A study of the expression of a protein proteinase inhibitor from sweet corn
<p>Sweet Corn Inhibitor (SCI), a small (11811Da.) protein from the seeds of opaque-2 corn is a potent and specific inhibitor of trypsin and the activated Hageman Factor (Factor βXIIa) of the human blood plasma coagulation system. With the eventual aim of obtaining insight into the structure-...
| Main Authors: | , |
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
1991
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| Subjects: |
| _version_ | 1826300785815715840 |
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| author | De Silva, H de Silva, H. A. Rohan |
| author2 | Lowe, G |
| author_facet | Lowe, G De Silva, H de Silva, H. A. Rohan |
| author_sort | De Silva, H |
| collection | OXFORD |
| description | <p>Sweet Corn Inhibitor (SCI), a small (11811Da.) protein from the seeds of opaque-2 corn is a potent and specific inhibitor of trypsin and the activated Hageman Factor (Factor βXIIa) of the human blood plasma coagulation system. With the eventual aim of obtaining insight into the structure- function relationships of the selective SCI-pXIIa interaction, a synthetic gene for SCI was cloned into <em>Saccharomyces cerevisiae</em> (yeast) and <em>Escherichia coli</em> (<em>E.coli</em>) expression systems in an attempt to obtain overexpression of the recombinant gene product. The establishment of functional expression, together with an isolation and purification procedure for SCI would provide a system for obtaining selected reactive-site mutants of SCI by cassette- and oligonucleotide-directed mutagenesis.</p> <p>A yeast secretion vector for a truncated form of SCI (tSCI) was constructed by cloning the gene for α-factor prepro-tSCI fusion, downstream to the α-mating factor (MFα1) promoter of yeast. Yeast transformants containing the expression vector failed to express and secrete the desired product.</p> <p>The synthetic gene encoding the complete SCI sequence was cloned into <em>E.coli</em> expression vectors that directed both cytoplasmic and periplasmic expression. In cytoplasmic expression, the SCI gene was cloned directly downstream to the powerful, inducible λ-phage PL- and <em>trc</em>-promoters. No expression was obtained with the latter. With the former, expression levels of up to 3% of the total bacterial protein were obtained. These levels were improved 3- to 4-fold on incorporation of the <em>E.coli dnaY<em> gene product. Solubilisation and refolding of the purified SCI inclusion bodies failed to yield the active, correctly folded product. Failure to obtain an N-terminal sequence indicated an incompletely processed N-terminal methionine. For periplasmic expression, SCI, fused in-frame to the signal sequence of OmpA, a major <em>E.coli</em> outer membrane protein, was cloned into the same λ-phage P<sub>L</sub> promoter vector. High levels (=10%) of expression of insoluble SCI were obtained. The nearly homogeneous product was obtained by a two-step procedure, involving ion-exchange chromatography, followed by hydrophobic interaction chromatography. Characterisation by N-terminal sequencing, SDS-PAGE and electrospray mass spectrometry, confirmed the presence of correctly processed SCI in the form of covalently associated dimers. Refolding studies are at present in progress.</em></em></p> |
| first_indexed | 2024-03-07T05:22:26Z |
| format | Thesis |
| id | oxford-uuid:df605ba7-4a1f-4968-92e6-4c8a06fa613c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:22:26Z |
| publishDate | 1991 |
| record_format | dspace |
| spelling | oxford-uuid:df605ba7-4a1f-4968-92e6-4c8a06fa613c2022-03-27T09:39:00ZA study of the expression of a protein proteinase inhibitor from sweet cornThesishttp://purl.org/coar/resource_type/c_db06uuid:df605ba7-4a1f-4968-92e6-4c8a06fa613cInhibitorsProtein-protein interactionsProteinaseSweet cornEnglishPolonsky Theses Digitisation Project1991De Silva, Hde Silva, H. A. RohanLowe, GLowe, G<p>Sweet Corn Inhibitor (SCI), a small (11811Da.) protein from the seeds of opaque-2 corn is a potent and specific inhibitor of trypsin and the activated Hageman Factor (Factor βXIIa) of the human blood plasma coagulation system. With the eventual aim of obtaining insight into the structure- function relationships of the selective SCI-pXIIa interaction, a synthetic gene for SCI was cloned into <em>Saccharomyces cerevisiae</em> (yeast) and <em>Escherichia coli</em> (<em>E.coli</em>) expression systems in an attempt to obtain overexpression of the recombinant gene product. The establishment of functional expression, together with an isolation and purification procedure for SCI would provide a system for obtaining selected reactive-site mutants of SCI by cassette- and oligonucleotide-directed mutagenesis.</p> <p>A yeast secretion vector for a truncated form of SCI (tSCI) was constructed by cloning the gene for α-factor prepro-tSCI fusion, downstream to the α-mating factor (MFα1) promoter of yeast. Yeast transformants containing the expression vector failed to express and secrete the desired product.</p> <p>The synthetic gene encoding the complete SCI sequence was cloned into <em>E.coli</em> expression vectors that directed both cytoplasmic and periplasmic expression. In cytoplasmic expression, the SCI gene was cloned directly downstream to the powerful, inducible λ-phage PL- and <em>trc</em>-promoters. No expression was obtained with the latter. With the former, expression levels of up to 3% of the total bacterial protein were obtained. These levels were improved 3- to 4-fold on incorporation of the <em>E.coli dnaY<em> gene product. Solubilisation and refolding of the purified SCI inclusion bodies failed to yield the active, correctly folded product. Failure to obtain an N-terminal sequence indicated an incompletely processed N-terminal methionine. For periplasmic expression, SCI, fused in-frame to the signal sequence of OmpA, a major <em>E.coli</em> outer membrane protein, was cloned into the same λ-phage P<sub>L</sub> promoter vector. High levels (=10%) of expression of insoluble SCI were obtained. The nearly homogeneous product was obtained by a two-step procedure, involving ion-exchange chromatography, followed by hydrophobic interaction chromatography. Characterisation by N-terminal sequencing, SDS-PAGE and electrospray mass spectrometry, confirmed the presence of correctly processed SCI in the form of covalently associated dimers. Refolding studies are at present in progress.</em></em></p> |
| spellingShingle | Inhibitors Protein-protein interactions Proteinase Sweet corn De Silva, H de Silva, H. A. Rohan A study of the expression of a protein proteinase inhibitor from sweet corn |
| title | A study of the expression of a protein proteinase inhibitor from sweet corn |
| title_full | A study of the expression of a protein proteinase inhibitor from sweet corn |
| title_fullStr | A study of the expression of a protein proteinase inhibitor from sweet corn |
| title_full_unstemmed | A study of the expression of a protein proteinase inhibitor from sweet corn |
| title_short | A study of the expression of a protein proteinase inhibitor from sweet corn |
| title_sort | study of the expression of a protein proteinase inhibitor from sweet corn |
| topic | Inhibitors Protein-protein interactions Proteinase Sweet corn |
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