Many proteins with special properties have been identified in aquatic organisms. Due to their peculiarity, some of these biomolecules could be used advantageously for some industrial and health care applications. However, the preparation of these biochemicals from its original source is highly impractical and generally unfeasible for commercial purposes. Nevertheless, the advances in DNA manipulation open the possibility of producing the recombinant proteins in different organisms to allow a viable production and extraction on a commercial scale. Among such particular proteins, the cunner-fish trypsin (CFT) is an enzyme with a high potential for exploitation by the food processing industry. The CFT is a cold adapted protease that has a proven ability to inactivate undesirable endogenous enzymes during the processing of some foodstuffs. Thus, the CFT gene was characterized and cloned in E. coli and Pichia pastoris for over-expression. A cDNA library from pancreatic mRNA was screened using a salmon trypsin cDNA probe. Positive clones harboring a cDNA insert of the predicted size were sequenced and a full-length cDNA was obtained that contained an ORF encoding the zymogen and a signal peptide. Multiple alignment of the gene sequence showed 85 to 90 per cent identities with other fish trypsins and the deduced amino acid sequence for the mature enzyme exhibited the entire characteristic features observed in trypsins. E. coli expressed the recombinant CFT (rCFT) at levels higher than 20% in the form of insoluble aggregates, while P. pastoris showed expression levels below 1%. Despite the low expression, the yeast rCFT appeared to be correctly folded and could be partially purified by immobilized nickel affinity chromatography. Trypsin-specific activity was confirmed in the purified rCFT after digestion with bovine enterokinase.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84289 |
| Date | January 2004 |
| Creators | Macouzet-García, Martin |
| Contributors | Simpson, B. K. (advisor), Lee, B. H. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002130874, proquestno: AAINQ98315, Theses scanned by UMI/ProQuest. |
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