The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36

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dc.contributor.advisorReid, Sharon Jen_ZA
dc.contributor.advisorLouw, Maureen Een_ZA
dc.contributor.advisorBerger, Een_ZA
dc.contributor.authorCrampton, Michael Craigen_ZA
dc.date.accessioned2015-11-04T10:37:26Z
dc.date.available2015-11-04T10:37:26Z
dc.date.issued2007en_ZA
dc.descriptionIncludes bibliographical references (leaves 103-126).en_ZA
dc.description.abstractThis 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.en_ZA
dc.identifier.apacitationCrampton, M. C. (2007). <i>The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/14641en_ZA
dc.identifier.chicagocitationCrampton, Michael Craig. <i>"The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2007. http://hdl.handle.net/11427/14641en_ZA
dc.identifier.citationCrampton, M. 2007. The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Crampton, Michael Craig AB - 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. DA - 2007 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2007 T1 - The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 TI - The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36 UR - http://hdl.handle.net/11427/14641 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/14641
dc.identifier.vancouvercitationCrampton MC. The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2007 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/14641en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMolecular and Cell Biologyen_ZA
dc.titleThe development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36en_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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