Purification and Crystallization of ATG9 HDIR-ATG101:ATG13 complex
Xuefeng Ren, Adam Yokom
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Abstract
Purification and Crystallization of ATG9 HDIR (828-839) fused ATG101 (1-198):ATG13 (1-197)
Steps
Expression
Transfect HEK GNTI cells at concentration of 2 × 106cells/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 0h 30m 0s at 37°C
Add mixture to cells. Let cells grow for 48h 0m 0s
Harvest Cells 500rpm,4°C
Wash pellet with cold PBS. Store pellet at -80C until purification or lyse immediately
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 17000rpm,4°C
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 1h 0m 0s at 4°C
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
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
