Recombinant Expression and Purification of AP4 Appendage Domains

Human AP4E1 (residues 881–1135^{18 (link)}) and AP4B1 (residues 612–739^{26 (link)}) appendage domains were expressed as GST-fusion proteins in BL21(DE3)pLysS cells (Invitrogen) for 16–20 h at 22 °C after induction with 0.4 mM IPTG. Proteins were purified in 20 mM HEPES pH 7.5, 200 mM NaCl, and 2 mM 2-Mercaptoethanol. Cells were lysed using a disruptor (Constant Systems Limited) and proteins were affinity purified using glutathione sepharose (GE Healthcare). Fusion proteins were eluted in buffer with 30 mM reduced glutathione and further purified by gel filtration on a Superdex S200 preparative column (GE Healthcare).

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Davies A.K., Itzhak D.N., Edgar J.R., Archuleta T.L., Hirst J., Jackson L.P., Robinson M.S, & Borner G.H. (2018). AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A. Nature Communications, 9, 3958.

Publication 2018

BL21(DE3)pLysS cells glutathione sepharose Superdex S200

2 mercaptoethanol Buffer Cells Gel filtration Glutathione Hepes Human Iptg Nacl Proteins Reduced glutathione Sepharose

Corresponding Organization :

Other organizations : University of Cambridge, Max Planck Institute of Biochemistry, Vanderbilt University

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Variable analysis

independent variables

Expression of AP4E1 (residues 881–1135) and AP4B1 (residues 612–739) appendage domains as GST-fusion proteins in BL21(DE3)pLysS cells

dependent variables

Purification of GST-fusion proteins

control variables

Expression of GST-fusion proteins for 16–20 h at 22 °C after induction with 0.4 mM IPTG
Purification of proteins in 20 mM HEPES pH 7.5, 200 mM NaCl, and 2 mM 2-Mercaptoethanol
Lysis of cells using a disruptor (Constant Systems Limited)
Affinity purification using glutathione sepharose (GE Healthcare)
Elution of fusion proteins in buffer with 30 mM reduced glutathione
Further purification by gel filtration on a Superdex S200 preparative column (GE Healthcare)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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