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Please use this identifier to cite or link to this item: http://hdl.handle.net/1942/19197

Title: An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation
Authors: Ta, Duy Tien
Steen Redeker, Erik
Billen, Brecht
Reekmans, Gunter
Sikulu, Josephine
Noben, Jean-Paul
Guedens, Wanda
Adriaensens, Peter
Issue Date: 2015
Citation: PROTEIN ENGINEERING DESIGN & SELECTION, 28 (10), p. 351-363
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.
Notes: Adriaensens, P (reprint author), Hasselt Univ, Inst Mat Res IMO, Biomol Design Grp, Agoralaan Bldg D, BE-3590 Diepenbeek, Belgium. peter.adriaensens@uhasselt.be
URI: http://hdl.handle.net/1942/19197
Link to publication: https://www.researchgate.net/publication/280871877_An_efficient_protocol_towards_site-specifically_clickable_nanobodies_in_high_yield_cytoplasmic_expression_in_Escherichia_coli_combined_with_intein-mediated_protein_ligation
DOI: 10.1093/protein/gzv032
ISI #: 000362837000006
ISSN: 1741-0126
Category: A1
Type: Journal Contribution
Validation: ecoom, 2016
Appears in Collections: Research publications

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