Mouse Fam132b DNA (NM_173395) was cloned into the pENTR4.LP shuttle vector54 (link) containing tandem tetracycline operators55 (link) after replacing its native secretion signal sequence with the one from mouse IgG, inserting a Kozak sequence and C-terminal addition of a G4S linker, and a C-tag via KpnI and EcoRI sites and purified (Maxiprep, Qiagen). The plasmid was transiently transfected in the Expi-CHO system (Gibco, Thermoscientific). Supernatants were harvested 13 days post transfection and the mouse ERFE protein was affinity purified via CaptureSelect C-tag affinity matrix (Life Technologies, Thermo Fisher Scientific) using 2 M MgCl2, 20 mM Tris, pH 7.0.
Pasricha S.R., Lim P.J., Duarte T.L., Casu C., Oosterhuis D., Mleczko-Sanecka K., Suciu M., Da Silva A.R., Al-Hourani K., Arezes J., McHugh K., Gooding S., Frost J.N., Wray K., Santos A., Porto G., Repapi E., Gray N., Draper S.J., Ashley N., Soilleux E., Olinga P., Muckenthaler M.U., Hughes J.R., Rivella S., Milne T.A., Armitage A.E, & Drakesmith H. (2017). Hepcidin is regulated by promoter-associated histone acetylation and HDAC3. Nature Communications, 8, 403.
Other organizations :
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Children's Hospital of Philadelphia, University of Groningen, Jenner Institute, John Radcliffe Hospital
Replacing the native secretion signal sequence of Mouse Fam132b DNA (NM_173395) with the one from mouse IgG
Inserting a Kozak sequence
Adding a C-terminal G4S linker and C-tag via KpnI and EcoRI sites
dependent variables
Purification of the mouse ERFE protein
control variables
Cloning Mouse Fam132b DNA (NM_173395) into the pENTR4.LP shuttle vector containing tandem tetracycline operators
Transient transfection in the Expi-CHO system
Harvesting supernatants 13 days post transfection
Affinity purification of the mouse ERFE protein via CaptureSelect C-tag affinity matrix using 2 M MgCl2, 20 mM Tris, pH 7.0
controls
Positive control: Not mentioned
Negative control: Not mentioned
Annotations
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