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Title: Towards an improved β-1,4-mannosyltransferase for biocatalysis : fusions to fluorescent partners and application for the development of high-throughput assays
Author: Alexandre, Julie Anne Christine
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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β-1,4-Mannosyltransferase plays an essential role in the biosynthesis of N-glycans. The application of the enzyme for biocatalysis is hampered by poor expression levels of the corresponding Saccharomyces cerevisiae gene, ALG1 and by very high specificity for phospholipid structures. The aim of the current project has been to investigate ways in which production of the enzyme can be improved and new substrate specificity can be studied. A series of β-1,4-mannosyltransferase fused to either the C-terminus of the green fluorescent protein (GFP) or the C- or N- terminus of the SNAP-tag were produced in Escherichia coli. Alternatively, improved expression levels of the gene fusions and stability of the chimeric proteins were sought by switching to a Pichia pastoris expression system. An N-terminal GFP fusion produced from a bacterial expression vector was the most promising and the corresponding gene fusion was used in directed evolution studies for improved expression levels. Key to these studies was the development of a new assay that could be used in a ‘high-throughput’ fashion. Screening for active enzyme was achieved using hierarchical methods: firstly, identification of colonies exhibiting good fluorescence indicated production of folded protein. Secondly, an affinity assay using GDP-sepharose was developed to isolate fluorescent mutants binding to GDP. A library of ALG1 mutants fused to GFP was generated by error-prone PCR and after screening, a number of promising candidates was isolated and characterised. None of these mutants gave reliably high expression levels than the wild-type gene. It was suggested that the levels of expression of the genes were not stable enough to identify any useful high-expression mutants.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available