Development of bacteriophage nanoparticles for glycomics analysis by genetic engineering
Thesis Type: Postgraduate
Institution Of The Thesis: Hacettepe Üniversitesi, Fen Bilimleri Enstitüsü, Biyomühendislik A.B.D., Turkey
Approval Date: 2014
Thesis Language: English
Student: GÖKSU GÜR
Principal Supervisor (For Co-Supervisor Theses): EDA ÇELİK AKDUR
Open Archive Collection: AVESIS Open Access Collection
Abstract:Complex carbohydrates (glycans) are attached to proteins and lipids by the process of glycosylation and play important roles in many biological processes. Altered glycosylation is known to cause diseases including cancer and retroviral infection. Carbohydratebased arrays, or "glycoarrays," have emerged in the last decade as a powerful tool in glycomics, especially in glycosylation-based diagnosis of diseases; however, to fully exploit the potential of glycans arrays, it is necessary to increase the diversity of glycans and to develop reliable and reproducible chemistries for immobilization of the carbohydrate probes onto solid support. Recently, the protein glycosylation locus (Pgl) discovered in Campylobacter jejuni was functionally transferred to Escherichia coli, conferring ability to glycosylate proteins. Additionally, our research group has recently demonstrated for the first time, N-glycosylation of phage particles (glycophages), simply by infecting the glycosylation competent E. coli with M13 phage displaying a glycan-acceptor protein. Considering that phage-patterned microarrays are a standard art in proteomics, the hypothesis of this study is that the glycophage particles can be exploited for the development of "glycophage arrays", to be used in pathogen or cancer diagnosis in the future. In this study, host cell engineering and six new bicistronic phagemids construction and design were planned (pBAD-MBPDQNAT-g3p::PglB, pBAD-MBPDQNAT-g3p::PglBmut, pBAD-MBP4xAQNAT-g3p::PglB, pBAD-MBP4xDQNAT-g3p::PglBmut, pBAD-MBP4xDQNAT-g3ptr::PglB, pBAD-MBP4xDQNAT-g3ptr::PglBmut), and the designed phagemids were constructed using standard genetic engineering techniques. The sequence of the constructed phagemids were confirmed with restriction digestion and DNA sequencing analysis. Next, because glycosylation potential of phage particles are increased if the waaL gene is deleted from E. coli TG1 host cell, the deletion of waaL gene was verified with flourescence-activated cell sorting (FACS) analysis. Thereafter, in order to increase the glycophage production efficiency and stability of phagemids, different parameters were tested; and (i) the truncated-g3p in the phagemid enabled better display of glycans on the phage compared to full-length g3p, (ii) different purification conditions were tested to get spesific signals and the higher anti-glycan signal was obtained from sarkosyl treated samples, (iii) effect of induction conditions on cell growth and phage production capacity were compared with nine different parameters and best results were detected in the case where, OD600=0.6 at the time of infection, 8x109 CFU/mL of helper phage is used for infection and induction period is 16 h. Finally, "glycophage-ELISA" method was developed for the first time, as a new tool in glycomics analysis. Indirect ELISA method was choosen as the suitable ELISA type; and %2 BSA-PBS (bovine serum albumine) buffer gave a more specific anti-glycan signal compared to other blocking buffers tested. The study is significant because it has overcome the current bottlenecks in glycan array construction and provide a relatively inexpensive, specific and stable glycan representation method, as well as introduce a simplified and universal purification technique that is not dependent on the carbohydrate. With the development of new glycosylation pathway encoding plasmids, the variety of glycophages can be extended in the future. The basis of glycophage array technology described here should help to expand the diversity of glycan libraries and provide a complement to the existing toolkit for high-throughput analysis of glycan–protein interactions.