Akta pure 25l system

SidJ and IQ mutant were incubated at a concentration of 35 μM in the presence or absence of a 1.2 molar ratio of CaM. 125 μL of solution was injected onto a Superdex 200 Increase 100/300 GL column (GE) and separated at 0.7 mL/min on an AKTA Pure 25L System (GE). UV traces were generated using R-Studio Software and 0.5 mL fractions were collected and analyzed by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue.

The molecular weight (MW) distribution of porcine PH extract was determined by a fast protein liquid chromatography (FPLC) (Sousa et al. 2020 (link)). An aliquot (100 µL) of filtered samples was injected in a AKTA pure 25 L system, from GE Healthcare Life Sciences (Freiburg, Germany), coupled with two gel filtration columns: Superdex 200 increase10/300 GL and Superdex peptide, 10/300 GL. The eluent used was 0.025 M phosphate buffer (pH 7.0), 0.15 M sodium chloride and 0.2 g/L of sodium azide. The flow rate was 0.5 mL/ min and elution was monitored at 280 nm. A MW standard curve was established using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and a whey peptide (1.2 kDa). The analysis was performed in duplicate and the results were expressed in milli Absorbance Units (mAU) per eluted volume (mL). The software used to evaluate the results was UNICORN 7.0.

E. coli BL21 cells containing a pE-SUMO fusion to the construct of interest (cloned using Gibson Assembly) were grown in LB with antibiotics at 37 °C to OD₆₀₀~0.9, and induced for 3 h with 0.5 mM IPTG. Cells were centrifuged and resuspended in lysis buffer (50 mM HEPES pH 7.2, 300 mM NaCl, 20 mM imidazole, Pierce^TM Protease Inhibitor Mini Tablets (Thermo), 1 mM TCEP, 0.5%Tx-100) and sonicated. Cell debris was removed by centrifugation (18,000 × g for 40 min at 4 °C), and the lysate was applied to a Nickel resin affinity column (HisPur Ni-NTA Resin). The column was washed with two column-volumes wash buffer (50 mM HEPES pH 7.2, 1 M NaCl, 20 mM Imidizole, 1 mM TCEP) and eluted with elution buffer (50 m M HEPES pH 7.2, 300 mM NaCl, 300 mM Imidizole, 1 mM TCEP). Eluted 6xHisSumo-Int was then run through a HiTrap Heparin HP 5 mL column, and pooled fractions were run on a Superose 6 Increase 10/300 GL column on an AKTA Pure 25 L system (GE Healthcare). Eluted 6xHisSumo-PexA was run on a Superose 6 Increase 10/300 GL column. To cleave the SUMO tag, 1 μL SUMO protease was added per 100 μg of protein and incubated overnight at 4 °C. The mixture was then bound to Novex His-Tag Dynabeads and the unbound fraction was collected and analyzed by SDS-PAGE visualized with Stain-Free technology (Bio-rad).