Preparation of high-mannose type N-glycans from recombinant beta-mannanases expressed in Pichia pastoris

Al Bajalan, Hussein Mahamood Ahmed; Sarizan, Nur Maisarah; Yahaya, Yasmin; Chai, Sin Yee; Needs, Sarah; Hasbullah, Siti Aishah; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Wormald, Mark; Allman, Sarah; Alonzi, Dominic and Mackeen, Mukram Mohamed (2017). Preparation of high-mannose type N-glycans from recombinant beta-mannanases expressed in Pichia pastoris. In: International Conference on Natural Products 2017, 15-16 Mar 2017, Swiss-Garden Beach Resort, Damai Laut, Perak, Malaysia.

Abstract

Asparagine (N)-linked glycosylation is a protein modification found in the majority of secreted glycoproteins This highly conserved process occurs in the endoplasmic reticulum (ER) of all eukaryotes involving the transfer of the Glc3Man9GlcNAc2 oligosaccharide/glycan by the enzyme oligosaccharyl transferase (OST) to the asparagine residue within the tripeptide sequence, asparagine-X-serine/threonine (X= any amino acid except proline) of newly synthesized polypeptides followed by chaperone-assisted protein folding to produce correctly folded secretory N-linked glycoproteins. The yeast Pichia pastoris (methylotrophic yeast) is a promising tool to potentially produce abundant yields of high-mannose type N-glycans such as Man8-14GlcNAc2. In this study, recombinant β-mannanases from Aspergillus flavus and Trichoderma viridans were expressed in Pichia pastoris and purified as described previously. The total pool of N-glycans were released from the supernatant of both mannanases using the hexoaminidase, peptide N-glycosidase F (PNGase F). The released N-glycans were then labelled with the fluorescent tag 2-aminobenzamide (2-AB) and analysed by high performance liquid chromatography with florescence detection (HPLC-FD). The results of this preliminary glycan profiling will subsequently be used for the preparative production of N-glycans after further characterisation using HPLC-based monosaccharide composition and enzymatic analyses (jack bean mannosidase and ER mannosidase I).

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