The recombinant expression and characterization of human neuron specific enolase
The recombinant expression and characterization of human neuron specific enolase
Enolase is one of the enzymes of glycolysis, evolutionarily one of the oldest energy pathways. Within vertebrates, it is found in three tissue-specific forms, of which neuron specific enolase (NSE) is one. This project has sought via sequence analysis and selective expression experimentation; i) to analyse the patterns of sequence variation within the human enolase isoenzymes and also differences in enolase sequence between species; ii) to relate these differences to a structural model of enolase, based upon the X-ray defined structure of yeast enolase 1; iii) to reconstruct a full length cDNA of human NSE from cDNA fragments, and prove the reconstruction by expression of the sequence; iv) to determine the major epitope regions of the human NSE molecule by a combination of restriction fragment and random fragment expression; v) to employ the NSE epitope data in the construction of a two site assay, based upon a precise knowledge of antibody binding sites on the NSE molecules. Sequence analysis has shown a higher fixation of mutations in the NNE sequences analysed, with orthologous comparisons between the muscle and neuronal enolase isoenzymes in rat and mouse showing a higher resistance to mutation fixation. From a reconstructed and proven full length cDNA of human NSE, the NSE protein has been shown to exhibit four major epitope regions, located predominantly at mapped loop and α-helix positions. By immunisation of Balb/c mice, each of the epitope regions has been shown to be immunogenic, an encouraging result for the prospect of monoclonal antibody production. Polyclonal antibodies from different species of animal have been affinity purified into epitope specific fractions, which in turn have been used in preliminary studies of two site assays for NSE, which have, in non-optimised tests, shown detection to below 0.5ng/ml. A conformational influence in NSE-antibody binding has also been observed.
University of Southampton
1992
Quinn, Gregory Bernard
(1992)
The recombinant expression and characterization of human neuron specific enolase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Enolase is one of the enzymes of glycolysis, evolutionarily one of the oldest energy pathways. Within vertebrates, it is found in three tissue-specific forms, of which neuron specific enolase (NSE) is one. This project has sought via sequence analysis and selective expression experimentation; i) to analyse the patterns of sequence variation within the human enolase isoenzymes and also differences in enolase sequence between species; ii) to relate these differences to a structural model of enolase, based upon the X-ray defined structure of yeast enolase 1; iii) to reconstruct a full length cDNA of human NSE from cDNA fragments, and prove the reconstruction by expression of the sequence; iv) to determine the major epitope regions of the human NSE molecule by a combination of restriction fragment and random fragment expression; v) to employ the NSE epitope data in the construction of a two site assay, based upon a precise knowledge of antibody binding sites on the NSE molecules. Sequence analysis has shown a higher fixation of mutations in the NNE sequences analysed, with orthologous comparisons between the muscle and neuronal enolase isoenzymes in rat and mouse showing a higher resistance to mutation fixation. From a reconstructed and proven full length cDNA of human NSE, the NSE protein has been shown to exhibit four major epitope regions, located predominantly at mapped loop and α-helix positions. By immunisation of Balb/c mice, each of the epitope regions has been shown to be immunogenic, an encouraging result for the prospect of monoclonal antibody production. Polyclonal antibodies from different species of animal have been affinity purified into epitope specific fractions, which in turn have been used in preliminary studies of two site assays for NSE, which have, in non-optimised tests, shown detection to below 0.5ng/ml. A conformational influence in NSE-antibody binding has also been observed.
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Published date: 1992
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Local EPrints ID: 461642
URI: http://eprints.soton.ac.uk/id/eprint/461642
PURE UUID: a94bd546-519e-42ec-9bfb-1b07e25ca72f
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Date deposited: 04 Jul 2022 18:51
Last modified: 04 Jul 2022 18:51
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Author:
Gregory Bernard Quinn
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