Kaltenbach, Miriam ; Stein, Viktor ; Hollfelder, Florian (2011)
SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.
In: Chembiochem : a European journal of chemical biology, 12 (14)
Artikel, Bibliographie
Kurzbeschreibung (Abstract)
Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2011 |
| Autor(en): | Kaltenbach, Miriam ; Stein, Viktor ; Hollfelder, Florian |
| Art des Eintrags: | Bibliographie |
| Titel: | SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution. |
| Sprache: | Englisch |
| Publikationsjahr: | 2011 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Chembiochem : a European journal of chemical biology |
| Jahrgang/Volume einer Zeitschrift: | 12 |
| (Heft-)Nummer: | 14 |
| Kurzbeschreibung (Abstract): | Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered. |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Protein Engineering of Ion Conducting Nanopores |
| Hinterlegungsdatum: | 14 Nov 2016 11:37 |
| Letzte Änderung: | 14 Nov 2016 11:37 |
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