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Figure 1
Purification and biochemical characterization of recombinant ALSV NS3-Hel. (a) Preliminary purification of ALSV NS3-Hel. Lane 1, supernatant from the bacterial lysis; lane 2, eluate from the Ni–NTA column containing 6×His-SUMO-tagged ALSV NS3-Hel; lane 3, overnight digestion by Ulp1 peptidase; lane 4, flowthrough from the Ni–NTA column, on which the cleaved 6×His-SUMO tag and undigested species were trapped. Lane M contains molecular-mass markers (labeled in kDa). (b) Left: final purification of ALSV NS3-Hel using ion-exchange chromatography. The nontagged ALSV NS3-Hel was eluted from the HiTrap Q HP column with a linear gradient of NaCl from 75 mM to 1 M. UV absorbance (280 nm) and the conductivity of the elution buffer are plotted as a function of elution volume. Right: an SDS–PAGE analysis of the eluate fractions corresponding to the UV absorbance peak. Lane M contains molecular-mass markers (labeled in kDa). (c) ATPase activity of ALSV NS3-Hel. The velocity of ATP hydrolysis is plotted as a function of the ATP concentration. The kinetic parameters Km and Vmax were calculated via nonlinear fitting to the Michaelis–Menten equation.

IUCrJ
Volume 7| Part 3| May 2020| Pages 375-382
ISSN: 2052-2525
https://doi.org/10.1107/S2052252520003632