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Title: Expression of recombinant snake venom metalloproteinases in Escherichia coli for antivenom production.
Author: Affleck, Louise Jane.
ISNI:       0000 0001 3398 8105
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 1998
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Snake Venom Metalloproteinases (SVMPs)/haemorrhagins are members of the adamalysin superfamily of large, multi-domain zinc-dependent metalloproteinases. In addition to being the main lethal component of crotaline and viperine venoms, the haemorrhagins are also a major antigenic component, promoting them as suitable antigens for antivenom production. A recombinant approach using Escherichia coli as the heterologous host was taken to produce proteins for antivenom production utilising the putative haemorrhagin EchI cDNA clone isolated from Echis pyramidum leakeyi (Epl) venom mRNA (Paine et al. 1994) as a template. EchI is 2062 bp in length, encodes an Open Reading Frame (ORF) of 617 amino acids and a mature protein predicted to be 46 kDa. Despite using two prokaryotic expression vectors and several E. coli expression strains, expression of the full-length, the ORF, the mature protein and the metalloproteinase domain failed. The metalloproteinase domain was found to be toxic to Ecoli which provided an explanation for the lack of expression of the metalloproteinase domain-containing constructs as discussed. However, expression of Echi-C (the disintegrin-like and cysteine rich domain) was achieved only in the pET32/AD494(DE3)pLysS vector host combination, albeit as insoluble Inclusion Bodies (IBs) containing unstable recombinant proteins. To overcome the toxicity of the metalloproteinase domain, expression of "truncated peptide", sequential, overlapping, approximately 100 amino acid regions derived from the EchI ORF was attempted using a pET32/AD494(DE3)pLysS combination. The N-terminal region of the metalloproteinase domain encoded by Pep 3 was shown to be a major toxic element of Echl to E. coli. IPTG induction of Peps, 4,5 and 6 resulted in insoluble, partially folded recombinant proteins. Preliminary analysis showed them to be inactive and possible reasons for the lack of bioactivity are discussed. Mice immunised with Peps 4,5 and 6 (individually and a cocktail mixture) produced antibodies reactive with whole Epl venom and native EchII (ELISA and immunoblot) and showed partial protection against the lethal effects of whole Epl venom in mice
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pharmacology & pharmacy & pharmaceutical chemistry