LRRK1 expression and purification

N-terminally tagged His6-Z-TEV-LRRK1(FL) was expressed in Sf9 insect cells. Insect cells were infected with baculovirus and grown at 27°C for 3 days.

Cells were harvested and then resuspended in lysis buffer (50mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2, 20 μM GDP, 0.5 mM Pefabloc and cOmplete EDTA-free protease inhibitor cocktail (Roche).

Cells were lysed using a Dounce homogenizer and clarified by centrifugation at 50000 rpm, 4°C, 01:28:00, and Ti70 rotor.

The supernatant was incubated for 1 hr with Ni-NTA agarose beads (Qiagen) equilibrated in lysis buffer. Beads were applied to a gravity column where they were extensively washed with lysis buffer, wash buffer ( 50mM HEPES pH 7.4, 500mM NaCl, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP), followed by elution in lysis buffer containing 300 mM imidazole ( 50mM HEPES pH 7.4, 500mM NaCl, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP, 300mM imidazole)

The eluted protein was diluted to 250 mM NaCl using dilution buffer (50mM HEPES pH 7.4, 0.5mM TCEP, 5% glycerol, 5mM MgCL2, 20uM GDP) and loaded onto a SP Sepharose column (Cytiva) equilibrated in buffer (50 mM HEPES pH 7.4, 250 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2, 20 μM GDP)

The protein was eluted using a 250 mM to 2.5 M NaCl gradient. Fractions containing LRRK1(FL) were pooled, diluted to ~500 mM NaCl, and incubated with TEV protease overnight at 4°C.

The protein was concentrated and put directly over a S200 size exclusion column (Cytiva) equilibrated in storage buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM MgCl2 and 20 μM GDP).

The protein was concentrated to ~5-6 μM and flash frozen in liquid nitrogen for storage. N-terminally tagged His6-Z-TEV-LRRK120-2015 was purified using the same protocol.