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Effects of co-expression of chaperone combinations on production of soluble plasmodial protein PfAdoMetDC in E. coli

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dc.contributor.advisor Shonhai, A.
dc.contributor.author Makhoba, Xolani Henry
dc.date.accessioned 2019-10-16T13:27:42Z
dc.date.available 2019-10-16T13:27:42Z
dc.date.issued 2015
dc.identifier.uri http://hdl.handle.net/10530/1858
dc.description A thesis submitted the Department of Biochemistry and Microbiology in fulfilment of the requirement for the degree of Doctor of Philosophy in the Faculty of Science and agriculture at the University of Zululand,2015. en_US
dc.description.abstract 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 ΔdnaK 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. en_US
dc.description.sponsorship University of Zululand Research Committee National Research Foundation (South Africa) Scarce Skills Development Fund Programme en_US
dc.language.iso en en_US
dc.publisher University of Zululand en_US
dc.subject chaperone combinations en_US
dc.subject plasmodial protein en_US
dc.subject PfAdoMetDC en_US
dc.title Effects of co-expression of chaperone combinations on production of soluble plasmodial protein PfAdoMetDC in E. coli en_US
dc.type Thesis en_US

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