Community Research and Development Information Service - CORDIS



Project ID: 322096
Funded under: FP7-PEOPLE
Country: Turkey

Final Report Summary - GLYCOPHAGEAPP (Development of GlycoPhage Arrays as a novel high-throughput method for glycomics)

Glycosylation is a widespread post-translational modification to proteins and plays important roles in health and disease, however knowledge on glycans and their role in metabolism is much more primitive than that of DNA and proteins. Traditional analytical tools has not been sufficient to ‘decode' the complex structural information in glycans. Recently, printed glycan microarrays (glycoarrays) have emerged as powerful, high-throughput tools for screening glycan-protein interactions, and have been developed by different research groups for disease detection, drug discovery, study of immunity and host-pathogen interactions. Still, the full potential of defined glycan microarrays will only be realized by (i) increasing the quantity and diversity of glycan structures and (ii) developing reliable and reproducible chemistries for the immobilization of chemically and structurally diverse glycan probes onto a solid support with retention of functionality. Hence, this project aims to develop novel glycan arrays (GlycoPhage Arrays), where various glycan epitopes displayed on filamentous phage particles are immobilized on a solid support in a spatially defined arrangement for highly parallel, high-throughput profiling of glycan-binding protein (GBP) specificities in experimental and clinical samples.

Towards this goal, firstly, the previously designed glycophage system developed by the Researcher, Asst. Prof. Dr. Eda Çelik (Akdur), was redesigned such that the phagemid is bicistronic (carries the oligosaccharyl transferase, PglB) and contains additional acceptor sites for glycan attachment and a truncated g3p. Moreover, a knockout of the waaL gene in Escherichia coli strain TG1 was created (E. coli TG1 delta-waaL), to increase the pool of available substrates for PglB. Second, various phage-displayed glycan structures using the redesigned glycophage system was produced. The host was transformed with the phagemid and a second plasmid encoding a glycan biosynthesis pathway and then infected with a helper phage, to produce the glycophages. The immunoblotting results confirmed that O-antigen polysaccharides and N-linked glycans were covalently linked to the phage-displayed glycan acceptor protein. Third, from these various, structurally diverse set of glycophages, spatially defined small arrays were created on microtiter plates, nitrocellulose membranes and microarray glass slides and screened for interaction with GBPs. Ultimately, the glycophages were found to possess significantly specific GBP-binding profiles when tested against negative controls such as synthetic glycans or non-specific glycophages. In the second period of the project, the efforts focused basically on (i) increasing the glycophage yield from the culture medium (ii) optimization of the glycophage purification process (iii) optimization of the arraying conditions in order to increase the signal-to-noise ratio (S:N) (iv) stability and reproducibility studies.

In summary, by deployment of phage display so that the displayed proteins carry a covalently linked glycan of interest (glycophages) and subsequently patterned in array format, the researcher has introduced a powerful novel technique for facile and scalable production, and purification of glycans compatible with microarray patterning. The project was successfully completed and two high-impact papers were published as planned, as well as seven conference proceedings at international meetings. Two lab-open-days were organized for high-school students. Her work appeared in many national newspapers and the website is also intended to disseminate the results of this on-going work.

Thankfully, this career integration grant has been a crucial stepping stone for the fellow´s career as well as her integration into the host institution. Dr. Çelik continued her collaborative studies with Cornell University and has formed new collaborations within the Department and also in Europe and USA, received seven new grants in total and additional funds from the host institution to renovate her laboratory space and travel grant to go for study visits to Cornell. The researcher formed her independent research group, currently including 4 PhD candidates and two MSc degree students. Dr. Çelik received the prestigious UNESCO-L’Oréal National Fellowship for Young Women in Science (2014) Award and also got an Assistant Professor position from the host institution.

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Life Sciences
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