title: Design and characteristics of a stable protein scaffold for specific binding based on variable lymphocyte receptor sequences.
abstract: Variable lymphocyte receptors (VLRs) serve as antigen binding proteins in jawless vertebrates. Their relatively low molecular weight makes VLRs an interesting alternative to antibodies in biotechnological applications. A typical VLR comprises several unique motifs called leucine-rich repeats (LRRs). Using consensus approach we designed a novel VLR protein (called dVLR) containing six LRR repeats based on a sea lamprey receptor sequence. The designed protein was expressed in Escherichia coli in a soluble, native form and showed very favorable biophysical properties. Recombinant dVLR is monomeric in solution and preserves its secondary structure within the pH range 3.0 to 11.0 and tertiary structure between pH 4.0 and 10.0. It undergoes reversible thermal denaturation in a broad pH range (4.0 to 10.0). The maximal denaturation temperature of 73.9°C is observed at pH 6.0, 0.3M NaCl. Chemical denaturation of dVLR at pH 7.5 is a cooperative two-state process with a midpoint at 3.3M GdmCl and a very high free energy change of unfolding in the absence of denaturant equal to 14.1kcal/mol. The biophysical properties of dVLR make it highly suitable for biotechnological applications such as generation of specific ligand-binding molecules.
authors.affiliation: Department of Protein Engineering, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wezner-Ptasińska
authors.forename: Daniel
authors.initials: D
authors.lastname: Krowarsch
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D056704:Adaptive Immunity; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D007798:Lampreys / Q000276:immunology; D000090263:Leucine-Rich Repeat Proteins; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011946:Receptors, Antigen / Q000737:chemistry*
chemical_list: D000090263:Leucine-Rich Repeat Proteins; D011506:Proteins; D011946:Receptors, Antigen