Purification of OPTN-GST

To purify OPTN-GST, Clone human OPTN cDNA in a pETDuet-1 vector with an C-terminal GST-tag.

After the transformation of the pETDuet-1 vector encoding OPTN-GST in E. coli Rosetta pLysS cells, grow the cells in 2xTY medium at 37°C until an OD₆₀₀ of 0.4 and then continue at 18°C.

Once the cells reached an OD₆₀₀ of 0.8, induce protein expression with 50micromolar (µM) IPTG for 16h 0m 0s at 18°C

Collect the cells by centrifugation and resuspend in lysis buffer.

Lysis buffer

A	B
HEPES pH 7.4	50 mM
NaCl	300 mM
MgCl2	2 mM
Glycerol	5%
Imidazole	10 mM
β-mercaptoethanol	2 mM
cOmplete EDTA-free protease inhibitors (Roche)
CIP protease inhibitor (Sigma)
DNase (Sigma)

Sonicate cell lysates.

Sonicate cell lysates twice for 0h 0m 30s (1/2).

Sonicate cell lysates twice for 0h 0m 30s (2/2).

Clear the lysates by centrifugation at 18000rpm,4°C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).

Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 2h 0m 0s at 4°C with gentle shaking to bind OPTN-GST.

Centrifuge the samples to pellet the beads and remove the unbound lysate.

Wash beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.

Wash buffer

A	B
HEPES pH 7.4	50 mM
NaCl	300 mM
DTT	1 mM

High-salt wash buffer

A	B
HEPES pH 7.4	50 mM
NaCl	700 mM
DTT	1 mM

Elute the proteins 2h 0m 0s with 20millimolar (mM) reduced L-glutathione in 50millimolar (mM) HEPES 7.4, 300millimolar (mM) NaCl, 1millimolar (mM) DTT buffer.

Collect the supernatant, filter through a 0.45 µm syringe filter, concentrate using a 50 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva).

Elute the proteins with SEC buffer.

SEC buffer

A	B
HEPES pH 7.4	25 mM
NaCl	150 mM
DTT	1 mM

Analyze fractions by SDS-PAGE and Coomassie staining.

Pool fractions containing purified OPTN-GST.

After concentrating the purified protein, aliquot the protein and snap-frozen it in liquid nitrogen. Store proteins at -80°C.