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The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36
Includes bibliographical references (leaves 103-126).
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2015
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| _version_ | 1867613165879033856 |
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| access_status_str | Open Access |
| author | Crampton, Michael Craig |
| author2 | Reid, Sharon J |
| author_browse | Crampton, Michael Craig Reid, Sharon J |
| author_facet | Reid, Sharon J Crampton, Michael Craig |
| author_sort | Crampton, Michael Craig |
| collection | Thesis |
| description | Includes bibliographical references (leaves 103-126). |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/14641 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:31:48.735Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2015 |
| publishDateRange | 2015 |
| publishDateSort | 2015 |
| 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:11427/14641 The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 Crampton, Michael Craig Reid, Sharon J Louw, Maureen E Berger, E Molecular and Cell Biology Includes bibliographical references (leaves 103-126). This study relates to the development of an alkaliphilic, thermo-tolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium harbors the endogenous genetic background to over-produce flagellin protein continuously. In order to harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene which codes for flagellin was inactivated on the chromosome giving rise to the B. halodurans BhFC0l mutant. This strain was non-motile as determined on motility plates and confirmed by PCR analysis. Motility was, however, restored through complementation of the expression vector carrying a functional hag gene. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions in order to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-l clade C gpl20 epitope were respectively incorporated into both of the permissive sites as in-frame fusions and found to be successfully displayed on the cell surface. The poly-His peptide was shown to be functional through metal binding and affinity purification studies. The display of the HIV-1 subtype C gp 120 V3 loop was also shown to be functional through immunological studies using peptide specific antibodies. Surface display of the poly-His and HIV-l epitope was shown to have improved metal binding and enhanced expression levels of the chimeric flagellin when the peptides were insel1ed at amino acid position 180 (pSECNC6). This specific site is the only insertion point that falls within the re-defined variable domain of the FliC protein from B. halodurans Alk36. 2015-11-04T10:37:26Z 2015-11-04T10:37:26Z 2007 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/14641 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Molecular and Cell Biology Crampton, Michael Craig The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 |
| thesis_degree_str | Doctoral |
| title | The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 |
| title_full | The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 |
| title_fullStr | The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 |
| title_full_unstemmed | The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 |
| title_short | The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 |
| title_sort | development of a flagellin surface display expression system in the gram positive bacterium bacillus halodurans alk36 |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/14641 |
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