Results and Discussion
3.3.5 Further purification of 6His-PfTFIIB
88 been described for HsTFIIB (Glossop et al. 2004). This requires additional investigation, and was not pursued further in this project.
89 A proportion of the 6His-PfTFIIB protein was bound to DEAE- Sepharose® resin and was only partially eluted with high salt concentrations from the resin (see lanes 5 and 6 of Figure 20B). The majority of the protein, however, was still uncaptured by the resin. Only limited binding of contaminant proteins to Q-Sepharose® was seen (not shown). No binding of Figure 20: 6His-PfTFIIB binds partially to weak anion-exchange resin.
A. PfTFIIB and the majority of the contaminants do not bind to SP Sepharose® resin. 100ng of 6His-PfTFIIB was loaded into the input lane (In), and an equivalent amount of the protein not bound (unbound, Ub). 1.5× of the input is loaded in each of the KCl elution lanes 3-5.
B. Partial binding of 6His-PfTFIIB to DEAE resin and partial elution at 400mM KCl. 100ng of 6His-PfTFIIB was loaded into the input lane (In), and 1.5× of the equivalent amount of the protein not bound (unbound, Ub). 1.5× of the input is loaded in each of the KCl elution lanes 3-5. Lane 6 contains 1/6th of the resin (R), eluted in 2×SDS loading buffer.
Molecular weight marker (Mw) - 2µl of Precision Plus Protein All Blue standard (Bio-Rad).
90 PfTFIIB or contaminant proteins to CM-Sepharose® was seen (not shown). The partial binding of PfTFIIB to DEAE resin suggests heterogeneity in the protein preparation. It is possible that some proportion of the protein may have aggregated. The protein contains regions which are predicted to be negatively charged predicted with pIs of less than 4.5 (B- linker and reader), as well as regions predicted to be positively charged, with predicted pIs of over 10. These may interact preferentially with one another over the binding of DNA.
It is known that DNA is strongly bound by the weak anion exchange resin, DEAE- Sepharose®. It was suggested that PfTFIIB may bind weakly to DNA, as it is known to associate in other TFIIB homologues, albeit in the presence of TBP. Additionally, the calculated pI of PfTFIIB (9.73), with several regions of pI above 10 (Table 4), suggests the protein may be highly positively charged, and therefore may bind DNA.
Aliquots of the 6His-PfTFIIB preparation were treated with a nuclease enzyme (Benzonase, Sigma-Aldrich®). The nuclease enzyme digests nucleic acids contaminating the protein preparation. Should 6His-PfTFIIB bind to DNA, which in turn is bound to the DEAE resin, then PfTFIIB in the treated protein preparation should no longer bind to the DEAE resin.
In Figure 21B and C, the results of attempting to bind nuclease treated protein preparations to the various Sepharose® resins is shown. It was found that the protein was consistently recovered in the unbound protein fraction of each experiment. Proteins were eluted with 500mM final KCl, similarly to previously described experiments. Notably, this is true also for the experiment with DEAE resin. Note that panel A in the figure is a positive control.
The recombinant HsTFIIB protein is known to bind to strong cation exchangers, such as SP- Sepharose®, and was used to confirm that the resin was functional. This strongly suggests that recombinant PfTFIIB was able to interact indirectly to DEAE-Sepharose® due to the presence of DNA.
91 Due to time constraints, further attempts for the purification of 6His-PfTFIIB were not undertaken.
Figure 21: Nuclease treated 6His-PfTFIIB does not bind to ion-exchange resins.
A. Positive control of 200ng of recombinant 6His-HsTFIIB, previously purified using Mono S Sepharose®. Eluted in 500mM KCl.
B. Nuclease treated PfTFIIB protein does not bind to the SP resin.
C. Nuclease treated PfTFIIB protein does not bind to either Q or DEAE Sepharose® resins.
Lane 1 is the preparation prior to nuclease treatment, and lane 2 is post-treatment, used with the resins.
Unbound protein (Ub) is equivalent of 50% the input (In) protein (100ng), hence the reduction in signal. Elution (E) with 500mM KCl, is equivalent to 63% of the input loaded. Treated input (TIn) is equivalent to In. Molecular weight marker (Mw) - 2µl of Precision Plus Protein All Blue
standard (Bio-Rad).
92 3.3.1 Summary of PfTFIIB expression and purification
A number of interesting features of PfTFIIB protein were found. Firstly, PfTFIIB may be efficiently expressed through a method of ‘auto-induction’, by modification of the cell culture growth media. This is useful not only due to the ease of expression, but also because this method is cost-effective as there is no need for IPTG-induction.
The purification of PfTFIIB shows the presence of a doublet. This may be related to the open and closed conformations that been studied in other TFIIB orthologous, (Elsby & Roberts 2004; Zheng et al. 2004; Glossop et al. 2004; Fairley et al. 2002) and may be implicated in transcription. This is supported by the conservation of the charged cluster domain, described previously (Chapter 3).
Due to the inability to purify 6His-PfTFIIB further, it was decided that the research group would re-clone the putative PfTFIIB protein into an expression vector containing two flanking tags, a 6His-tag, and a GST-tag, to allow for a more stringent purification (Gertrud Talvik, current investigation).
Finally, it is intriguing that evidence from the binding of 6His-PfTFIIB to DEAE resin, and the subsequent abolishment of this upon treatment with nuclease, suggests that PfTFIIB may bind to DNA in a manner not yet described for the protein. Current work in the research group appears to support DNA-binding activity in recombinant PfTFIIB (Gertrud Talvik, unpublished observations).
93 Expression of recombinant PfTLP protein