title: Cytoplasmic versus periplasmic expression of site-specifically and bioorthogonally functionalized nanobodies using expressed protein ligation.
abstract: Site-specific functionalization of nanobodies after introducing bioorthogonal groups offers the possibility to biofunctionalize surfaces with a uniformly oriented layer of nanobodies. In this paper, expressed protein ligation (EPL) was used for site-specific alkynation of the model nanobody NbBcII10. In contrast to EPL constructs, which are typically expressed in the cytoplasm, nanobodies are expressed in the periplasm where its oxidizing environment ensures a correct folding and disulfide bond formation. Different pathways were explored to express the EPL constructs in the periplasm but simultaneously, the effect of cytoplasmic expression on the functionality of NbBcII10 was also evaluated. By using Escherichia coli SHuffle®T7 cells, it was demonstrated that expression of the EPL complex in the cytoplasm was readily established and that site-specifically mono-alkynated nanobodies can be produced with the same binding properties as the non-modified NbBcII10 expressed in the periplasm. In conclusion, this paper shows that periplasmic expression of the EPL complex is quite challenging, but cytoplasmic expression has proven to be a valuable alternative.
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, BE-3590 Diepenbeek, Belgium. Electronic address: brecht.billen@uhasselt.be.
authors.forename: Brecht
authors.initials: B
authors.lastname: Billen
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), BE-1050 Brussels, Belgium. Electronic address: cvincke@vub.ac.be.
authors.forename: Cécile
authors.initials: C
authors.lastname: Vincke
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, BE-3590 Diepenbeek, Belgium. Electronic address: rebekka.hansen@uhasselt.be.
authors.forename: Rebekka
authors.initials: R
authors.lastname: Hansen
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel (VUB), BE-1090 Brussels, Belgium. Electronic address: ndevoogd@vub.ac.be.
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), BE-1050 Brussels, Belgium. Electronic address: svmuylde@vub.ac.be.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, BE-3590 Diepenbeek, Belgium; Applied and Analytical Chemistry, Institute for Materials Research (IMO), Hasselt University, BE-3590 Diepenbeek, Belgium. Electronic address: peter.adriaensens@uhasselt.be.
authors.forename: Peter
authors.initials: P
authors.lastname: Adriaensens
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, BE-3590 Diepenbeek, Belgium. Electronic address: wanda.guedens@uhasselt.be.
authors.forename: Wanda
authors.initials: W
authors.lastname: Guedens
mesh_terms: D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000235:genetics
chemical_list: D061905:Single-Domain Antibodies
keywords: Click chemistry; CuAAC; Expressed protein ligation; Nanobodies; Periplasmic expression and extraction