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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 | Source: | 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 | Keywords: | bioorthogonal chemistry; CuAAC; cytoplasmic expression; intein-mediated protein ligation; VCAM1-targeting nanobody | Document URI: | http://hdl.handle.net/1942/19197 | Link to publication/dataset: | 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 | ISSN: | 1741-0126 | e-ISSN: | 1741-0134 | DOI: | 10.1093/protein/gzv032 | ISI #: | 000362837000006 | Rights: | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com | Category: | A1 | Type: | Journal Contribution |
Appears in Collections: | Research publications |
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gzv032.pdf | Published version | 928.79 kB | Adobe PDF | View/Open |
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