Superose 6 increase

PA28αβ was purified as described (60 (link)). Briefly, recombinant PA28αβ was expressed in BL21-STAR E. coli and purified using strong anion exchange (HiTrapQ and MonoQ, GE Life Sciences), followed by hydroxyapatite (CHT-II, Bio-Rad), and finished with SEC (Superose 6 Increase, GE Life Sciences). Recombinant PA28γ was expressed in Rosetta E. coli and purified using Ni-NTA affinity resin (Qiagen) and followed with SEC (Superose 6 Increase, GE Life Sciences), as previously described (10 (link)). PA28γ-K188E was created using QuikChangeII Site Directed Mutagenesis Kit (Agilent) and purified using methods like PA28γ, as previously described (31 (link)). PA28γ-α, PA28γ-2XCys, PA28αβ2XLys, and PA28γΔIDR constructs were designed as G-Blocks with N-terminal 6XHis Tags (Integrated DNA Technologies) and cloned into pET11a plasmids. pET11a plasmids with successfully cloned G-blocks were transformed into BL21-STAR E. coli and purified using the PA28γ Ni-NTA purification methods. Concentrations were determined using a Bradford assay with bovine serum albumin as the reference protein.

SUR2 subunits were expressed using the BacMam system as described previously with minor modifications^{19 (link)}. Briefly, cells were harvested 48 h post-infection and membrane pellets were purified as described previously^{22 (link)}. For purification, membrane pellets were homogenized in TBS (20 mM Tris and 200 mM NaCl) and then solubilized in 1% GDN and 0.05% CHS for 30 min at 4 °C. Unsolubilized material was removed by centrifugation at 100,000 g for 30 min. The supernatant was supplemented with 1 mM ATP and 1 mM MgCl₂ and loaded onto Streptactin Beads 4FF (Smart Lifesciences). The beads were washed with buffer A (TBS with 50 µM GDN and 1 mM ATP) plus 1 mM MgCl₂ and protein was eluted with buffer A plus 10 mM desthiobiotin. GFP tags were removed by PreScission protease. To purify the SUR2B protein for Mg-nucleotides + Lev state and Mg-nucleotide-bound state, proteins were supplemented with 2 mM ADP + 2 mM MgCl₂ and concentrated by 100-kDa cut-off concentrator (Sartorius) and loaded onto Superose 6 increase (GE Healthcare) running in TBS with 50 μM GDN, 2 mM ADP and 2 mM MgCl₂. To purify the protein of SUR2B in the Mg-nucleotides + P1075 state, protein was loaded onto Superose 6 increase running in buffer A with 1 mM MgCl₂.

SUR2 subunits were expressed using the BacMam system as described previously with minor modifications^{23 (link)}. Briefly, cells were harvested 48 h post-infection and membrane pellets were purified as described previously^{26 (link)}. For purification, membrane pellets were homogenized in TBS (20 mM Tris and 200 mM NaCl) and then solubilized in 1% GDN and 0.05% CHS for 30 min at 4°C. Unsolubilized material was removed by centrifugation at 100,000×g for 30 min. The supernatant was supplemented with 1 mM ATP and 1 mM MgCl₂ and loaded onto Streptactin Beads 4FF (Smart Lifesciences). The beads were washed with buffer A (TBS with 50 µM GDN and 1 mM ATP) plus 10 mM MgCl₂ and protein was eluted with buffer A plus 10 mM desthiobiotin. GFP tags were removed by PreScission protease. To purify the SUR2A protein, proteins were concentrated by 100-kDa cut-off concentrator (Sartorius) and loaded onto Superose 6 increase (GE Healthcare) running in TBS with 50 μM GDN, 1 mM ATP. SUR2B is purified similarly to SUR2A.