title: Dual-affinity avidin molecules.
abstract: A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.
authors.affiliation: NanoScience Center, Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland.
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Henri R
authors.initials: HR
authors.lastname: Nordlund
authors.forename: Jarno
authors.initials: J
authors.lastname: Hörhä
authors.forename: Thomas K M
authors.initials: TK
authors.lastname: Nyholm
authors.forename: David E
authors.initials: DE
authors.lastname: Hyre
authors.forename: Tuomas
authors.initials: T
authors.lastname: Kulomaa
authors.forename: Eevaleena J
authors.initials: EJ
authors.lastname: Porkka
authors.forename: Ari T
authors.initials: AT
authors.lastname: Marttila
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
authors.forename: Olli H
authors.initials: OH
authors.lastname: Laitinen
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
mesh_terms: D000818:Animals; D001360:Avidin / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002645:Chickens; D002846:Chromatography, Affinity; D005456:Fluorescent Dyes; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013696:Temperature; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; D001360:Avidin