Purification protocol of Mouse (Mus Musculus) E1-like enzyme ATG7

Infect 1L of Sf9 cells growing in Sf921 medium with Penicillin/Streptomycin at 1-1.5 mil/ml cells/volume at 99-100% viability in log phase with Virus 1 (V1) according to viral titer.

(NOTE: protein yield from 1 lt culture is high, e.i. 32 mg/1lt culture).

Monitor infection and harvest cells when viability goes to 97-98%.

Resuspend cell pellet corresponding to 1L in 50mL; gently stir at 4°C until pellet dissolves avoiding bubbling.

Additionally, mechanically lyse the cells passing them through a pre-cooled douncer for 3x (10x pestle A followed by 10x pestle B).

Clear the lysate by spinning it in a Backman centrifuge, at 25K using a Ti45 Rotor for 0h 45m 0s at 4°C.

Inject the supernatant onto a 5ml HT column pre-equilibrated with Buffer A at 1ml/min flow rate to allow protein binding.

Wash the column for 5CV with Buffer A at 2 ml/min flow rate to remove unspecific bound proteins.

Elute protein through a step elution gradient in 50mM, 75mM, 100mM, 150mM, 200mM and 300mM Imidazole concentration. Perform the elution at 1ml/min flow rate.

Collect a sample from peak fractions of each elution step and check them on a SDS-PAGE (see gel in the "Guidelines" section). Pool and concentrate fractions corresponding to the peak containing the protein of interest (usually 200mM and/or 300mM Imid.) by spinning them down at4°C using a 30 kDa cut-off Amicon Filter to 2 ml in a 5810R centrifuge (Eppendorf).

Inject 2mL onto a S200_16/60 column at 44°C pre-equilibrated in buffer containing 25mM Hepes pH=7.5, 150mM NaCl and 1mM DTT (see profile in the "Guidelines" section).

Check fractions on a 10% SDS-PAGE (see gel in the "Guidelines" section), pool and concentrate down those containing the protein of interest by spinning them at 4°C using a 30kDa cut-off Amicon Filter in a 5810R centrifuge (Eppendorf). Protein elutes at around 72.5ml, a retention volume corresponding to that of a dimeric globular protein of ~150-160 kDa.

Measure protein absorbance at A₂₈₀ is measured with Spectrophotometer against Size Exclusion Chromatography buffer.

Aliquot the protein, snap freeze it in liquid Nitrogen and store it at -80°C.