title: An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.
abstract: In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium Faculty of Food Technology and Biotechnology, Can Tho University of Technology, Can Tho, Vietnam.
authors.forename: Duy Tien
authors.initials: DT
authors.lastname: Ta
authors.affiliation: Maastricht Science Programme, Maastricht University, Maastricht 6200 MD, The Netherlands.
authors.forename: Erik Steen
authors.initials: ES
authors.lastname: Redeker
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium.
authors.forename: Brecht
authors.initials: B
authors.lastname: Billen
authors.affiliation: Applied and Analytical Chemistry, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium.
authors.forename: Gunter
authors.initials: G
authors.lastname: Reekmans
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium.
authors.forename: Josephine
authors.initials: J
authors.lastname: Sikulu
authors.affiliation: Biomedical Research Institute (Biomed) and School of Life Sciences, Transnationale Universiteit Limburg, Hasselt University, Agoralaan-Building C, Diepenbeek BE-3590, Belgium.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Noben
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium.
authors.forename: Wanda
authors.initials: W
authors.lastname: Guedens
authors.affiliation: Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium Applied and Analytical Chemistry, Institute for Materials Research (IMO), Hasselt University, Agoralaan-Building D, Diepenbeek BE-3590, Belgium peter.adriaensens@uhasselt.be.
authors.forename: Peter
authors.initials: P
authors.lastname: Adriaensens
mesh_terms: D000480:Alkynes / Q000737:chemistry; D002686:Chitin / Q000378:metabolism; D057930:Click Chemistry; D003593:Cytoplasm / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D047668:Inteins; D015202:Protein Engineering / Q000379:methods*; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics*; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics
chemical_list: D000480:Alkynes; D061905:Single-Domain Antibodies; D019010:Vascular Cell Adhesion Molecule-1; D002686:Chitin
keywords: CuAAC; VCAM1-targeting nanobody; bioorthogonal chemistry; cytoplasmic expression; intein-mediated protein ligation