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A P P L I C A T I O N N O T E
METHODS
Protein expression and purification
All proteins were recombinantly expressed in E. coli and purified using an optimized protocol
described previously
(2)
. Strep-Tactin®XT Resin was used to purify the membrane protein, which was
solubilized in copolymer nanodiscs from the Cube Synthetic Nanodisc Screening Kit MAXI.
Prometheus Panta experiments
To identify the most effective copolymer for stabilizing and isolating membrane proteins in their
active states, a series of miniaturized expression and purification experiments was conducted using
16 copolymers of the NativeMP
TM
platform on the three protein classes represented by WbaP, PglB
and PglC (Table 1). A er each miniaturized purification, 10 µL of sample was directly loaded into
Prometheus Standard Capillaries, and the isothermal (25 °C) DLS measurements were conducted
using the Prometheus Panta. Subsequently, a thermal ramp (1.5 °C/min, 25-95 °C) was applied to
each 10 µL sample, with the nanoDSF, DLS, and turbidity data being collected simultaneously.
