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

[DESCRIPTION]
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.

[GUIDELINES]
General information
Insert: Homo sapiens ATG3, NP_071933.2; Expression system: E.Coli Rosetta pLyss; plasmid origin: Sascha Martens Lab, Addgene 169079, lab internal construct database number SMC861; backbone: pET-Duet1; plasmid resistance: Ampicillin; tags & cleavage sites: N-term 6xHis, followed by Tobacco Etch Virus (TEV) cleavage site, ATG3 ORF. Ext coeff: 45840 M-1 cm-1 , MW 35.8 kDa.

[STEPS]
SECTION: Protein Purification
9. Cells are lised via sonication: thaw pellet corresponding to 1 L culture. All following steps are to be executed at 4 °C or on ice.

SECTION: Protein Purification
10. Lyse cells by sonicating them using an immersion tip Sonicator (2x 30 s). Note: adjust times and intensity according to the available instrument.

SECTION: Protein Purification
11. Clear lysate by spinning it down in a Beckman centrifuge at 40000 x g, 30 min, 4 °C.
SECTION: Protein Purification
12. Load supernatant onto 2ml Ni-NTA column at 4 °C  pre-equilibrated in Buffer A. 

SECTION: Protein Purification
13. Wash column with 50 mL to remove unspecific bound proteins.
SECTION: Protein Purification
14. Elute protein of interest with 300mM Imidazole concentration. Collect fractions.


SECTION: Protein Purification
15. Check fractions of each step on a SDS-PAGE. Pool and concentrate those containing the protein of interest by spinning at 4 °C down in a 10kDa cut-off Amicon Filter to 2 mL.
 
SECTION: Protein Purification
17. Inject 2 mL onto a S75_16/600 column operating at 4 °C  and pre-equilibrated in buffer containing SEC Buffer (see profile below). 

SECTION: Protein Purification
18. Check fractions on a 10% SDS-PAGE. Pool and concentrate those containing the protein of interest down at 4 °C in a 10kDa cut-off Amicon Filter. 


  

SECTION: Protein Purification
19. Measure protein Absorbance A280 using a Spectrophotometer blanking against SEC buffer (MW = 35,864 kDa ; Extinction coefficient = 45840M-1cm-1).
SECTION: Protein Purification
20. Resuspend protein in Glycerol to a final concentration of 30 % (v/v). Store the protein batch at -20 °C . Estimated protein yield: 10 mg. Protein activity is kept for 18 months.

SECTION: Protein Expression
1. 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. 

SECTION: Protein Expression
2. The following day, inoculate a 5 mL with 1-2 colonies and grow   at 37 °C shaking. 

SECTION: Protein Expression
3. The following day, use 5 mL to inoculate 1 L at 37 °C until an OD600 (Optical Density at 600nm) of 0.4 is reached. 

SECTION: Protein Expression
4. Cool down the culture to 18 °C and grow until OD600 = 0.8. 

SECTION: Protein Expression
5. Induce protein expression with 200 micromolar (µM) and keep shaking for a further 960 min at 18 °C. 

SECTION: Protein Expression
6. Pellet cells at 4000 rpm, 15 min, 4 °C in a Sorvall RC6+ centrifuge (Thermo Scientific), discard supernatant and resuspend pellets in ice cold lysis buffer (25 ml/1 lt culture).

SECTION: Protein Expression
7. Flash freeze resuspended pellets in liquid nitrogen and store at -80 °C until purification.

SECTION: Protein Purification
8. Perform His-Trap affinity purification followed by Size Exclusion Chromatography.  
SECTION: Protein Purification
16. Cleave His tag overnight with TEV protease in dialysis against buffer containing 0mM Imidazole.