Producing Folded Recombinant Proteins

All proteins were produced by transient transfection using human embryonic kidney HEK293E cells as described (11 (link)) to ensure posttranslational modifications such as disulfide bonds and glycans were added. Proteins were purified using their His₆ tag using a bespoke supernatant loading rig and 96-well Ni²⁺-nitrilotriacetic acid filter plates (15 (link)). Heat-labile immunoreactivity to demonstrate folding was confirmed by heat-denaturing the proteins for 10 min at 90 °C before capture on a streptavidin-coated plate via their biotin tag and determination of immunoreactivity by ELISA as described (19 (link)).

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Sun Y., Vandenbriele C., Kauskot A., Verhamme P., Hoylaerts M.F, & Wright G.J. (2015). A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand. Molecular & Cellular Proteomics : MCP, 14(5), 1265-1274.

Publication 2015

Biotin Cells Disulfide Elisa Embryonic Glycans His6 tag Human Kidney Nitrilotriacetic acid Posttranslational modifications Proteins Streptavidin Transfection Transient

Corresponding Organization : KU Leuven

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Variable analysis

independent variables

Transient transfection using human embryonic kidney HEK293E cells

dependent variables

Heat-labile immunoreactivity to demonstrate folding

control variables

Posttranslational modifications such as disulfide bonds and glycans were added
Proteins were purified using their His6 tag using a bespoke supernatant loading rig and 96-well Ni2+-nitrilotriacetic acid filter plates

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