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Title: | Cytoplasmic versus periplasmic expression of site-specifically and bioorthogonally functionalized nanobodies using expressed protein ligation | Authors: | BILLEN, Brecht Vincke, Cécile HANSEN, Rebekka Devoogdt, Nick Muyldermans, Serge ADRIAENSENS, Peter GUEDENS, Wanda |
Issue Date: | 2017 | Source: | PROTEIN EXPRESSION AND PURIFICATION, 133, p. 25-34 | 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. | Notes: | Guedens, W (reprint author), Hasselt Univ, Inst Mat Res IMO, Biomol Design Grp, BE-3590 Diepenbeek, Belgium. brecht.billen@uhasselt.be; cvincke@vub.ac.be; rebekka.hansen@uhasselt.be; ndevoogd@vub.ac.be; svmuylde@vub.ac.be; peter.adriaensens@uhasselt.be; wanda.guedens@uhasselt.be | Keywords: | nanobodies; expressed protein ligation; periplasmic expression and extraction; click chemistry; CuAAC | Document URI: | http://hdl.handle.net/1942/23468 | ISSN: | 1046-5928 | e-ISSN: | 1096-0279 | DOI: | 10.1016/j.pep.2017.02.009 | ISI #: | 000401684400004 | Rights: | © 2017 Elsevier Inc. All rights reserved. | Category: | A1 | Type: | Journal Contribution | Validations: | ecoom 2018 |
Appears in Collections: | Research publications |
Files in This Item:
File | Description | Size | Format | |
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Artikel Brecht_PREP_cytoplasmic versus periplasmic expression.pdf Restricted Access | Published version | 1.48 MB | Adobe PDF | View/Open Request a copy |
YPREP_5054_edit_report (1).pdf | Peer-reviewed author version | 580.22 kB | Adobe PDF | View/Open |
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