Browsing by Author "Makhoba, Xolani Henry"

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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.
    (2011) Makhoba, Xolani Henry; Shonhai, A.
    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.
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    Effects of co-expression of chaperone combinations on production of soluble plasmodial protein PfAdoMetDC in E. coli
    (University of Zululand, 2015) Makhoba, Xolani Henry; Shonhai, A.
    Plasmodium falciparum S-adenosylmethionine decarboxylase (PfAdoMetDC) plays an important role during the synthesis of polyamines, such as putrescine, spermidine and spermine. Polyamines play a major role in the survival of the malaria parasite. They are critical components of cell growth and division, particularly in rapidly proliferating cells that include cancerous cells and numerous parasites. Hence, PfAdoMetDC protein is regarded as an ideal drug target for malaria. Even though E. coli is the most established expression system for recombinant protein expression, the production of this malarial drug target protein in E. coli still remains an obstacle towards development of compounds or drugs to prevent its function. In this study, an approach that improves the quality of PfAdoMetDC protein produced in E. coli was developed based on the use of molecular chaperones as co-expression partners. For structural studies aimed at designing drugs or compounds obtaining a pure protein is crucial. Therefore, PfAdoMetDC was co-expressed with six different combinations of molecular chaperones. E. coli BL21 (DE3) starTM cells were transformed with plasmids in six combinations: combination 1 (pBB535) encoding DnaK with DnaJ; combination 2 (pBB535-Pf70J) encoding PfHsp70 with DnaJ; combination 3 (pBB535-KPfJ) encoding KPf with DnaJ; combination 4 (pBB542) encoding DnaK with DnaJ and GroEL; combination 5 (pBB542-Pf70JE) encoding PfHsp70 with DnaJ and GroEL; and combination 6 (pBB542-KPfJE) encoding KPf with DnaJ and GroEL. The effects of these combinations on the conformation of purified PfAdoMetDC protein was assessed by limited proteolysis and also by conducting activity assays to evaluate the activity of the protein. The PfAdoMetDC protein expressed with supplementation of PfHsp70+DnaJ and KPf+DnaJ combinations was not completely degraded by proteinase K and its activity was higher compared University of Zululand ii to the protein that was not supplemented with molecular chaperones. Almost similar results were observed on PfAdoMetDC co-expressed with DnaJ+GroEL, combined with the respective Hsp70s. The protein was not completely degraded, suggesting that PfAdoMetDC protein was protected by these chaperones during its folding. PfAdoMetDC protein, produced using E. coli Ī”dnaK strains, showed the highest activity compared to co-expressed protein with molecular chaperones. It was, however, completely degraded by proteinase K. This suggests that Hsp70 molecular chaperones are important for the fold and stability of proteins. To further confirm any possible cooperation amongst these molecular chaperones, MDH aggregation suppression assays were conducted to mimic what might be taking place inside the cell. The recombinant proteins DnaK, KPf, PfHsp70, GroEL and DnaJ were evaluated for their ability to suppress MDH aggregation, both as individual chaperones and as combined chaperones. There were indications that these molecular chaperones may be cooperative during protein folding. This study also revealed that the biotechnological tools developed in this study may be useful to other recombinant proteins, and not only those from plasmodium falciparum.