Avidin mutants

Inventors

Nordlund, Henri Rainer • Laitinen, Olli Heikki • HYTOENEN VESA PEKKA, null • Hytönen, Vesa Pekka • Kulomaa, Markku Sakari

Assignees

Aidian Oy

Abstract

Two circularly permuted avidin monomers arc designed. The circularly permuted monomers are fused and the resulting fusion peptides (dcAvd) form a pseudo-tetrameric dual-chain avidin, which is biologically active in biotin binding and shows similar structural characteristics as wild-type avidin. The dcAvd makes the development of dual-affinity avidins possible by allowing the adjustment of the ligand binding properties in the half of the binding sites differently than in the rest of the sites. The present invention provides further a single-chain avidin (scAvd) where two dcAvd-molecules are fused together via a linker to form a single polypeptide with four binding sites for biotin or other ligand.

Core Innovation

The invention relates to engineering avidin fusion proteins by constructing a dual-chain avidin (dcAvd) from two circularly permuted avidin monomers (cpAvd). The cpAvd monomers assemble into a pseudo-tetrameric architecture having four biotin-binding sites. This design enables adjustment of ligand-binding affinity independently in different binding-site subsets.

A dual-chain avidin (dcAvd) is defined as a fusion of two circularly permuted avidin monomers (cpAvd), where the original C-terminal amino acid and the original N-terminal amino acid are joined directly or via a linker to create a new C-terminus and a new N-terminus. The cpAvd monomers are selected from defined circularly permuted avidin monomer types (cpAvd5→24, cpAvd6→25, and cpAvd4→23), and the resulting dcAvd binds biotin or other ligand.

A further architecture is described in which a single-chain avidin (scAvd) is formed by fusing two dual-chain avidin (dcAvd) molecules via a linker, yielding four binding sites in one polypeptide. The document also characterizes quaternary structure assembly and ligand-binding behavior of these constructs, including biotin dissociation behavior and affinity measurements using ligands such as 2-iminobiotin and HABA.

Claims Coverage

Independent claim clm-00001 covers a dual-chain avidin (dcAvd) formed by fusing two circularly permuted avidin monomers (cpAvd) with defined permutation types and new termini created by joining original termini directly or via a linker, where the dcAvd binds biotin or other ligand. A second inventive feature covers a dual-chain pseudo-tetrameric avidin assembled from two dual-chain avidin molecules.

Across the independent claim(s) explicitly provided, the inventive scope centers on a dcAvd architecture built from defined circularly permuted avidin monomers with newly created termini, directly or via a linker, that bind biotin or other ligands, and on forming a pseudo-tetrameric avidin architecture by assembling two dcAvd molecules.

Stated Advantages

Documented Applications