Susana Geifman
Keywords: DRNTU::Science::Biological sciences
Issue Date: 2013
Source: How, J., Zhang, A., Phillips, M., Reynaud, A., Lu, S. Y., Pan, L. X., et al. (2013). Comprehensive Analysis and Identification of the Human STIM1 Domains for Structural and Functional Studies. PLoS ONE, 8(1).
Series/Report no.: PLoS ONE
Abstract: STIM1 is a Ca2+ sensor within the ER membrane known to activate the plasma membrane store-operated Ca2+ channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca2+ signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca2+ sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies.
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0053979
Rights: © 2013 The Author(s).
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Journal Articles

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