TITLE: Expression and purification protocols of Homo sapiens GST-ATG4B recombinant protein
AUTHORS: Dorotea Fracchiolla

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes expression and purification procedures for obtaining GST-tagged human recombinant Autophagy specific de-ubiquitinase ATG4B (AuTophaGy-related protein).</div></div>

[STEPS]
?. [Protein expression]
Transform  into one aliquot () of chemically competent E.coli Rosetta pLyss cells according to standard protocol.
[mini DNA plasmid SMC1392 (~100ng/µl)]
50 µl
?. [Protein expression]
Plate cells on Amp/Cam Ty (or LB) plate and grow  at .
37 °C
?. [Protein expression]
The following day, pick 2-3 colonies to inoculate a pre-culture of ~ and grow  at .
[LB (Amp/Cam)]
37 °C
?. [Protein expression]
In the morning, use 4 x  to inoculate 4 x .
[pre-culture]
[LB (Amp/Cam)]
?. [Protein expression]
Grow cultures shaking at  until OD600 (Optical Density) = 0.4 and then switch to .
37 °C
18 °C
?. [Protein expression]
Induce cultures when cooled down and OD600 =0.9 is reached with  and carry protein expression  at .
[IPTG]
18 °C
?. [Protein expression]
The following day, harvest cells by spinning at  in a Sorvall RC6+ centrifuge (Thermo Scientific).
Centrifuge: 4000 33, 10 min, 4 10
?. [Protein expression]
Resuspend cell pellets in ~/, flash freeze in liquid nitrogen and store at .
[ice cold lysis buffer]
[cell culture]
-80 °C
?. [Protein purification]
Wash  stored in 20% Ethanol (Pierce or Glutathione Sepharose 4B) in milliQ water and finally equilibrate in cold WB1. Perform spinning at  in a 5810R centrifuge (Eppendorf).
[slurry GSH beads]
Centrifuge: 4000 33, 3 min, 4 10
?. [Protein purification]
Thaw frozen resuspended cell pellets in  water bath and move  as soon as thawed.
-80 Room temperature
on ice
?. [Protein purification]
Sonicate cells 2x30s 50% cycle, 70% Power at  using a tip sonicator.
4 °C
?. [Protein purification]
Collect Lys sample for gel loading.
?. [Protein purification]
Clear lysate in a Beckmann Ultracentrifuge for .
Centrifuge: 25000 33, 45 min, 4 10
?. [Protein purification]
Collect supernatant sample.
?. [Protein purification]
Incubate supernatant with .
[GSH beads pre-equilibrated in WB1]
?. [Protein purification]
Perform binding at  for , rolling.
4 °C
?. [Protein purification]
Wash beads 2x in WB1.
Each wash can be performed by gently inverting the tube a few times or by rolling the tube for 10 minutes at 4°C-> spin at 4000 rpm for 3 min at 4°C.
?. [Protein purification]
Take "sup W1" sample. Take "beads WB1". Wash 2x in WB2.
?. [Protein purification]
Take "sup W2" sample. Take "beads WB2". Wash 2x in WB3.
?. [Protein purification]
Take "sup WB3" sample. Take "beads WB3".
?. [Protein purification]
Check all fractions on a 12% SDS-gel stained with Coomassie Brilliant Blue.
?. [Protein purification]
Collect sample before cleavage.
?. [Protein purification]
Incubate beads for  (or less) or  rolling at  with 1 vial Thrombin (SERVA) to cleave off the GST-tag.
4 °C
Alternatively, GST tag can be left on and protein is eluted with 50mM reduced L-Glutathione solution in WB3 pH=7.5-8. Elution for GST-tagged proteins is usually performed for 1-2 hours at 4°C, rolling.
?. [Protein purification]
If cleavage is performed: spin beads down and collect “Elution sample" at  (brake=2) in a 5810R centrifuge (Eppendorf).
4 °C
?. [Protein purification]
Filter elution through a 0.2µm filter and concentrate down to  using a 10kDa cut-off Amicon filter.
2 mL
?. [Protein purification]
Keep concentration steps short and pipette regularly to avoid protein precipitation on the filter.
?. [Protein purification]
In the meanwhile, collect Beads after cleavage sample and check cleavage on a 12% SDS-gel stained with Coomassie Brilliant Blue.
?. [Protein purification]
Inject  onto a S75_16/600 Gel Filtration column pre-equilibrated in Gel Filtration Buffer.
[concentrated protein]
?. [Protein purification]
Check fractions containing the protein of interest on gel, collect and pool. Concentrate down to reach the desired concentration, flash freeze in liquid nitrogen and store at  until use.ATG4B, MW=44,3 kDa, Epsilon=65890 M-1 cm-1
-80 °C
?. [Protein purification]
Sonicate cells for  50% cycle, 70% Power. (1/2)
?. [Protein purification]
Sonicate cells for  50% cycle, 70% Power. (2/2)
?. [Protein purification]
Wash beads in WB1. (1/2)
?. [Protein purification]
Wash beads in WB1. (2/2)