Purification of GST-LC3B

To purify GST-LC3B, insert human LC3B cDNA in a pGEX-4T1 vector. Note, the last five amino acids of LC3B are deleted to mimic the cleavage by ATG4.

After the transformation of the pGEX-4T1 vector encoding GST-LC3B in E.coli Rosetta (DE3) pLysS cells, grow the cells in LB medium at 37°C until an OD₆₀₀ of 0.8-1 and induce the protein expression with 1millimolar (mM) IPTG for 4h 0m 0s at 37°C.

Collect cells by centrifugation and resuspend in lysis buffer.

Lysis buffer:

A	B
HEPES pH 7.5	50 mM
NaCl	300 mM
MgCl2	2 mM
βmercaptoethanol	2 mM
cOmplete EDTA-free protease inhibitors (Roche)
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 lysates by centrifugation at 140000x g,4°C in a Beckman Ti45 rotor.

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 GST-LC3B.

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 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 75 16/600 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 GST-LC3B protein.

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