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Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in sing...

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Autores principales: Anderson, George P., Teichler, Daniel D., Zabetakis, Dan, Shriver-Lake, Lisa C., Liu, Jinny L., Lonsdale, Stephen G., Goodchild, Sarah A., Goldman, Ellen R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975481/
https://www.ncbi.nlm.nih.gov/pubmed/27494523
http://dx.doi.org/10.1371/journal.pone.0160534
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author Anderson, George P.
Teichler, Daniel D.
Zabetakis, Dan
Shriver-Lake, Lisa C.
Liu, Jinny L.
Lonsdale, Stephen G.
Goodchild, Sarah A.
Goldman, Ellen R.
author_facet Anderson, George P.
Teichler, Daniel D.
Zabetakis, Dan
Shriver-Lake, Lisa C.
Liu, Jinny L.
Lonsdale, Stephen G.
Goodchild, Sarah A.
Goldman, Ellen R.
author_sort Anderson, George P.
collection PubMed
description Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.
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spelling pubmed-49754812016-08-25 Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein Anderson, George P. Teichler, Daniel D. Zabetakis, Dan Shriver-Lake, Lisa C. Liu, Jinny L. Lonsdale, Stephen G. Goodchild, Sarah A. Goldman, Ellen R. PLoS One Research Article Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. Public Library of Science 2016-08-05 /pmc/articles/PMC4975481/ /pubmed/27494523 http://dx.doi.org/10.1371/journal.pone.0160534 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Anderson, George P.
Teichler, Daniel D.
Zabetakis, Dan
Shriver-Lake, Lisa C.
Liu, Jinny L.
Lonsdale, Stephen G.
Goodchild, Sarah A.
Goldman, Ellen R.
Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein
title Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein
title_full Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein
title_fullStr Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein
title_full_unstemmed Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein
title_short Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein
title_sort importance of hypervariable region 2 for stability and affinity of a shark single-domain antibody specific for ebola virus nucleoprotein
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975481/
https://www.ncbi.nlm.nih.gov/pubmed/27494523
http://dx.doi.org/10.1371/journal.pone.0160534
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