Recombinant human SMOCs produced by in vitro refolding: calcium-binding properties and interactions with serum proteins

Protein Expression and Purification
Marko NovinecBrigita Lenarcic

Abstract

Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. In this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K(d) values in the 0.7-1 microM range), while the other had lower affinity (K(d) values 20-25 microM). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and memb...Continue Reading

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Citations

Oct 27, 2010·Expert Review of Proteomics·Dimitrios IliopoulosAspasia Tsezou
Jan 18, 2012·The International Journal of Biochemistry & Cell Biology·Amy D Bradshaw
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Jan 25, 2018·Environmental Technology·Ishak MoghrabiDaniel Levacher
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Sep 4, 2020·Science Translational Medicine·Magdalene K MontgomeryMatthew J Watt

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