| dc.description.abstract | The bacterium Vibrio cholerae is one of the most well-known contaminants of water and food sources across the world, and a cause of the disease cholera. It is particularly notorious for having outbreaks in underdeveloped parts of the world, or in areas affected by natural disasters or war, where access to clean drinking water is poor. Each year, cholera is estimated to cause 100 000 deaths. In addition to having the ability to colonize the human intestine, V. cholerae is known to colonize surfaces containing the polysaccharide chitin, found in the exoskeletons of several marine invertebrates. Colonization is believed to involve the adhesin N-acetyl glucosamine binding protein A (GbpA), which V. cholerae secretes into its aquatic environment. GbpA is a multifunctional protein with four domains, three of which have previously been structurally determined by x-ray crystallography. These are the N-terminal domain (D1), and the two middle domains (D2-3). D1 belongs to a class of enzymes called lytic polysaccharide monooxygenase (LPMO), which are known to cleave polysaccharides like chitin or cellulose. This domain is also known to interact with mucins, which are glycoproteins found in the human intestine. The functions of D2 and D3 are not known, but it has been hypothesized that they may play a role in anchoring V. cholerae to its substrate. The structure of the small C-terminal domain (D4) remains unknown, but it has been reported to also bind to chitinous surfaces. This thesis presents the results of interaction studies on GbpA with chitin, carried out by nuclear magnetic resonance (NMR) spectroscopy and electron microscope (EM) imaging. GbpA was produced using VmaxTM as an expression host. VmaxTM is a strain of Vibrio natriegens and a non-toxic relative of V. cholerae, possessing similar secretion pathways useful for GbpA expression and functionality. VmaxTM has previously been shown to express GbpA in high yields, which was confirmed in in this thesis. GbpA was also isotope-labelled with nitrogen-15 (15N) for NMR spectroscopy experiments. Although the NMR experiments yielded some inconclusive results, interactions between chitin and GbpA could be clearly observed in the EM-images obtained. Additionally, D4 was expressed alone with a glutathione S-transferase (GST) tag, using both Escherichia coli and VmaxTM as expression hosts. Although D4 was successfully isolated, extensive optimization of expression is required to acquire sufficient yields for v structure determination by NMR spectroscopy. An alternative method for isolating D4 was tested, where cleavage sites for TEV and HRV 3C proteases were inserted into the flexible linker separating D3 and D4, which would allow for GbpA to be expressed in high yields, from which D4 would be separated. Unfortunately, these cleavage sites could not be recognized by the proteases used, even though the same proteases could successfully cleave other proteins possessing the same cleavage sites. Predictions of the three-dimensional fold of D4 was made using AlphaFold and RoseTTAFold. | nob |