TITLE: Expression and purification protocol of human PI3KC3-C1 (±mCherry)
AUTHORS: Dorotea  Fracchiolla

[DESCRIPTION]
This protocol describes the procedures for expression and purification of the active autophagy-specific human PI3KC3-C1 kinase complex.

[STEPS]
SECTION: Infection/expression/harvest
1. Infect 1 L culture of Sf9 cells growing in Sf921 medium with antibiotics Penicillin/Streptomycin at 1-1.5 mil/ml cells/volume at 99-100% viability in log phase with Virus 1 (V1), volume according to viral titer. Baculovirus is obtained by transfection of Sf9 cells with policystronic constructs coding for the (GST±mCherry)-PI3KC3-C1 complex

SECTION: Infection/expression/harvest
2. Monitor infection and harvest cells when viability goes to 97-98%.
 

26. To harvest spin down the culture at 4000 rpm, 4°C, 00:15:00 in a Sorvall RC6+ centrifuge (Thermo Scientific).
SECTION: Infection/expression/harvest
3. To harvest spin down the culture at 4000 rpm, 14 min, 4 °C in a Sorvall RC6+ centrifuge (Thermo Scientific).

SECTION: Infection/expression/harvest
4. Pour off the supernatant without disturbing the cell pellet.
SECTION: Infection/expression/harvest
5. Gently wash the pellet 1x in cold PBS buffer on ice.
SECTION: Infection/expression/harvest
6. Centrifuge again and remove PBS.  

SECTION: Infection/expression/harvest
7. Flash freeze cell pellet in liquid nitrogen and store at-80 °C until purification.

SECTION: Protein purification
8. All steps are to be carried on ice or at 4 °C . 
Re-suspend the cell pellet corresponding to 1 L culture in50 mL ice cold Lysis buffer; gently stir at 4 °Cavoiding bubbling until pellet dissolves.

SECTION: Protein purification
9. Mechanically lyse the cells passing them through a pre-cooled 40ml vol. douncer repeating for 3x: 10x pestle A followed by 10x pestle B.
SECTION: Protein purification
10. Clear the lysate by spinning it at 25000 rpm in a Ti45 Rotor for 45 minat 4 °C using Beckman centrifuge.  

SECTION: Protein purification
11. In the meanwhile, wash 3-5 mL  mL slurry GSH beads stored in 20% Ethanol (Pierce or Glutathione Sepharose 4B) with milliQ water and finally equilibrate them in cold WBI. Spin beads at 4000 rpm, 3 min, 4 °C, brake=2 in a 5810R centrifuge (Eppendorf).  

SECTION: Protein purification
12. Allow lysate incubation with beads for 60 min  4 °C, gently rolling the tube.

SECTION: Protein purification
13. Wash beads 2x with a fixed volume of WBI.
 

SECTION: Protein purification
14. Collect sample for "sup WI" and "beads WBI".
SECTION: Protein purification
15. Wash beads 2x in WBII. Collect sample for "sup WII" and "beads WBII".
SECTION: Protein purification
16. Wash beads 2x in WBIII. Collect sample for "sup WIII" and "beads WBIII".
SECTION: Protein purification
17. Check all fractions on a 10% SDS-gel stained with Coomassie Brilliant Blue (see attached pdf below for example gel).
SECTION: Protein purification
18. Collect sample for gel electrophoresis before cleavage.
SECTION: Protein purification
19. Incubate beads 3 min gently rolling at 4 °C with C3 protease to cleave off the GST-tag.

SECTION: Protein purification
21.  Spin down beads and collect the supernatant containing cleaved/eluted protein. Filter elution through a 0.2µm filter syringe to remove beads and concentrate down to 0.5 mL using a 30kDa cut-off Amicon filter.
 


SECTION: Protein purification
22. Spin down the sample in a table top centrifuge for 10 min at 4 °C to pellet eventual aggregates, collect supernatant and inject 0.5 mL sample onto a S6_10/300 Gel Filtration column pre-equilibrated in Gel Filtration Buffer at 4 °C .

SECTION: Protein purification
23. After the run, check fractions containing the protein of interest on gel and pool them. Concentrate down to reach the desired concentration, flash freeze in liquid nitrogen and store at -80 °C until use.
 
      

SECTION: Protein purification
20. The following day, collect sample after cleavage and run samples on gel to confirm cleavage (see attached pdf below).