Impact of GFP tagging on Robo1-heparin interaction

Authors
Zhang, Fuming
Moniz, Heather A.
Walcott, Benjamin
Moremen, Kelley W.
Wang, Lianchun
Linhardt, Robert J.
ORCID
https://orcid.org/0000-0003-2219-5833
No Thumbnail Available
Other Contributors
Issue Date
2014-01-01
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Degree
Terms of Use
In Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/
Full Citation
Impact of GFP tagging on Robo1-heparin interaction, F. Zhang, B. Walcott, H. A. Moniz, K. W. Moremen, L. Wang, R. J. Linhardt, Glycoconjugate Journal, 31, 299–307, 2014.
Abstract
Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.
Description
Glycoconjugate Journal, 31, 299–307
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
Publisher
Relationships
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Glycoconjugate Journal
https://harc.rpi.edu/
Access
https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1007/s10719-014-9522-1