title: Design and characterization of structured protein linkers with differing flexibilities.
abstract: Engineered fusion proteins containing two or more functional polypeptides joined by a peptide or protein linker are important for many fields of biological research. The separation distance between functional units can impact epitope access and the ability to bind with avidity; thus the availability of a variety of linkers with different lengths and degrees of rigidity would be valuable for protein design efforts. Here, we report a series of designed structured protein linkers incorporating naturally occurring protein domains and compare their properties to commonly used Gly4Ser repeat linkers. When incorporated into the hinge region of an immunoglobulin G (IgG) molecule, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen-binding domains, in contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein and tetratricopeptide repeats. This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the construction of antibodies with enhanced binding properties or other fusion proteins.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Klein
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Siduo
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Rachel P
authors.initials: RP
authors.lastname: Galimidi
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Keeffe
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA bjorkman@caltech.edu.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Bjorkman
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D057446:Hydrodynamics; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D018583:Pliability; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins
keywords: fusion proteins; hydrodynamic radius; linker design; size-exclusion chromatography