title: SNAP display: in vitro protein evolution in microdroplets.
abstract: SNAP display is based on the covalent reaction of the DNA repair protein AGT (O(6)-alkylguanine DNA alkyltransferase, the "SNAP-tag") with its substrate benzylguanine (BG). Linear, BG-labelled template DNA is encapsulated in water-in-oil emulsion droplets with a diameter of a few micrometres (i.e. 1 mL of emulsion contains ∼10(10) compartments). Each droplet contains only a single DNA copy, which is transcribed and translated in vitro. The expressed AGT fusion proteins attach to their coding DNA via the BG label inside the droplet, which ensures that a specific genotype-phenotype linkage is established. Subsequently, the emulsion is broken and protein-DNA conjugates, which constitute a DNA-tagged protein library, selected via affinity panning. This method will prove a useful addition to the array of in vitro display systems, distinguished by the stability of DNA as the coding nucleic acid and the covalent link between gene and protein.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Miriam
authors.initials: M
authors.lastname: Kaltenbach
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D004247:DNA / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D046210:Microfluidic Analytical Techniques; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D013379:Substrate Specificity
chemical_list: D019151:Peptide Library; D006147:Guanine; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase