Jul 20, 2022
Purification and Crystallization of ATG9 HDIR-ATG101:ATG13 complex
- Adam Yokom1,
- Xuefeng Ren1
- 1University of California, Berkeley
Protocol Citation: Adam Yokom, Xuefeng Ren 2022. Purification and Crystallization of ATG9 HDIR-ATG101:ATG13 complex. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbkdjlzp/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: July 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 66461
Keywords: ASAPCRN
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Purification and Crystallization of ATG9 HDIR (828-839) fused ATG101 (1-198):ATG13 (1-197)
Expression
Expression
Transfect HEK GNTI cells at concentration of 2 × 106 cells/ml
Dilute PEI with Warm Hybridoma-SFM(1X), In a separate tube, dilute DNA with Hybridoma-SFM(1X)
Add PEI to DNA dilution. Incubate mixture for 00:30:00 at 37 °C
30m
Add mixture to cells. Let cells grow for 48:00:00
2d
Harvest Cells 500 rpm, 4°C, 00:10:00
10m
Wash pellet with cold PBS. Store pellet at -80C until purification or lyse immediately
Purification
Purification
Resuspended pellet in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol) with 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific, Waltham, MA)
Clarify lysate for 17000 rpm, 4°C, 00:35:00
35m
Wash GST Sepharose 4B resin into lysis buffer (without Triton)
Load supernatant onto GST Sepharose resin using a gravity column setup
Rock supernatant with equilibrated resin for 01:00:00 at 4 °C
1h
Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
Elute with lysis buffer plus 25 mM glutathione for GST resin
Add purified His6-TEV protein to cleave GST tag at 4°C overnight
Dilute 5x into SP Buffer buffer A: 30 mM MES pH 6.0, 3 mM beta-mercaptoethanol
Load sample onto a HiTrap SP HP 5 ml column (GE healthcare, Piscataway, NJ)
Elute from the SP column on a 70 ml linear gradient from 0–500 mM NaCl in SP buffer A. The cleavage sample was eluted at the buffer conductivity of ~ 25 mS/cm.
After each fraction was analyzed by SDS gel. Fractions containing ATG9 HDIR-ATG101:ATG13 were pooled concentrated in Amicon Ultra15 concentrator (MilliporeSigma, Burlington, MA) and exchanged into 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP for crystallization.
Crystallization
Crystallization
ATG9 HDIR-ATG101:ATG13 complex at 6 mg/ml in 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP was used as the protein stock
Crystallization was carried out by sitting-drop vapor diffusion using an automated liquid-handling system (Mosquito, TTP LabTech, UK) at 288 K in 96-well plates
The protein solution was mixed with the reservoir buffer composed of 0.1 M HEPES pH 7.5, 0.2 M NaCl, 12% PEG8000 with a ratio of 1:1
Crystal trays were checked daily using a light microscopy for crystal growth
Large crystals were obtained in 2–4 days. Crystals were cryo-protectedin 28% glycerol/reservoir buffer and frozen in liquid N2