In Vivo Biotinylated scFv Fragments

Biotinylation of proteins is a rare process in almost all living organisms, whereas biotin-associated enzymes are key-enzymes for manifold essential metabolic pathways related to carboxyl transfer within cells. In vivo biotinylation of proteins via genetic engineering is highly attractive due to the high affinity between biotin and avidin or streptavidin and its applicability in various fields. Most importantly, biotinylation of antibodies and antibody fragments offers opportunities for automation and high throughput methods requiring specific binding partners. Here, a time and cost efficient protocol is described for in vivo biotinylation of single chain fragment variable (scFv) at constant rates in the prokaryotic host E. coli, purification of those scfv, and determination of biotinylation efficiency.