Protocol to identify host-viral protein interactions between coagulation-related proteins and their genetic variants with SARS-CoV-2 proteins.

Here, we describe a bioinformatics pipeline that evaluates the interactions between coagulation-related proteins and genetic variants with SARS-CoV-2 proteins. This pipeline searches for host proteins that may bind to viral protein and identifies and scores the protein genetic variants to predict the disease pathogenesis in specific subpopulations. Additionally, it is able to find structurally similar motifs and identify potential binding sites within the host-viral protein complexes to unveil viral impact on regulated biological processes and/or host-protein impact on viral invasion or reproduction. For complete details on the use and execution of this protocol, please refer to Holcomb et al. (2021).

An Optimized Purification Design for Extracting Active ADAMTS13 from Conditioned Media

ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies.