Optimization of the recombinant overexpression and purification of human Nedd4-1 in Escherichia coli.

The E3 ubiquitin ligase Nedd4-1 is a structurally complex, multidomain enzyme that plays a crucial role in maintaining proteostasis and regulating the cellular stress response. Nedd4-1's complex regulatory mechanism involves both intermolecular interactions (with upstream E2 conjugating enzymes and substrates) and intramolecular interactions that govern its function. Additionally, Nedd4-1 has received increased attention recently due to a small-molecule N-arylbenzimidazole 2 (NAB2) that prevents proteotoxicity and restores organelle trafficking associated with synucleopathies in a Nedd4-1-dependent manner. To study the enzymology of Nedd4-1, it is essential to employ recombinant Nedd4-1 in its native and untagged form to ensure high-fidelity biophysical characterization. In this study, we develop an efficient and optimized methodology for enhanced expression and purification of untagged active Nedd4-1 from E. coli over three affinity column steps. This strategy employs a nine-residue poly-histidine tag, glutathione S-transferase, and proteolytic cleavage with the TEV fusion protease, His6-MBP-uTEV3, providing 3.9 mg per liter of culture of high-purity (≥95%), active, stable, and storable untagged Nedd4-1. Additionally, the NAB2-Nedd4-1 interaction was re-evaluated using the untagged enzyme via two orthogonal techniques: microscale thermophoresis (MST) and surface plasmon resonance (SPR). Steady-state SPR analysis (χ2 = 0.543) estimated a Kdapp of 169 μM. While binding was constrained by the solubility limits of NAB2, these results suggest that the previously reported nanomolar affinity may be an overestimate resulting from affinity tag interference or immobilization-induced artifacts. This data highlights the potential importance of this method for accessing high-purity, stable untagged Nedd4-1 for biophysical characterization and mechanistic enzymology.