Robust and facile purification of full-length, untagged human Nedd4 as a recombinant protein from Escherichia coli.
Nedd4 is an E3 ubiquitin ligase that has received increased attention due to its role in the maintenance of proteostasis and in cellular stress responses. Investigation of Nedd4 enzymology has revealed a complex enzymatic mechanism that involves intermolecular interactions with upstream E2 conjugating enzymes and with substrates and intramolecular interactions that serve to regulate Nedd4 function. Thus, it is imperative that investigations of Nedd4 enzymology that employ recombinant enzyme be conducted with Nedd4 in its native, untagged form. We report herein an optimized, facile method for purification of recombinant human Nedd4 in its full-length form as a stable and active recombinant enzyme. Specifically, Nedd4 can be purified through a two-step purification which employs glutathione-S-transferase and hexahistidine sequences as orthogonal affinity tags. Proteolytic cleavage of Nedd4 was optimized to enable removal of the affinity tags with TEV protease, providing access to the untagged enzyme in yields of 2-3 mg/L. Additionally, investigation of Nedd4 storage conditions reveal that the enzyme is not stable through freeze-thaw cycles, and storage conditions should be carefully considered for preservation of enzyme stability. Finally, Nedd4 activity was validated through three activity assays which measure ubiquitin chain formation, Nedd4 autoubiquitination, and monoubiquitin consumption, respectively. Comparison of the method described herein with previously reported purification methods reveal that our optimized purification strategy enables access to Nedd4 in fewer chromatographic steps and eliminates reagents and materials that are potentially cost-prohibitive. This method, therefore, is more efficient and provides a more accessible route for purifying recombinant full-length Nedd4.
Hatstat, AK; McCafferty, DG
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