The cloning , expression and characterisation of bacterial chitin-binding proteins from pseudomonas aeruginosa , serratia marcescens, photorhabdus luminescens and photorhabdus asymbiotica.
Larragy, Ruth (2011) The cloning , expression and characterisation of bacterial chitin-binding proteins from pseudomonas aeruginosa , serratia marcescens, photorhabdus luminescens and photorhabdus asymbiotica. PhD thesis, Dublin City University.
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It is well recognised that most proteins are subject to post translational modifications and that these modifications can have specific effects on the biological properties and functions of these proteins. The majority of proteins secreted by cells are modified by the attachment of oligosaccharide chains. This glycosylation event has been shown to impact correct protein folding, protein stability, solubility, to aid in cell recognition and to help regulate cell processes. In order to gain a deeper understanding into the impact of altered glycosylation patterns on cellular processes and cell recognition it is necessary to develop new technologies to profile the glycan species displayed on the surface of protein molecules.
The present study was dedicated to the development of prokaryotic chitin-binding proteins as novel carbohydrate-binding molecules. Prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus asymbiotica and Photorhabdus luminescens were cloned, over-expressed in E. coli and purified to homogeneity via (His)6 affinity tags. The activity and specificity of these proteins was tested using a number of insoluble substrates; chitin, chitosan and crystalline cellulose. The ability of these proteins to bind to protein linked glycans was tested using Enzyme linked lectin assays (ELLAs). None of the proteins exhibited any ability to bind glycoproteins in this assay format. A novel N-acetylglucosamine binding assay was developed using CBP21 and the ability to immobilise active CBP21 on a sepharose surface was also demonstrated. Sugar inhibition studies indicated that CBP21 may be capable of binding to mannan and galactan polymers. A site-directed mutagenesis of CBP21 was carried out on the putative binding domain residues to alter the affinity of CBP21. Residues Y54, E55, P56, Q57, S58, E60, T111, H114 and D182 were mutated to alanine, expressed, purified and characterised. The mutation H114A was shown to negatively impact on β-chitin affinity, the Q57A mutant had an increased affinity for chitosan with the proteins Y54A, T111A and D182A displaying an increased affinity for Cellulose. Furthermore it was shown that the putative C-terminal binding domain of CbpD is a chitin-binding domain and that the putative chitin-binding proteins CbpA and CbpL are capable of binding to both α- and β-chitin.
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