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dc.contributor.authorHoulihan, Gillianen
dc.contributor.authorGatti-Lafranconi, Pietroen
dc.contributor.authorLowe, Daviden
dc.contributor.authorHollfelder, Florianen
dc.date.accessioned2015-09-02T15:40:49Z
dc.date.available2015-09-02T15:40:49Z
dc.date.issued2015-06-30en
dc.identifier.citationHoulihan et al. Protein Engineering, Design and Selection (2015) 28 (9): 269-279. doi: 10.1093/protein/gzv029en
dc.identifier.issn1741-0126
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/250438
dc.description.abstractIn vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.
dc.description.sponsorshipG.H. was supported by a CASE studentship from the Engineering and Physical Sciences Research Council and MedImmune and the Marie-Curie Research Training Network ENEFP. F.H. is a starting investigator of the European Research Council. Funding to pay the Open Access publication charges for this article was provided by the Engineering and Physical Sciences Research Council.
dc.languageEnglishen
dc.language.isoenen
dc.publisherOxford University Press
dc.rightsAttribution 2.0 UK: England & Wales*
dc.rights.urihttp://creativecommons.org/licenses/by/2.0/uk/*
dc.subjectalternative scaffolden
dc.subjectantibodyen
dc.subjectDARPinen
dc.subjectdirected evolutionen
dc.subjectin vitro compartmentalisationen
dc.subjectSNAP displayen
dc.subjecttrade-offen
dc.titleDirected evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection processen
dc.typeArticle
dc.description.versionThis is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/protein/gzv029en
prism.endingPage279
prism.publicationDate2015en
prism.publicationNameProtein Engineering, Design and Selectionen
prism.startingPage269
prism.volume28en
dc.rioxxterms.funderEPSRC
dcterms.dateAccepted2015-05-07en
rioxxterms.versionofrecord10.1093/protein/gzv029en
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2015-06-30en
dc.contributor.orcidHollfelder, Florian [0000-0002-1367-6312]
dc.identifier.eissn1741-0134
rioxxterms.typeJournal Article/Reviewen


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Attribution 2.0 UK: England & Wales
Except where otherwise noted, this item's licence is described as Attribution 2.0 UK: England & Wales