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Fig. 1 | Microbial Cell Factories

Fig. 1

From: Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system

Fig. 1

The E. coli hemolysin system for secretion of nanobodies. a Schematic representation of the HlyB, HlyD and TolC components of the Hly secretion system that spans the inner membrane (IM), the periplasmic space with the peptidoglycan (PG) layer, and outer membrane (OM) of E. coli. TolC is a trimeric OM protein with a large periplasmic domain; HlyB is an ATPase and forms a dimer in the IM. HlyD is a trimeric adaptor IM protein that interacts with HlyB and with the periplasmic domain of TolC. For simplicity, a longitudinal section of the Hly-protein complex is represented showing only two subunits of HlyD and a continuous open channel for protein export. The HlyBD complex recognises the C-terminal domain of HlyA (C-HlyA) in the bacterial cytosol to export the fusion protein with the nanobody (Nb) VHH domain to the extracellular medium. b Plasmids pEHLYA5 and pVDL9.3 used in this work for the secretion of Nb-HlyA fusions on E. coli bacteria (TolC+). Plasmid pEHLYA5 is used to generate fusion of the VHH sequence with an N-terminal His-tag and C-HlyA secretion signal. The linker region between the VHH and C-HlyA sequences includes tags for immunodetection (HA-tag, E-tag) and a human rhinovirus 3C protease recognition site. Plasmid pVDL9.3 encodes HlyB and HlyD components. Expression of the VHH-HlyA, HlyB and HlyD is controlled under the Plac promoter in both plasmids

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