{"id":5877,"date":"2025-12-04T12:27:03","date_gmt":"2025-12-04T12:27:03","guid":{"rendered":"https:\/\/www.sygnaturediscovery.com\/?post_type=case-study&p=5877"},"modified":"2026-02-05T16:53:01","modified_gmt":"2026-02-05T16:53:01","slug":"in-vivo-biotinylation-of-a-secreted-protein-from-mammalian-cells","status":"publish","type":"case-study","link":"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/in-vivo-biotinylation-of-a-secreted-protein-from-mammalian-cells\/","title":{"rendered":"In vivo biotinylation of a secreted protein from mammalian cells"},"content":{"rendered":"\n

Biotinylation of proteins\u00a0is a powerful tool for protein science and our recent case study on the\u00a0in vivo<\/em> biotinylation of intracellular proteins by co-expression with BirA ligase<\/a>\u00a0described how this method is now used routinely at Sygnature Discovery.<\/h3>\n\n\n\n

A number of our mammalian expressed proteins, however, are secreted proteins and BirA is an intracellular protein. A method for biotinylation within the\u00a0secretory pathway<\/a>\u00a0was reported in 2008. This method involved the co-expression of BirA and the target protein from a bicistronic plasmid, with BirA being fused to the signal leader peptide from a mouse immunoglobulin heavy chain; the idea being that both the target protein and BirA translocate to the endoplasmic reticulum (ER) where biotinylation occurs.<\/p>\n\n\n\n

To provide further options for our clients\u2019 projects, we decided to try a similar approach in our proprietory HEK293-6E cell expression system. We fused BirA to an IgK secretary leader signal in a separate plasmid for co-transfection and expression with the target protein. This trans approach was adopted to allow us to produce both non-biotinylated and biotinylated forms of the protein, if needed.<\/p>\n\n\n\n

We chose a heterodimeric, secreted cytokine protein we have previously expressed and biotinylated successfully in vitro, as a case study protein to test this approach. One of the proteins was tagged with the short AviTag required for site specific biotinylation.<\/p>\n\n\n\n

Co-transfection and expression<\/h3>\n\n\n\n

HEK293 cells were co-transfected with three plasmids expressing proteins with secretory signal signals: the two target heterodimeric proteins (\u03b1 + \u03b2) and BirA. The BirA expression plasmid was transfected at a 10-fold lower level than the two target plasmids and cells were grown, supplemented with biotin. Expression was also performed without BirA present.<\/p>\n\n\n\n

Purification<\/h3>\n\n\n\n

Cells were spun down and the supernatant was purified by nickel chelate affinity chromatography, showing two clear bands in the nickel eluate.<\/p>\n\n\n\n

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