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The development of a molecular chaperone-based system to improve the heterologous production of Plasmodium falciparum AdoMetDC protein in E. coli.

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dc.contributor.advisor Shonhai, A.
dc.contributor.author Makhoba, Xolani Henry
dc.date.accessioned 2011-06-14T11:18:00Z
dc.date.available 2011-06-14T11:18:00Z
dc.date.issued 2011
dc.identifier.uri http://hdl.handle.net/10530/598
dc.description Submitted in fulfillment of the requirements for the degree of MSc. in Biochemistry in the Faculty of Science and Agriculture University of Zululand, 2011. en_US
dc.description.abstract S-Adenosylmethionine decarboxylase/ornithine decarboxylase from Plasmodium falciparum (PfAdoMetDC/ODC) has been described as an ideal antimalarial drug target. However, the production of this large bifunctional protein to facilitate its biochemical characterization is hampered by the poor yields of recombinant protein. It has previously been proposed that co-expression of target recombinant malaria proteins with molecular chaperones of malarial origin in Escherichia coli could improve the yield of the target recombinant proteins. A cytosolic heat shock protein 70 (Hsp70) from Plasmodium falciparum (PfHsp70-1), has been previously shown to exhibit chaperone function when heterologously expressed in E. coli cells whose endogenous Hsp70 function was compromised. PfHsp70-1 presumably protected the E. coli cells against thermal stress by preventing protein aggregation. In the current study, PfAdoMetDC and PfAdoMetDC/ODC were expressed along with PfHsp70-1 in E. coli BL21 (DE3) star cells to improve the yield and quality of the PfAdoMetDC/ODC proteins. E. coli BL21 (DE3) star cells were transformed with plasmid constructs pMRBAD/PfHsp70-1 (encoding PfHsp70-1) and either pASK-IBA/PfAdoMetDC (encoding PfAdoMetDC, either as wild type or codon harmonized version) or pASK-IBA/PfAdoMetDC/ODC (encoding PfAdoMetDC/ODC, either as wild type or codon harmonized version), followed by induction to facilitate the production of the chaperone and the target proteins, respectively . Protein expression and solubility studies were conducted followed by purification of the PfAdoMetDC and PfAdoMetDC/ODC proteins using strep-tectin column. The co-expression of PfAdoMetDC/ODC with PfHsp70-1 did not necessarily improve the production of the former. Although there is no evidence that PfHsp70-1 improved the solubility of PfAdoMetDC protein, it greatly improved the purity of PfAdoMetDC protein obtained following elution through the strep-tectin column. Whilst PfAdoMetDC were expressed from a codon harmonized coding sequence alone (expressed in the absence of PfHsp70-1) was purified associated with contaminants, the same protein purified after co-expression with PfHsp70-1 was obtained at a much higher purity level. This could have been due to PfHsp70-1 shielding nascent PfAdoMetDC, thus preventing its association with the contaminating species. On the other hand, PfHsp70-1 improved the expression and yield of PfAdoMetDC/ODC (wild type), but it had no effect on the yield of the protein expressed of the plasmid haboring the codon harmonized version. Thus PfHsp70-1 may have facilitated the folding and production of the PfAdoMetDC/ODC (wild type) protein whose folding could have been impeded due to potential delayed translation due to codon mismatch. en_US
dc.description.sponsorship The National Research Foundation (NRF) and the University of Zululand research committee. en_US
dc.language.iso en en_US
dc.subject S-Adenosylmethionine decarboxylase/ornithine decarboxylase en_US
dc.subject Antimalarial drug en_US
dc.subject Plasmodium falciparum AdoMetDC protein en_US
dc.title The development of a molecular chaperone-based system to improve the heterologous production of Plasmodium falciparum AdoMetDC protein in E. coli. en_US
dc.type Thesis en_US


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