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.
Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive ...antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks.
RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO2 and purified up to 40 mg/L. The progressive protein accumulation in the culture medium was monitored with an immunobinding assay in order to identify the optimal collection time. Successively, a two-step chromatographic protocol enabled its selective purification in the monomeric state. RBD quality assessment was positively evaluated by SDS-PAGE, Western Blotting and Mass Spectrometry, while Bio-Layer Interferometry, flow cytometer and ELISA tests confirmed its functionality. This effective protocol for the RBD production in transient eukaryotic system can be immediately extended to the production of RBD mutants.
•SARS CoV-2 RBD was obtained at high yields from transient expression in ExpiCHO cells.•RBD accumulation in culture media was monitored by BioLayer Interferormetry.•Protein quality assessment confirmed that RBD is monomeric and functional.•The obtained RBD was a reliable reagent for testing biological samples.
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