2.3 Results
2.3.4 Enzymatic characterisation of TcMCA5
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.
2.3.4 Enzymatic characterisation of TcMCA5
the urea method. As seen in Table 2.2, the concentration of recombinant TcMCA5 in each eluted fraction was higher when using the sarkosyl method compared to that of the urea method. This is consistent with what was observed in Fig. 2.16, panels A and B, that although the intensities of the visualised bands are similar, the concentration of enzymatically active recombinant TcMCA5 is higher using the sarkosyl method. This might be due to the fact that the fractions eluted from the urea method are still in 8 M urea at a pH of 4.3.
Table 2.2: Concentrations of purified recombinant TcMCA5 after on-column refolding using sarkosyl and urea methods based on the hydrolysis of Z-Gly-Gly-Arg-AMC.
Calculated concentration (nM)
Sarkosyl method Urea method
Fraction 2 - 147.77
Fraction 3 2582.52 174.29
Fraction 4 6100.73 121.74
Fraction 5 5129.48 98.10
Fraction 6 3745.87 85.18
Fraction 7 2128.20 -
Fraction 8 1474.38 -
Figure 2.17: Sarkosyl and urea solubilised, refolded and purified recombinant TcMCA5 hydrolysis of Z-Gly-Gly-Arg-AMC. Samples of the on-column refolded and purified TcMCA5 eluted from the (A) sarkosyl and (B) urea methods were incubated with MCA assay buffer containing the fluorogenic substrate (20 µM) at 37°C for 10 min and the resulting fluorescence was measured at Ex360nm and Em406nm. The fluorescence readings represent the average of triplicate experiments and are plotted as arbitrary fluorescence units.
2.3.4.2 Gelatin containing SDS-PAGE
Enzymatic activity can be visualised using a gelatin containing SDS-PAGE gel, also known as a zymogram (Heussen and Dowdle, 1980). Gelatin is the most commonly used substrate for peptidases and activity is evident as clear bands against the amido black stained background (Manchenko, 2003). Samples from the on-column refolding
and purification fractions, using both the urea and sarkosyl methods, of recombinant TcMCA5 were analysed on a gelatin containing SDS-PAGE gel. A positive control in the form of papain was included in each zymogram. Since TcMCA5 is a cysteine peptidase, a cysteine peptidase activity buffer was used as well as a previously described MCA assay buffer (Moss et al., 2007). No TcMCA5 proteolytic activity was seen in either of the assay buffers; however papain remained active in both buffers (results not shown).
In order to increase the sensitivity of detection, a longer period of incubation in assay buffer was performed (Lantz and Ciborowski, 1994). This, however, made no difference as there was still no evidence of proteolytic activity for TcMCA5, and in addition a high level of diffusion was observed for the papain control (Fig. 2.18).
Figure 2.18: Gelatin-containing 12.5% non-reducing SDS-PAGE gel to demonstrate proteolytic activity of recombinantly expressed, solubilised, refolded and purified TcMCA5 using longer incubation times. Samples from the fractions eluted using the urea and sarkosyl solubilisation and on-column refolding methods were electrophoresed on a 12.5% non-reducing SDS-PAGE gel containing 0.1% (w/v) gelatin.
After incubation in MCA buffer at 37°C for 16 h, the gel was stained with amido black. Papain (10 µg/ml) was included as a positive control. Note that kDa values of the molecular weight marker are approximate under non-reducing conditions.
2.3.4.3 Autocatalytic processing of purified recombinant TcMCA5
Moss and co-workers (2007) showed that TbMCA2 was dependent on Ca2+ ions for autocatalytic cleavage. Since solubilisation and on-column refolding of recombinant TcMCA5 using urea yielded a lower concentration of lower molecular weight proteins/cleavage products, if Ca2+ is required for autocatalytic activity, incubation with Ca2+ should result in a higher concentration of the cleavage products. This, however, is not the case for the fractions eluted using the urea method (Fig. 2.19), where no additional cleavage products were visualised. The presence of a high concentration of urea in the fractions obtained from the urea method might be preventing any
measurable enzyme activity. The eluted fractions three and four from the sarkosyl method showed an increase in the number of lower molecular weight proteins/cleavage products (Fig. 2.19) when compared to the fractions shown in Fig. 2.16, panel B.
Figure 2.19: Analysis of CaCl2 induced autocatalytic cleavage of recombinant TcMCA5. After on-column refolding and purification by urea and sarkosyl methods, samples of the eluted fractions were incubated with 0.1 M CaCl2 at 37°C for 30 min before being electrophoresed on a 12.5% reducing SDS-PAGE gel and stained with Coomassie blue R-250.