Results and Discussion
Chapter 4 Conclusions
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Chapter 4
130 many residues implicated in DNA and TFIIB binding are conserved, there is far less conservation of the TFIIA interaction sites. These interaction sites lie adjacent to the long (~130bp) LCR, suggesting that TLP may have reduced affinity for TFIIA. This stands in contrast to PfTBP which has retained conservation of these TFIIA interaction sites.
However, the identification of two distinct TFIIA γ-subunits expressed in P. falciparum (Robert Milton and Thomas Oelgeschläger, unpublished) may indicate that PfTBP and PfTLP interact with alternate forms of PfTFIIA.
Expression and purification of recombinant PfTFIIB
The over-expression of recombinant 6His-PfTFIIB was found to be toxic to the E. coli host cells. A method of expressing the protein was developed, whereby protein was expressed through auto-induction by release of catabolite repression. This method allowed for a large quantity of PfTFIIB protein to be expressed for purification. Purification attempts were hampered by the apparent DNA-binding activity of the recombinant protein, where interactions with DNA reduced the availability of purified protein. Additionally, affinity purification with a 6His-tag was not effective in generating sufficiently pure PfTFIIB for future assays. Finally, purification of PfTFIIB shows the consistent presence of a doublet of protein, and while this may be indicative of the open and closed conformations studied in other TFIIB orthologues, the clear heterogeneity of the protein preparation was of concern.
The inability to purify 6His-PfTFIIB sufficiently has led to current work in the lab to express the protein with flanking GST- and 6His-tags.
Expression and purification of recombinant PfTLP
Expression of recombinant PfTLP was found to be extremely toxic to the host E. coli cells, leading to cessation of growth in cells induced to express the protein. By codon optimising the gene for expression in E. coli, and expressing the gene at low-temperatures, this toxicity was alleviated. The cause of the toxicity of the gene is most likely due to the over-utilisation
131 of codons rare in E. coli, as well as the A+T richness of the gene. The high pI of the protein also led to premature translational termination of the protein, and so the accumulation of GST-6His peptides as a significant contaminant in the protein preparation. Expression of the double-tagged GST-6His-PfTLP allowed for sequential affinity-purification of the protein.
However, heterogeneity of the protein led to a significant reduction of recovered purified protein, as large quantities of the protein were found to bind irreversibly to agarose-based resins. Finally, a preparation of sufficiently purified 6His-PfTLP protein was obtained for DNA-binding assays.
DNA-binding potential of PfTLP and PfTFIIB
Immobilised template assays and EMSAs indicate that PfTLP is able to bind to DNA, with greater affinity for the putative Plasmodium promoters of the kahrp and gbp-130 genes, as compared to the Ad2ML probe. Evidence from polyacrylamide and agarose EMSAs suggest that PfTLP binds to multiple locations on the DNA-probe. This is in contrast to findings that PfTBP binds to distinct TATA-box like sequences in these same probes (Ruvalcaba-Salazar et al. 2006). Other eukaryotic TLPs characterised do not have affinity to the TATA-box (Akhtar & Veenstra 2011), and this may be an indication that PfTLP has sequence specificity alternate to that of PfTBP.
Finally, the suggestion that PfTFIIB has binding activity is supported by agarose EMSA, although the underlying mechanism, the specificity of this binding, and the functional relevance requires further investigation.
Overall conclusions and future work
This research project was undertaken to investigate the similarity and/or divergence of PfTFIIB and PfTLP to their well-characterised orthologous in human. To this end, in silico analysis of the proteins was performed and has found that the putative Plasmodium
132 orthologues share many of the main structural and, potentially, functional features of the classically studied molecules.
Expression of the epitope-tagged molecules was also performed, and the conditions for expression of soluble forms of the molecules in E.coli BL21-CodonPlus RIL cells established. However, future work into optimising the expression conditions of recombinant PfTLP is required, in order to reduce the expression of truncated species, and so increase the yield of purified protein.
The methodology for the affinity purification of the molecules was investigated. It was found that the addition of only a single His-tag is insufficient for the purification of recombinantly expressed protein, due to very low expression levels and multiple contaminants present after metal-ion affinity purification. The addition of a second affinity-purification tag, in this case GST, allows for far superior purification, and is recommended in the expression of other recombinant putative Plasmodium GTFs.
The initial investigation into the DNA-binding potential of PfTLP and PfTFIIB has revealed that both molecules appear to have affinity for DNA. The specificity of this binding, as well as the development of optimal binding conditions will be the focus of future work.
Additionally, the interplay of various recombinant P. falciparum GTFs will also be investigated, in the modulation of both DNA-binding affinity and specificity.
Future work in the research group will focus on determining the sites of PIC assembly, through the mapping of sites bound by PfTBP with PfTFIIA and/or PfTFIIB, as well as PfTLP PfTFIIA and/or PfTFIIB, in characterised putative Plasmodium promoters, as well as by a SELEX strategy. The work presented in this research project has provided a basis for the expression and purification of these factors, as well as provided valuable insights into the possible structures and functions of PfTLP and PfTFIIB.
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