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Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris
Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and dele...
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2016
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| _version_ | 1867613305180258304 |
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| access_status_str | Open Access |
| author | Hüsler, Jennifer |
| author2 | Klump, Horst H |
| author_browse | Hüsler, Jennifer Klump, Horst H |
| author_facet | Klump, Horst H Hüsler, Jennifer |
| author_sort | Hüsler, Jennifer |
| collection | Thesis |
| description | Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Cucurbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/20454 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:34:00.978Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2016 |
| publishDateRange | 2016 |
| publishDateSort | 2016 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/20454 Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris Hüsler, Jennifer Klump, Horst H Brandt, Wolf F Maeder, Dennis Biochemistry Squash inhibitors are the smallest natural serine protease inhibitors. Their compact, rigid nature has enabled detailed examination of their 3D structure by NMR and X-ray crystallography. Being of a convenient size to synthesise chemically, the effects on activity of selective substitutions and deletions within the sequence have also been investigated. Thus, this family of inhibitors is considered useful as a model system for the study of protein-protein interactions. Cucurbita maxima trypsin inhibitor I (CMTI I) may be thought of as representative of the squash inhibitors, for which there is detailed structural and functional information available. It is a 29 amino acid protein, with the tri-disulphide bridging pattern common to all squash inhibitors. There are only a few examples of squash inhibitors being produced by recombinant DNA technology. As this technique offers a relatively cheap way of producing large amounts of these proteins, further investigation is required. Problems have been experienced with the expression of disulphide-rich proteins in E. coli, as the cytosol of this microorganism is not conducive to their folding. Furthermore extraction of these proteins from the peri plasmic space is often required, resulting in a reduction in yield. To overcome these shortcomings, the methylotrophic yeast Pichia pastoris was investigated as an alternative means of expression, although at the inception of this work, no disulphide-rich proteins of this size had been expressed in P. pastoris. It was a challenge to investigate the feasibility of producing squash inhibitors in this expression host and to compare the activity of the recombinant inhibitor to that of native CMTI I. 2016-07-19T14:20:47Z 2016-07-19T14:20:47Z 1996 Master Thesis Masters Master of Science http://hdl.handle.net/11427/20454 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Biochemistry Hüsler, Jennifer Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris |
| thesis_degree_str | Master's |
| title | Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris |
| title_full | Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris |
| title_fullStr | Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris |
| title_full_unstemmed | Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris |
| title_short | Biosynthesis of Cucurbita maxima trypsin inhibitor I in the methylotropic yeast Pichia pastoris |
| title_sort | biosynthesis of cucurbita maxima trypsin inhibitor i in the methylotropic yeast pichia pastoris |
| topic | Biochemistry |
| url | http://hdl.handle.net/11427/20454 |
| work_keys_str_mv | AT huslerjennifer biosynthesisofcucurbitamaximatrypsininhibitoriinthemethylotropicyeastpichiapastoris |