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Title: Expression, mutagenesis and properties of bacteriophage K1E endosialidase
Author: Bryant, J. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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A full length endoE gene construct has been generated from the partial clones used to sequence the gene. Recombinant endoE has been expressed and purified as a fusion protein that is catalytically active and possesses the same kinetic characteristics as the bacteriophage enzyme. The mature, active endoE is 76 kDa in size and is probably produced by the post-translational cleavage of a 90 kDa primary translation product. Recombinant endoE has been purified to homogeneity, in milligram quantities, with the fusion protein partner removed for further studies of the enzyme. The role of the C-terminus of endoE was studied by expression of C-terminally truncated proteins. These were expressed from 3' truncated endoE gene constructs generated either by exonuclease III-mediated nested deletion or by PCR. The site of proteolytic processing has been located to within 18 amino acids and further truncation beyond this cleavage site renders the enzyme inactive and more susceptible to proteolytic degradation. The endoE sequence contains two copies of the sialidase motif and one P-loop motif. The importance of these motifs in enzyme activity was studied by site-directed mutagenesis, replacing part or all of the motifs with alanine residues. These indicated that the P-loop motif was not involved either in binding or hydrolysis of polysialic acid and that the sialidase motifs appear to be necessary to allow production of correctly folded, soluble protein but are not involved directly in catalysis. The kinetic characteristics of the binding interaction of the endoE fusion protein and polysialic acid have been studied and compared to those of C-terminally truncated endoE fusion proteins. As a first step in investigating the use of endoE as a reagent in cell biological studies, the subcellular localisation of polysialic acid and the effects of chloroquine and brefeldin A treatment on its localisation in cultured MCF-7 breast tumour cells have been studied.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available