Optimization of the heterologous expression of folate metabolic enzymes of Plasmodium falciparum

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dc.contributor.advisor Louw, Abraham Izak en
dc.contributor.coadvisor Birkholtz, Lyn-Marie en
dc.contributor.postgraduate Goolab, Shivani en
dc.date.accessioned 2013-09-06T15:43:19Z
dc.date.available 2011-09-22 en
dc.date.available 2013-09-06T15:43:19Z
dc.date.created 2011-09-06 en
dc.date.issued 2011-09-22 en
dc.date.submitted 2011-03-30 en
dc.description Dissertation (MSc)--University of Pretoria, 2011. en
dc.description.abstract Malaria is a fatal tropical disease affecting billions of people in impoverished countries world-wide. An alarming fact is that a child in Africa dies of malaria every 30 seconds that amounts to 2500 children per day (www.who.int/features/factfiles). Malaria is caused by the intraerythrocytic forms of Plasmodium species, notably P. falciparum, P. vivax, P. ovale and P. malariae (Hyde 2007). The spread of drug-resistant strains, failure of vector control programs, rapid growth rate of the parasite, and lack of a vaccine have further exacerbated the effects of malaria on economic development and human health. It is therefore imperative that novel drug targets are developed or current antimalarial drugs optimized (Foley and Tilley 1998). One such target is folate biosynthesis, given that folates and their derivatives are required for the survival of organisms (Muller et al. 2009). DHFR and DHPS are currently the only folate targets exploited however, their antifolate drugs are almost useless against parasite resistant strains. As such, guanosine-5’triphosphate cyclohydrolase I (GTPCHl) among other antifolate candidates are considered for intervention (Lee et al. 2001). Knock-out studies (of P. falciparum gtpchI) resulted in the suppression of DHPS activity (Nzila et al. 2005). Additionally, gtpchI amplified 11-fold in P. falciparum strains resistant to antifolates due to mutations in dhps and dhfr and this may be a mechanism for the compensation of reduced flux of folate intermediates (Kidgell et al. 2006; Nair et al. 2008). Over-expression of P. falciparum proteins in E. coli remains a challenge mainly due to the A+T rich Plasmodium genome resulting in a codon bias. This results in the expression of recombinant proteins as insoluble proteins sequestered in inclusion bodies (Carrio and Villaverde 2002; Mehlin et al. 2006; Birkholtz et al. 2008a). Comparative expression studies were conducted of native GTPCHI (nGTPCHI), codon optimized GTPCHI (oGTPCHI) and codon harmonized (hGTPCHI) in various E. coli cell lines, using alternative media compositions and co-expression with Pfhsp70. The nGTPCHI protein did not express because the gene consisted of codons rarely used by E. coli (codon bias). The expression levels of purified hGTPCHI were a greater in comparison to oGTPCHI using the different expression conditions. This is because codon-harmonization involves substituting codons to replicate the codon frequency preference of the target gene in P. falciparum, as such the translation machinery matches that of Plasmodium (Angov et al. 2008). Furthermore, greater expression levels of GTPCHI were achieved in the absence of Pfhsp70 due to expression of a possible Nterminal deletion product or E. coli protein. Purification conditions could be improved to obtain homogenous GTPCHI and further analysis (mass spectrometry and enzyme activity assays) would be required to determine the nature of soluble GTPCHI obtained. To improve the expression of soluble proteins the wheat germ expression system was used as an alternate host. However, GTPCHI expression was not effective, possibly due to degradation of mRNA template or the absence of translation enhancer elements. en
dc.description.availability unrestricted en
dc.description.department Biochemistry en
dc.identifier.citation Goolab, S 2010, Optimization of the heterologous expression of folate metabolic enzymes of Plasmodium falciparum, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/23647 > en
dc.identifier.other C11/9/164/ag en
dc.identifier.upetdurl http://upetd.up.ac.za/thesis/available/etd-03302011-181338/ en
dc.identifier.uri http://hdl.handle.net/2263/23647
dc.language.iso en
dc.publisher University of Pretoria en_ZA
dc.rights © 2010 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. en
dc.subject Folate biosynthesis en
dc.subject Recombinant protein expression en
dc.subject Guanosine 5’ triphosphate en
dc.subject Codon harmonization en
dc.subject Malaria en
dc.subject UCTD en_US
dc.title Optimization of the heterologous expression of folate metabolic enzymes of Plasmodium falciparum en
dc.type Dissertation en


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