2.3 Results
2.3.3 Recombinant expression, solubilisation, refolding and purification of TcMCA5
The single putative full length metacaspase gene (UniProtKB ID: G0UUY6) identified in chromosome 9 of T. congolense strain IL 3000 (TcMCA5) codes for a protein of 533 amino acids in length with an expected molecular weight of 59 kDa and a pI of 8.42 as predicted by the Compute pI/Mw tool on the ExPASy server (Gasteiger et al., 2005).
2.3.3.1 Expression optimisation of recombinant TcMCA5
The PROSO II sequence-based solubility prediction calculator predicted that TcMCA5 had a 92.5% chance of being insoluble when over-expressed in E. coli (Smialowski et al., 2006). Protein solubility is determined by its primary structure (Smialowski et al., 2006). It has been demonstrated that proteins which possess a higher percentage of aromatic amino acid residues and stretches of 20 or more hydrophobic amino acid residues, are more likely to be insoluble when over-expressed in E. coli (Christendat et al., 2000). Research done by Bertone and co-workers (2001) indicated that proteins whose hydrophobic amino acid composition was greater than 16% were more likely to be insoluble. The TcMCA5 protein is comprised of 22.1% hydrophobic amino acids. In addition to the disordered C-terminal extension caused by the large amount turn forming Pro residues, it follows that TcMCA5 would be insoluble when over-expressed, based on the basic analysis of its primary structure.
Expression of the M7pET28a clones 1 to 3 and M13pET28a clone 2 was performed at 37°C, with and without IPTG induction. Following expression without IPTG induction, a prominent protein band at approximately 69.8 kDa was observed after staining with Coomassie blue R-250 (Fig. 2.13, panel A) and identified using the mouse anti-6xHis IgG HRPO and 4-chloro-1-naphthol∙H2O2 detection system (Fig. 2.13, panel B) in the insoluble pellet fraction. Two additional prominent, lower molecular weight proteins at 41.4 and 37.3 kDa were present for each clone but were not detected in the western blot by the mouse anti-6xHis IgG HRPO conjugate (Fig. 2.13, panel B). Due to the fact that the lower molecular weight proteins from the recombinant TcMCA5 expression were not detected by the mouse anti-6xHis IgG HRPO conjugate this led to the conclusion that they might be the result of autocatalytic cleavage of the recombinant TcMCA5. The M7pET28 clone 2 was selected for large scale expression.
Figure 2.13: Analysis of recombinantly expressed TcMCA5 in pET-28a.Samples of the soluble and insoluble fractions of the expression of recombinant TcMCA5, from the M7pET28a clones 1 to 3 and M13pET28a clone 2, were electrophoresed on two 12.5%
reducing SDS-PAGE gels with one (A) stained with Coomassie blue R-250 and the other (B) transferred onto nitrocellulose, blocked with 5% (w/v) milk-TBS and incubated with mouse anti-6xHis IgG [1:1 000 in 0.5% (w/v) BSA-PBS]. Goat anti-mouse IgG HRPO conjugate [1:12 000 in 0.5% (w/v) BSA-PBS] and 4-chloro-1-naphthol∙H2O2 were used as the detection system.
Proteins expressed within inclusion bodies can vary from being completely misfolded to mostly native protein (Bowden et al., 1991; Ventura and Villaverde, 2006). The presence of the two lower molecular weight proteins/cleavage products, lends to the fact that the expressed recombinant TcMCA5 may be in a partially native state and has retained some degree of activity. Moss and co-workers (2007) demonstrated that purified recombinant TbMCA2, which shares a 34% sequence similarity with TcMCA5, displayed 93, 91 and 70% inhibition by 100 µM leupeptin, antipain and N-tosyl-L-lysyl chloromethylketone (TLCK) respectively. When recombinant TcMCA5 expression by IPTG induction was carried out at 37°C in the presence of 100 µM leupeptin, antipain and TLCK, no inhibition of TcMCA5 autocatalytic activity was observed (results not shown).
Recombinant expression at lower temperatures is thought to allow for slower transcription and translation which would result in more time for the recombinant protein to refold into its enzymatically active form, resulting in lower levels of inclusion body formation (Vera et al., 2006; Burgess, 2009). No expression of recombinant TcMCA5 was observed when incubated at 25, 15 or 4°C without IPTG induction (results not shown).
