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http://hdl.handle.net/1942/19197
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DC Field | Value | Language |
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dc.contributor.author | TA, Duy Tien | - |
dc.contributor.author | STEEN REDEKER, Erik | - |
dc.contributor.author | BILLEN, Brecht | - |
dc.contributor.author | REEKMANS, Gunter | - |
dc.contributor.author | Sikulu, Josephine | - |
dc.contributor.author | NOBEN, Jean-Paul | - |
dc.contributor.author | GUEDENS, Wanda | - |
dc.contributor.author | ADRIAENSENS, Peter | - |
dc.date.accessioned | 2015-09-23T09:54:36Z | - |
dc.date.available | 2015-09-23T09:54:36Z | - |
dc.date.issued | 2015 | - |
dc.identifier.citation | PROTEIN ENGINEERING DESIGN & SELECTION, 28 (10), p. 351-363 | - |
dc.identifier.issn | 1741-0126 | - |
dc.identifier.uri | http://hdl.handle.net/1942/19197 | - |
dc.description.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. | - |
dc.description.sponsorship | This work was funded by the Fund for Scientific Research of Flanders (FWO-Vlaanderen) via the project G.0581.12N. | - |
dc.language.iso | en | - |
dc.rights | © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com | - |
dc.subject.other | bioorthogonal chemistry; CuAAC; cytoplasmic expression; intein-mediated protein ligation; VCAM1-targeting nanobody | - |
dc.title | An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation | - |
dc.type | Journal Contribution | - |
dc.identifier.epage | 363 | - |
dc.identifier.issue | 10 | - |
dc.identifier.spage | 351 | - |
dc.identifier.volume | 28 | - |
local.format.pages | 13 | - |
local.bibliographicCitation.jcat | A1 | - |
dc.description.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 | - |
local.type.refereed | Refereed | - |
local.type.specified | Article | - |
dc.identifier.doi | 10.1093/protein/gzv032 | - |
dc.identifier.isi | 000362837000006 | - |
dc.identifier.url | 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 | - |
item.contributor | TA, Duy Tien | - |
item.contributor | STEEN REDEKER, Erik | - |
item.contributor | BILLEN, Brecht | - |
item.contributor | REEKMANS, Gunter | - |
item.contributor | Sikulu, Josephine | - |
item.contributor | NOBEN, Jean-Paul | - |
item.contributor | GUEDENS, Wanda | - |
item.contributor | ADRIAENSENS, Peter | - |
item.fulltext | With Fulltext | - |
item.fullcitation | TA, Duy Tien; STEEN REDEKER, Erik; BILLEN, Brecht; REEKMANS, Gunter; Sikulu, Josephine; NOBEN, Jean-Paul; GUEDENS, Wanda & ADRIAENSENS, Peter (2015) An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation. In: PROTEIN ENGINEERING DESIGN & SELECTION, 28 (10), p. 351-363. | - |
item.accessRights | Open Access | - |
crisitem.journal.issn | 1741-0126 | - |
crisitem.journal.eissn | 1741-0134 | - |
Appears in Collections: | Research publications |
Files in This Item:
File | Description | Size | Format | |
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gzv032.pdf | Published version | 928.79 kB | Adobe PDF | View/Open |
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