Results and Discussion
3.3.3 Expression of recombinant Plasmodium TFIIB can be achieved from the release of catabolite repression in non-induced cell cultures
81 derived from bovine milk and it is known that tryptone powder may be contaminated with traces of lactose. Lactose has been extensively studied in the activation of transcription from the Lac operon (Perlman et al. 1969) as part of the innate metabolism regulation pathways in E. coli. The Lac operon has been incorporated into the E. coli BL21 strains in order to regulate the expression of T7 polymerase which in turn drives protein expression from plasmids containing a T7 promoter, in this case pET11d plasmids. The presence of lactose in the media may thus account for the presence of protein expressed in the non-induced sample.
The Lac operon is repressed through the action of the catabolite activator protein (CAP) naturally expressed in E. coli in the presence of glucose (Perlman et al. 1969). It was reasoned that the addition of glucose to the growth media would repress the leaky expression of T7 polymerase, and therefore inhibit 6His-PfTFIIB expression. Should the expression of T7 and/or 6His-PfTFIIB negatively affect the growth of the cells, it is likely that the transformation of cells on plates without glucose may select against cells which express high levels of the proteins. For this reason, E. coli BL21-CodonPlus-RIL cells were transformed and plated onto media that contained 1% (w/v) glucose.
3.3.3 Expression of recombinant Plasmodium TFIIB can be achieved from
82 Diauxic growth is characterised by the presence of two exponential and stationary growth phases. An initial exponential growth phase occurs as the cells grow on the easily metabolised glucose. As the glucose is depleted the cells enter a short stationary phase as they adjust their metabolic machinery for the utilisation of a secondary nutrient source. A second exponential phase begins as they start to metabolise the alternative energy source.
This experiment sought to investigate whether the lacUV promoter driven T7 polymerase expression could be repressed by the supplementation of the growth media with glucose.
Additionally, as the cell culture is depleted of glucose, whether the lifting of catabolite- repression would result in the expression of T7 polymerase, and thus recombinant PfTFIIB.
In this experiment, both the overnight cultures used to inoculate the expression cultures, as well as the expression cultures were supplemented with 1% glucose to repress leaky expression from the lacUV operon. Two cell cultures were grown, and the growth of the cells measured by spectrophotometry. One culture was harvested late into the first
Figure 16: Diauxic growth of transformed E. coli cells in growth media supplemented with 1%glucose.
E. coli BL21-codonPlus® (DE3)-RIL cells carrying the expression vector pET11d-6His-PfTFIIB were grown and harvested at the late initial exponential phase or early in the second exponential phase. Optical densities of the cultures are plotted against time.
83 exponential growth phase, when catabolite repression should be in place. A second culture was allowed to grow past the first stationary phase, at which point the glucose-derived catabolite repression would be lifted. As expected, the cell culture allowed to continue to grow past an initial exponential phase displayed a diauxic growth curve (Figure 16).
PfTFIIB protein is expressed through the release of catabolite repression
Expressed 6His-PfTFIIB protein was purified from cleared cell lysates and analysed by SDS-PAGE as described above. It is clear in the comparison of the protein purified from the culture harvested late in the first exponential phase, and that which was grown to the beginning of the second exponential phase (Figure 17), that protein is expressed through
‘auto-induction’, due to the release of catabolite-repression. Additionally, due to the presence of glucose in the media, the rate of cell-growth was increased, and resulted in higher
Figure 17: Increased expression of recombinant PfTFIIB protein in E. coli BL21-codonPlus®
(DE3)-RIL cells through the release of catabolite repression.
E. coli cells carrying the expression vector pET11d-6His-PfTFIIB were grown and harvested either at the late initial exponential phase (Panel A), or early second exponential phase (Panel B). Protein was purified by nickel-affinity purification from cleared cell lysates and analysed by SDS-PAGE. Equivalent amounts of cleared lysate input (In, 1/100 vol.), and the protein fraction not bound to the Ni-beads (unbound, Ub), and the protein fraction eluted from the Ni-beads with 2×SDS-loading buffer (E, ½ vol.) were loaded. A black arrow indicates the expected size of 6His-PfTFIIB protein at 44kDa. Molecular weight marker (Mw) – 5µl of Precision Plus Protein All Blue standard (Bio-Rad).
84 cell-density and increased harvested cell mass. These results clearly show that PfTFIIB may be efficiently expressed through auto-induction. The identity of 6His-PfTFIIB was confirmed by mass spectrometry.
Having successfully determined the conditions necessary for 6His-PfTFIIB expression, accumulation of cell mass of cells which had expressed the protein was performed.
Discussion of expression of 6His-PfTFIIB
There is clear growth reduction upon induction with 1mM IPTG in the 6His-PfTFIIB expressing cells as compared to the non-induced cell culture, (Figure 14). This suggests that the induction of the cell cultures with IPTG led directly to either a reduction in cell growth and/or cell death. Upon induction with IPTG the cells are required to utilise many cell- resources in order to synthesise both the T7 polymerase, as well as 6His-PfTFIIB proteins.
The metabolic burden of heterologous gene expression and protein synthesis may deter cell growth or prove to be toxic, and this is a known issue with heterologous gene expression the BL21(DE3) E. coli cells (Rosano & Ceccarelli 2014; Bentley et al. 1990; Dumon-Seignovert et al. 2004). This protein over-production may result in cell death. It is also known that individual cells may vary widely both in their sensitivity to an inducing agent, as well as in their protein expression. It is suggested that since the protein expression per gram of cell mass in both cultures was the same, the cells which survived induction by IPTG ultimately expressed similar average quantities of protein per cell, however, due to the decrease of harvestable cell mass from these induced cell cultures, it was decided to instead make use of the auto-induction method of protein expression.