Consequently, expression of insoluble recombinant TcMCA5 was attempted at 37°C and 30°C with IPTG induction at various concentrations, the results of which are shown in Fig. 2.14, panel A and B respectively. Expression of 69.8 kDa recombinant TcMCA5,
as well as the two lower molecular weight proteins/cleavage products at approximately 41.4 and 37.3 kDa, was highest at 1 mM IPTG at 37°C when comparing induction at 1, 0.7, 0.5 and 0.3 mM IPTG at both temperatures. At 30°C (Fig. 2.14, panel B), the expression of recombinant TcMCA5 is significantly less than at 37°C (Fig. 2.14, panel A) with the lower molecular weight proteins/cleavage products still present. After the purification of recombinant TcMCA5, antibodies were raised in chickens and were used to determine if they would recognise the recombinantly expressed TcMCA5 in the 1 mM ITPG expression samples at 37°C in a western blot format (Fig. 2.15). In addition to the 69.8 kDa full length recombinant TcMCA5, the two lower molecular weight proteins/cleavage products at approximately 41.4 and 37.3 kDa were recognised by the chicken anti-TcMCA5 IgY. Due to the high concentration of primary antibody used, 100 µg/ml, additional bands were recognised.
Figure 2.14: Expression by IPTG induction of recombinant TcMCA5 in pET-28a.
Six different IPTG concentrations (1, 0.7, 0.5, 0.3, 0.1 and 0 mM) at temperatures (A) 37°C and (B) 30°C was employed in the optimisation of the expression of recombinant TcMCA5 in the pET-28a expression vector. Samples were electrophoresed on a 12.5% reducing SDS-PAGE gel and stained with Coomassie blue R-250. NI: non-induced control sample.
Figure 2.15: Western blot analysis of recombinantly expressed TcMCA5 in pET-28a. Expression samples from the expression of M7pET28a at 37°C, induced at 1 mM IPTG, were electrophoresed on two 12.5% reducing SDS-PAGE gels with one (A) stained with Coomassie blue R-250 and the other (B) transferred onto nitrocellulose, blocked with 5% (w/v) low fat milk-TBS and incubated with chicken anti-TcMCA5 IgY from chicken 1, week 7 [100 µg/ml in 0.5% (w/v) BSA-PBS]. Rabbit anti-chicken IgY HRPO conjugate [1:15 000 in 0.5% (w/v) BSA-PBS] and Pierce™ ECL western blotting substrate were used as the detection system. MW: PageRuler™ prestained protein marker. NI: non-induced control sample.
2.3.3.2 Solubilisation, refolding and purification of recombinantly expressed TcMCA5
Some degree of native conformation of the recombinant TcMCA5 protein is present within the inclusion bodies as is evident due to the products of autocatalytic cleavage/lower molecular weight proteins (Fig. 2.14 and 2.15). Solubilisation of the inclusion bodies using urea and sarkosyl methods was performed and the recombinantly expressed TcMCA5 was subsequently refolded and purified on a nickel chelate column. Refolding and purification using the urea method is shown in Fig. 2.16, panel A, where the eluted proteins were relatively pure with the two lower molecular weight proteins/cleavage products being eluted in fractions 2 to 5. A small amount of the N-terminal 6xHis tag remained attached to the lower molecular weight proteins/cleavage products as they are only eluted upon the application of the elution buffer after extensive washing of the nickel chelate resin. They were however not detected using the anti-6xHis IgG antibody in Fig. 2.13. The smearing observed in the samples from the wash steps was caused by the high concentrations of urea present in the samples.
Even after extensive washing of the nickel chelate resin after the unbound fraction had been collected, in the refolding and purification using the sarkosyl method, the first two eluted fractions contained large amounts of undesired bacterial host proteins including the two lower molecular weight proteins/cleavage products (Fig. 2.16, panel B). From
fractions 3 to 10, purified TcMCA5 was obtained along with low concentrations of the two lower molecular weight proteins/cleavage products.
Figure 2.16: Nickel affinity on-column refolding and purification of solubilised recombinant TcMCA5 using sarkosyl and urea methods. Samples from the soluble and insoluble expression fractions as well as from the (A) sarkosyl method and the (B) urea method of on-column refolding and affinity purification were electrophoresed on a 12.5%
reducing SDS-PAGE gel and stained with Coomassie blue R-250.
Comparing panels A and B in Fig. 2.16, solubilisation and on-column refolding using the urea and sarkosyl methods respectively, it is evident that in the presence of urea, recombinant TcMCA5 has less activity as visualised by the low concentration of the two lower molecular weight products/cleavage products. This could be due to the fact that the eluted fractions are still in 8 M urea and need to undergo a stepwise dialysis to remove the denaturant. In addition, there was less contamination by bacterial host proteins when using urea. Solubilisation and on-column refolding using sarkosyl resulted in significant amounts of pure recombinant TcMCA5 protein being eluted along with the lower molecular weight proteins/cleavage products. Bacterial host proteins were only eluted in fractions 1 and 2 and thereafter pure recombinant TcMCA5 was eluted which did not require dialysis prior to use in enzymatic assays.