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Title: Structure and function of a neuraminidase from Streptococcus pneumoniae
Author: Soma, Shital
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2000
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The aim of this project was to characterise a neuraminidase from Streptococcus pneumoniae by relating its amino acid sequence to the enzymatic activity of the protein, leading to the production of mutated neuraminidases that could be tested as protective immunogens. The sequence of the cloned neuraminidase gene (nan A), was compared to other bacterial neuraminidases to identify conserved residues, and also utilising crystallography data, predictions were made of the residues likely to be important in catalysis. Three residues, glutamic acid (E) 647, arginine (R) 663 and tyrosine (Y) 752 were chosen for further study. To assess the importance of these residues in catalysis, conservative substitutions of these residues (E647 > Q, R663 > H and Y752 > F) were made and the subsequent effect of enzyme activity measured. The wild-type and mutated neuraminidase genes were cloned into the expression vector pQE30 and purified by Ni-NTA affinity chromatography. The purified neuraminidases were assayed for enzyme activity using a colorimetric assay. The E647 > Q and Y752 > F mutations resulted in reduction in specific activity to a level below the detection range of the assay (H mutation resulted in a decrease in activity to 2% of that of the wild type. The protective effects of the mutated neuraminidases in mice was investigated by immunization followed by challenge with virulent Streptococcus penumoniae. Mice immunized with heat-inactivated wild-type neuraminidase survived for 90 hours after challenge, mice immunized with (E647 > Q, Y752 > F or R663 > H survived for 97, 191 or 140 hours respectively. The antibody levels in the sera of the mice immunized with wild type or E647 > Q neuraminidases were measured before and after immunization, the antibody levels were increased following immunization, however no relationship was detected between antibody levels and the survival time of the individual mice.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available