LRRK2 expression and purification

Transfect the constructs encoding 3xFlag-LRRK2, 3xFlag-LRRK2(I2020T), 3xFlag-RCKW or 3xFlag-GFP-LRRK2 into Expi293F cells according to manufacturer instructions.

Express the proteins for 72h 0m 0s following induction according to manufacturer instructions.

Harvest the cells by centrifugation (400x g,0h 0m 0s, 0h 4m 0s) and lyse by 3 freeze-thaw cycles in lysis buffer.

Remove the cellular debris by centrifugation at 15000x g,0h 0m 0s for 1h 0m 0s at 4°C.

Mix the clarified lysate with anti-FLAG M2 resin for 2h 0m 0s while rotating at 4°C.

Wash the resin with 3x10 bed volumes of lysis buffer.

Elute the proteins with lysis buffer supplemented with 0.2mg/mL 3xFlag peptides.

Remove the N-terminal 3xFlag tag by incubation with the GST tagged Prescission Protease (0.01U/µL) while rotating at 4°C.

Remove the GST tagged Prescission Protease subsequently by Glutathione Sepharose.

Assess the purity of the proteins by SDS-PAGE and Western blotting.

Dialyze the purified proteins at 4°C against the dialysis buffer.

After dialysis, clarify the proteins by centrifugation at 17000x g,0h 0m 0s for 0h 10m 0s at 4°C.

Determine the protein concentration by SDS-PAGE using Bovine Serum Albumin (BSA) as standard and used without freezing in liposome binding and tubulation experiments.