Development and expression of a factor X-derived recombinant protein for ELISA-based detection of Emicizumab

Emicizumab is a therapeutic antibody used in the management of Hemophilia A. It mimics the function of coagulation Factor VIII by bridging activated Factor IX (FXa) and Factor X (FX), thereby facilitating the activation of FX and promoting blood coagulation. Due to its prolonged half-life in patient plasma following administration, accurate detection of Emicizumab is clinically important. Enzyme-linked immunosorbent assay (ELISA) is a valuable method for monitoring Emicizumab levels, but it requires proteins that bind with high affinity to the antibody. In this study, we engineered and expressed a recombinant fragment of Factor X containing the EGF-like 2 domain, designed to bind Emicizumab. A gene fragment encoding the EGF-like 2 domain of Factor X, previously identified as an Emicizumab-binding region, was codon-optimized and cloned into pUC57, then subcloned into the pET28a expression vector. Escherichia coli DH5α and E. coli Shuffle T7 (DE3) strains were used as cloning and expression hosts, respectively. The recombinant protein was purified and verified using 10 % SDS-PAGE, Bradford assay, and Western blot. Following expression, the recombinant protein was purified and quantified, with concentrations of 119 μg/mL for native and 81 μg/mL for denatured protein. These values correspond to total recoveries of 255 μg and 65 μg, respectively, from 50 mL cultures. These findings validate the use of this bacterially expressed FX-derived protein as a candidate component for incorporation into ELISA-based platforms aimed at Emicizumab detection.