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Design and characterization of structured protein linkers with differing flexibilities

Klein, Joshua S. and Jiang, Siduo and Galimidi, Rachel P. and Keeffe, Jennifer R. and Bjorkman, Pamela J. (2014) Design and characterization of structured protein linkers with differing flexibilities. Protein Engineering, Design and Selection, 27 (10). pp. 325-330. ISSN 1741-0126. PMCID PMC4191447. doi:10.1093/protein/gzu043.

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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 Gly_4Ser repeat linkers. When incorporated into the hinge region of an immunoglobulin G (IgG) molecule, flexible Gly_4Ser 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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Jiang, Siduo0000-0002-2143-4030
Keeffe, Jennifer R.0000-0002-5317-6398
Bjorkman, Pamela J.0000-0002-2277-3990
Additional Information:© 2014 The Author. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received July 2, 2014; revised July 2, 2014; accepted August 22, 2014. Edited by Lynne Regan. We thank Maria Politzer for assistance with sub-cloning and plasmid preparations and the Caltech Protein Expression Center for producing antibody-linker constructs. This work was supported by a grant from the Bill and Melinda Gates Foundation through the Grand Challenges in Global Health Initiative, the Director’s Pioneer Award [1DP1OD006961-01 to P.J.B.] and the National Institutes of Health HIVRAD [P01Al100148 to P.J.B.]. Funding to pay the Open Access publication charges for this article was provided by NIDA/National Institutes of Health.
Funding AgencyGrant Number
Bill and Melinda Gates FoundationUNSPECIFIED
National Institute on Drug AbuseUNSPECIFIED
Subject Keywords:fusion proteins; hydrodynamic radius; linker design; size-exclusion chromatography
Issue or Number:10
PubMed Central ID:PMC4191447
Record Number:CaltechAUTHORS:20141017-103700879
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:50490
Deposited By: Tony Diaz
Deposited On:17 Oct 2014 17:46
Last Modified:10 Nov 2021 18:56

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