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Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression
Dissertation (MSc(Biochemistry))--University of Pretoria, 2006.
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University of Pretoria
2013
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| _version_ | 1867613704876457984 |
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| access_status_str | Open Access |
| author2 | Louw, Abraham Izak |
| author_browse | Louw, Abraham Izak |
| author_facet | Louw, Abraham Izak |
| collection | Thesis |
| dc_rights_str_mv | © 2004, 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(Biochemistry))--University of Pretoria, 2006. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/24333 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:40:23.116Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| 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:2263/24333 Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression Louw, Abraham Izak upetd@ais.up.ac.za Coetzee, Linda Malariotherapy Malaria research Plasmodium falciparum UCTD Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. Malaria causes nearly 3 million deaths annually. The parasite species responsible for the most lethal forms of malaria is P. falciparum (Miller et.al., 2002). Its destructive effect is most evident in the developing African countries, which lack the infrastructure and financial support to effectively control the disease. The only viable means of control at this stage is the use of antimalarial chemotherapy and –prophylaxis, but these drugs are losing their effectivity due to parasite resistance. This underlines the need for new, safe, efficient and cheap drugs as a solution to the African malaria problem. Within the validated folate metabolic pathway of P. falciparum, the identification of three new genes has provided new options for drug inhibition (Lee et.al., 2001). One of these genes encodes the bifunctional dihydrofolate synthase-folylpolyglutamate synthase (DHFS-FPGS), which is unique to P. falciparum (Salcedo et.al., 2001). When compared to human folylpolyglutamate synthase (FPGS), the parasite enzyme is an attractive drug target for selective inhibition due to the additional DHFS activity and low sequence similarity. However, to assess the value of DHFS-FPGS as a drug target and rationally design new drugs against the enzyme, large amounts of enzyme are needed for activity studies and structural determination. The heterologous expression of malaria genes often result in low expression levels, due to its high A+T content and codon bias. To circumvent this problem, a modified P. falciparum dhfs-fpgs, adapted to E.coli codon preferences and with a lower A+T content was synthesised in this study for increased expression. A two-step overlap-extension PCR method was optimised for the synthesis of the 1586bp dhfs-fpgs from only 1 pmol each of partially overlapping oligonucleotides. The use of partially overlapping oligonucleotides, lower amounts of starting material and fewer PCR cycles cut the costs of gene synthesis and the optimisation increased PCR efficiency, when compared to other gene synthesis reports (Carpenter et al., 1999; Zhang et al., 2002). The correct sequences could be obtained from the sequencing of as little as three clones. The successfully constructed dhfs-fpgs gene was expressed in a variety of E. coli expression hosts and vector systems. In all the systems, expression levels of the synthetic gene were much higher than for the native P. falciparum gene. Functional complementation of a DHFS-FPGS deficient E. coli strain verified that the DHFS and FPGS activities were encoded by the synthetic gene, that complementation was achieved to a greater extent than for native P. falciparum dhfs-fpgs and that a synthetic tagless and C-terminal Histidine-tagged DHFS-FPGS had the highest levels of DHFS and FPGS activity. Preliminary purification studies for these two constructs were performed for optimised enzyme isolation, to be followed by activity assays. These optimisations will also serve as basis for future large-scale isolation strategies to obtain sufficient amounts of protein for the structural determination of the enzyme, which would be vital to drug target verification, drug development and –design, thus paving the way for a new generation of antifolate malaria therapy. Biochemistry unrestricted 2013-09-06T17:15:51Z 2005-05-19 2013-09-06T17:15:51Z 2004-04-24 2006-05-19 2005-05-05 Dissertation Coetzee, L 2004, Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression, MSc(Biochemistry) dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/24333 > http://hdl.handle.net/2263/24333 http://upetd.up.ac.za/thesis/available/etd-05052005-115717/ © 2004, 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 application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf University of Pretoria |
| spellingShingle | Malariotherapy Malaria research Plasmodium falciparum UCTD Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression |
| title | Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression |
| title_full | Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression |
| title_fullStr | Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression |
| title_full_unstemmed | Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression |
| title_short | Plasmodium falciparum dihydrofolate synthase (DHFS-FPGS) : gene synthesis and recombinant expression |
| title_sort | plasmodium falciparum dihydrofolate synthase dhfs fpgs gene synthesis and recombinant expression |
| topic | Malariotherapy Malaria research Plasmodium falciparum UCTD |
| url | http://hdl.handle.net/2263/24333 http://upetd.up.ac.za/thesis/available/etd-05052005-115717/ |