May 24, 2023
Expression and purification protocol of human PI3KC3-C1 (±mCherry)
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Dorotea Fracchiolla 2023. Expression and purification protocol of human PI3KC3-C1 (±mCherry). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59mz4g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 30, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63453
Keywords: PI3KC3-C1, kinase, expression, Sf9, purification, autophagy, ASAPCRN
Abstract
This protocol describes the procedures for expression and purification of the active autophagy-specific human PI3KC3-C1 kinase complex.
Materials
Proteins' parameters
| A | B | C | D | |
| Addgene # | Protein | MW (kDa) | Epsilon (M-1cm-1) | |
| 187988 | GST-C3cs-mCherry-ATG14L | 109 | 135110 | |
| 187987 | GST-C3cs-ATG14L | 81,7 | 100730 | |
| 187990 | Vps15 | 155 | 159630 | |
| 187989 | Vps34 | 102 | 115630 | |
| 187991 | Beclin | 52 | 54890 | |
| 187992 | GST-PI3KC3-C1 | 390,7 | 430880 | |
| 187831 | GST-mCherry-PI3KC3-C1 | 418 | 315250 |
Molecular Weights and Extinction coefficients of all subunits and their complexes.
Materials and Reagents
- Sf9 insect cells
- SF921 medium with antibiotics 100 IU/ml Penicillin and 100 μg/ml Streptomycin
- sterile cell culture hood
- 27°C shaker incubator
- sterile flasks and pipettes
- douncer 40 mL
- Virus coding for GST-C3-ATG14/Vps34/Vps15/BECN1 (SMC1326/SM014, Addgene ) or GST-C3-mCherry-ATG14/Vps34/Vps15/BECN1 (SMC1327/SM015, Addgene ). Note:All the CDSs are codon-optimized for insect cell expression system (purchased from GenScript). The final poli-cystronic constructs were cloned via Golden Gate approach by the Vienna BioCenter Core Facilities (VBCF) Protech Facility.
Buffers for Protein Purification
LysisBuffer: 50mM Hepes pH=7.5, 300mM NaCl, 0.5% CHAPS(stock 10% CHAPS in water, filtered), Benzonase (1μl/50ml lysisbuffer), 1mM MgCl2, 1mM DTT, 1xProteaseInhibitors/50ml lysis buffer (EDTA-freeCIPtablet) + 300µl CIP (Sigma).
Wash Buffer I: 50mM HepespH=7.5, 300mM NaCl, 0.5% CHAPS,1mM DTT;
Wash Buffer II: 50mM HepespH=7.5, 500mM NaCl, 1mM DTT;
Wash Buffer III: 50mM HepespH=7.5, 300mM NaCl, 1mM DTT;
Gel Filtration Buffer: 25mM Hepes pH=7.5, 200mMNaCl, 1mMDTT.
Note: all purification buffers are filtered and degassed. Reducing agent (DTT, Dithiothreitol) is added after degassing step.
Columns: 5 ml GSH beads,GE Healthcare or Pierce
- S6_10/300
Gels:10%SDS-PAGE
Infection/expression/harvest
Infection/expression/harvest
14m
14m
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
Monitor infection and harvest cells when viability goes to 97-98%.
Note
Always check reporter gene fluorescence under the microscope to monitor the viral infection: when all alive cells are brightly fluorescent and only few dead -> harvest!
To harvest spin down the culture at 4000 rpm, 4°C, 00:14:00 in a Sorvall RC6+ centrifuge (Thermo Scientific).
14m
Pour off the supernatant without disturbing the cell pellet.
Gently wash the pellet 1x in cold PBS buffer on ice.
Centrifuge again and remove PBS.
Flash freeze cell pellet in liquid nitrogen and store at-80 °C until purification.
Protein purification
Protein purification
45m
45m
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 °C avoiding bubbling until pellet dissolves.
Mechanically lyse the cells passing them through a pre-cooled 40ml vol. douncer repeating for 3x: 10x pestle A followed by 10x pestle B.
Clear the lysate by spinning it at 25000 rpm in a Ti45 Rotor for 00:45:00 at 4 °C using Beckman centrifuge.
45m
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, 4°C, 00:03:00 , brake=2 in a 5810R centrifuge (Eppendorf).
3m
Allow lysate incubation with beads for 01:00:00 4 °C , gently rolling the tube.
1h
Wash beads 2x with a fixed volume of WBI.
Note
Each wash can be performed by gently inverting the tube a few times at 4 °C -> spin at 4000 rpm for 00:03:00 .
Collect sample for "sup WI" and "beads WBI".
Wash beads 2x in WBII. Collect sample for "sup WII" and "beads WBII".
Wash beads 2x in WBIII. Collect sample for "sup WIII" and "beads WBIII".
Check all fractions on a 10% SDS-gel stained with Coomassie Brilliant Blue (see attached pdf below for example gel).
Collect sample for gel electrophoresis before cleavage.
Incubate beads Overnight gently rolling at 4 °C with C3 protease to cleave off the GST-tag.
3m
The following day, collect sample after cleavage and run samples on gel to confirm cleavage (see attached pdf below).
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.
Note
Centrifugation steps are kept short 00:05:00 to avoid protein local concentration/aggregation on the filter. Gently pipette the sample to resuspend the protein between centrifugation steps.
Spin down the sample in a table top centrifuge for 00:10:00 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 .
10m
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.
Note
Gel Filtration GST_mCh_PI3KC3_C1.pdf SEC_PI3KC3_C1.pdf GSTPI3KC3_C1 purification_2020.06.16.pdf