Effects of co-expression of chaperone combinations on production of soluble plasmodial protein PfAdoMetDC in E. coli
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Date
2015
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University of Zululand
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
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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.
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.
Keywords
chaperone combinations, plasmodial protein, PfAdoMetDC