Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions |
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Authors: | Nyasha J. Makoni Evan C. Garrad Adela Redzic Michael R. Nichols |
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Affiliation: | Department of Chemistry and Biochemistry, University of Missouri-St. Louis, St. Louis, Missouri, USA |
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Abstract: | The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization. |
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Keywords: | ASC caspase-1 ExpiCHO-S Inflammasome NLRP3 |
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