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Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium
Dissertation (MSc (Microbiology))--University of Pretoria, 2023.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2023
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| _version_ | 1867613599125471232 |
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| access_status_str | Open Access |
| author2 | Theron, Jacques |
| author_browse | Theron, Jacques |
| author_facet | Theron, Jacques |
| collection | Thesis |
| dc_rights_str_mv | © 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc (Microbiology))--University of Pretoria, 2023. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/90583 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:38:42.081Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/90583 Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium Theron, Jacques u11272164@tuks.co.za Crampton, Michael Roth, Robyn Fisher, Matthew Escherichia coli Protein expression Secretion signal sequence General secretion pathway Protein transport UCTD Dissertation (MSc (Microbiology))--University of Pretoria, 2023. Secreted proteins offer the potential to reduce purification cost to enzyme and therapeutic proteins. While many bacterial transport signal sequences have been tested, few have been used across species. This study explores the effectiveness of a Gram-positive secreted protein signal sequence (SPss) used in a Gram-negative production platform (Escherichia. coli BL21(DE3)). A pET bacterial expression plasmid was constructed with a secreted protein signal sequence (SPss) from a secreted protein originating in Clostridium perfringens and combined with a common marker protein (Green Fluorescent Protein). The construct (SPss-GFP) was expressed in E. coli and tested under a variety of temperatures (25°C, 30°C, 37°C), IPTG concentrations (1μM, 10μM, 100μM, and 1000μM), and two growth media (Lysogeny Broth and an enzyme-release complex medium) to ascertain the ideal expression conditions. The results indicated that the SPss-GFP protein was present in the periplasm after expression, but transport was sporadic with most experiments showing only cytoplasmic GFP. More periplasmic protein was present when a control plasmid, expressing the full-length secreted iv protein from C. perfringens was used. The parameters that resulted in the highest levels of GFP was 100μM IPTG at 30°C grown in the enzyme-release medium. High levels of expression and transport were also observed at 1000μM, 37°C and at 1000μM, 30°C. Reduced cell concentration and rate of growth was observed during expression of SPss-GFP, but no parameter was found to be the cause and requires further investigation. Mass spectrophotometry indicated cleavage of SPss but much of the GFP remained in the cytoplasm. Based on the results obtained in this study, it is concluded that protein folding kinetics and limited interaction with accessory transport factors are the main problems with protein transport in this system. Microbiology and Plant Pathology MSc (Microbiology) Unrestricted 2023-05-08T09:22:35Z 2023-05-08T09:22:35Z 2023-09 2023 Dissertation * S2023 http://hdl.handle.net/2263/90583 DOI: https://doi.org/10.25403/UPresearchdata.22717702.v1 10.25403/UPresearchdata.22717702 en © 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Escherichia coli Protein expression Secretion signal sequence General secretion pathway Protein transport UCTD Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium |
| title | Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium |
| title_full | Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium |
| title_fullStr | Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium |
| title_full_unstemmed | Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium |
| title_short | Characterisation of a Gram-positive secreted protein signal sequence (SPss) : localisation and processing during transport in a Gram-negative bacterium |
| title_sort | characterisation of a gram positive secreted protein signal sequence spss localisation and processing during transport in a gram negative bacterium |
| topic | Escherichia coli Protein expression Secretion signal sequence General secretion pathway Protein transport UCTD |
| url | http://hdl.handle.net/2263/90583 |