The lectin Concanavalin A is processed <i>in planta </i>by a splicing-mediated circular permutation of its initial single-chain precursor, pro-Concanavalin A. Active (carbohydrate-binding) protein conformations can be purified by dextran-affinity chromatography. This thesis demonstrates <i>in vitro</i> splicing by asparaginyl endopeptidase of two cofolded polypeptide fragments (A- and B-chains) corresponding to Concanavalin A precursors. This ligation of A- and B-chains was enzyme-, temperature- and pH-dependent. The 9-residue extension at the C-terminus of A-chain is essential for splicing. To test whether correct cofolding and an intervening spacer are required for maturation, recombinant A- and B-chains without spacer sequence were purified and refolded separately. Asparaginyl endopeptidase was again absolutely required for ligation of non-cofolded A- and B-chains. Correct folding is crucial to form an active structure, but is less important for enzyme-mediated splicing. Protein splicing of two-chain forms of precursors was clearly evident from the large decrease in electrophoretic mobility of ligated product. However, interpretation of proteolytic cleavage patterns was difficult. <i>In vivo</i> maturation could be reconstituted <i>in vitro</i> using recombinant pro-Concanavalin A (single-chain, active) with asparaginyl endopeptidase. Enzyme could also splice synthetic peptides corresponding to processing-sequences of precursor proteins. Two short peptides were ligated to form a new longer peptide as indicated by reverse-phase chromatography. It was generally observed that increasing the pH from 5 to 7.5 changed the balance away from proteolytic cleavages (hydrolysis) and towards protein (or peptide) ligation (aminolysis). Promotion of splicing at higher pH indicates that availability of the unprotected (nucleophilic) form of the attacking terminal a-amino group is a major factor in determining product formation. Other factors (substrate conformation, concentrations, temperature and incubation time) may also influence the outcome.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:637901 |
| Date | January 2003 |
| Creators | Li, M. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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