Expression and purification MBP-ATG9 Constructs
Adam Yokom
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Abstract
Expression and purification from HEK cells of ATG13, ATG101 and FOLDON-ATG9A proteins
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.
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)
Wash Amylose resin with lysis buffer, ~2mL/1 Liter of cells
Clarify lysate by centrifugation 17000rpm,4°C
Load supernatant flow thru gravity column onto washed Amylose resin
Rock supernatant with equilibrated resin for 1h 0m 0s at 4°C
Wash with 5 column volumes of 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 40 mM Maltose for amylose resin
Concentrate elution and inject onto pre-equilibrated S200 10/30 column (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA)
Pool peak fractions, concentrate, snap freeze, and store at -80C
