Directed evolution of anti-HER2 DARPins by SNAP display reveals stability-function trade-offs in the selection processReportar como inadecuado


Directed evolution of anti-HER2 DARPins by SNAP display reveals stability-function trade-offs in the selection process


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Publication Date: 2015-06-30

Journal Title: Protein Engineering, Design and Selection

Publisher: Oxford University Press

Volume: 28

Issue: 9

Pages: 269-279

Language: English

Type: Article

Metadata: Show full item record

Citation: Houlihan, G., Gatti-Lafranconi, P., Lowe, D., & Hollfelder, F. (2015). Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process. Protein Engineering, Design and Selection, 28 (9), 269-279.

Description: This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/protein/gzv029

Abstract: In 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.

Keywords: alternative scaffold, antibody, DARPin, directed evolution, in vitro compartmentalisation, SNAP display, trade-off

Sponsorship: G.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.

Identifiers:

This record's URL: http://dx.doi.org/10.1093/protein/gzv029https://www.repository.cam.ac.uk/handle/1810/250438

Rights: Attribution 2.0 UK: England & Wales

Licence URL: http://creativecommons.org/licenses/by/2.0/uk/





Autor: Houlihan, GillianGatti-Lafranconi, PietroLowe, DavidHollfelder, Florian

Fuente: https://www.repository.cam.ac.uk/handle/1810/250438



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