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Title:	Preparation of high-mannose type n-glycans from recombinant β-mannanases expressed in pichia pastoris
Authors:	Hussein Mahmood Ahmed Al'Bajalan (P79492)
Supervisor:	Mukram Mckeen, Prof. Dr.
Keywords:	N-glycans Biological process Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	15-Jan-2019
Description:	N-glycans play important roles in fundamental biological processes such as the structural modulation of proteins, cell-cell signaling and interactions, pathogen-host recognition, and tumour progression. Their biological significance creates a demand for efficient and reliable methods of N-glycan preparation for structural and functional studies. The chemical synthesis of N-glycans such as Glc1-3Man5-9GlcNAc2 with many hydroxyl groups is a complex and expensive process due to the demanding protection chemistry required to control regio- and stereo-specificity. Synthesis of N-glycans by enzymatic approaches using glycosyl transferases and glycosidases is also challenging because of their respective substrate and specificity limitations. Alternatively, a non-synthesis approach via isolation of N-glycans from natural sources, typically from plant, hen egg yolk and mammalian cells is relatively accessible. However, this approach is time consuming and low yielding from long growth or culture periods. Therefore, in this study, two alternative scalable and biotechnology-friendly approaches to produce glucosylated N-glycans of limited commercial availability from natural sources were explored. These approaches involved the use of crude extracts from: (1) the alg8 N-glycosylation mutant of Saccharomyces cerevisiae to produce accumulated levels of monoglucosylated high-mannose type N-glycans (HMNGs) and (2) recombinant β-mannanase glycoproteins Man AF and Man TV that are highly overexpressed in methylotrophic Pichia pastoris (with and without glucosidase inhibitor treatment). This study showed that these approaches were unsuccessful in obtaining monoglucosylated N-glycans but yielded preparative amounts of non-glucosylated HMNGs from the glycoprotein overexpression system. The recombinant β-mannanase glycoproteins of both species were successfully overexpressed and secreted into the supernatant at 30 oC with 0.5% MeOH induction. N-glycan release from these glycoproteins in the supernatant (crude extract) using peptide N-glycosidase F (PNGase F) provided higher yields than the chemical approach (hydrazinolysis). In addition, higher recovery of released N-glycans was obtained using graphitised carbon black (GCB) compared to hydrophilic interaction liquid chromatography (HILIC) and solvent extraction methods. The released N-glycans from the supernatant of Man AF and Man TV were analysed using HPLC-FD after pre-column glycan derivatisation withthe 2-aminobenzamide (2-AB) fluorescent tag. Although HMNGs were detected in both Man AF and Man TV, this study focused on recovering HMNGs from the Man TV because of the presence of hypermannosylation in Man AF. The most abundant HPLC-FD peaks of significance from the supernatant of Man AF at retention times 43.08 (GU value 10.05), 45.23 (GU value 11.19) and 47.09 (GU value11.87) minutes were identified using Glycan Unit (GU) analysis (calculated using an external a glycan molecular weight ladder) with reference to the GlycoStore database. This was complemented with biochemical characterisation using enzymatic hydrolysis (jack bean α-mannosidase: +ve, and β-glucanase: -ve) and lectin analysis with concanavalin A (eluate: +ve). These three peaks were subsequently assigned as Man8GlcNAc2-2AB, Man9GlcNAc2-2AB and Man10GlcNAc2-2AB. In conclusion, the scalable preparation of HMNGs could be obtained using the overexpression of glycoproteins in the Pichia pastoris yeast expression system.,'Certification of Master's/Doctoral Thesis' is not available,Ph.D.
Pages:	223
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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