TITLE: Expression and purification protocol of Homo sapiens E2-like enzyme ATG3
AUTHORS: Eleonora Turco, Dorotea Fracchiolla

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes expression and purification procedures for obtaining human recombinant autophagy E2-like enzyme ATG3 (ATG, AuTophaGy-related protein) of the ATG8 ubiquitin-like conjugation system.</div></div>

[STEPS]
?. [Protein Purification]
Cells are lised via freeze/thaw cycles and sonication: thaw pellet corresponding to  culture by freeze/thawing in  water bath. All following steps are to be executed at  or on ice.
1 L
0 Room temperature
4 °C
?. [Protein Purification]
Lyse cells by sonicating them using an immersion tip Sonicator (2x ). Note: adjust times and intensity according to the available instrument.
?. [Protein Purification]
Clear lysate by spinning it down in a Beckman centrifuge at .
Centrifuge: 40000 34, 30 min, 4 10
?. [Protein Purification]
Filter supernatant through a 0.45 µm filter and inject onto a 5ml HT column operating at   pre-equilibrated in Buffer A at 1ml/min flow rate.
4 °C
?. [Protein Purification]
Wash column with  at 2 ml/min flow rate to remove unspecific bound proteins.
[of Buffer A]
?. [Protein Purification]
Elute protein of interest through a step elution gradient in 50mM, 75mM, 100mM, 150mM, 200mM and 300mM Imidazole concentration. Perform elution at 1ml/min flow rate. Collect peak fractions at each step.
?. [Protein Purification]
Check peak fractions of each step on a SDS-PAGE (see gel below). Pool and concentrate those containing the protein of interest (usually 100mM and 150mM Imidazole step) by spinning at  down in a 10kDa cut-off Amicon Filter to .
4 °C
[final volume]
Centrifugation steps are kept short ( ) to avoid protein local concentration/aggregation on the filter.
?. [Protein Purification]
Inject  onto a S75_16/600 column operating at   and pre-equilibrated in buffer containing SEC Buffer (see profile below).
[protein]
4 °C
?. [Protein Purification]
Check fractions on a 10% SDS-PAGE (see gel below). Pool and concentrate those containing the protein of interest down at  in a 10kDa cut-off Amicon Filter.
4 °C
Centrifugation steps are kept short ( ) to avoid protein local concentration/aggregation on the filter.
?. [Protein Purification]
Measure protein Absorbance A280 using a Spectrophotometer blanking against SEC buffer (MW = 35,864 kDa ; Extinction coefficient = 45840M-1cm-1).
?. [Protein Purification]
Resuspend protein in Glycerol to a final concentration of . Store the protein batch at  . Estimated protein yield: . Protein activity is kept for 18 months.
[for glycerol]
-20 °C
[per 1 lt culture]
?. [Protein Expression]
Transform plasmid DNA (Addgene 169079, SMC861) into E.Coli Rosetta pLyss cells and plate on Ampicillin/Chloramphenicol LB agar plate for  growth at .
37 °C
?. [Protein Expression]
The following day, inoculate a  with 1-2 colonies and grow  at  shaking.
[LB + Amp/Cam pre-culture ]
37 °C
?. [Protein Expression]
The following day, use  to inoculate  at  until an OD600 (Optical Density at 600nm) of 0.4 is reached.
[pre-culture]
[LB medium + Amp/Cam]
37 °C
?. [Protein Expression]
Cool down the culture to  and grow until OD600 = 0.8.
18 °C
?. [Protein Expression]
Induce protein expression with  and keep shaking for a further  at .
[IPTG]
18 °C
?. [Protein Expression]
Pellet cells at  in a Sorvall RC6+ centrifuge (Thermo Scientific), discard supernatant and resuspend pellets in ice cold lysis buffer (25 ml/1 lt culture).
Centrifuge: 4000 33, 15 min, 4 10
?. [Protein Expression]
Flash freeze resuspended pellets in liquid nitrogen and store at  until purification.
-80 °C
?. [Protein Purification]
Perform His-Trap affinity purification followed by Size Exclusion Chromatography.