Expression and affinity purification of the recombinant Plasmodium

4.2 Results

4.2.5 Expression and affinity purification of the recombinant Plasmodium

103 The imidazole concentrations required to elute the respective Plasmodium PMT proteins expressed here off the TALON^® (Co²⁺) resin were summarised in Table 4.5 below.

Table 4.5 Imidazole concentrations required for elution of bound recombinant Plasmodium PMT proteins off the TALON^® (Co²⁺) resin

Growth conditions rPfPMT

[Imidazole] (mM) rPvPMT [Imidazole] (mM)

LB 30°C 25 25

LB 30°C + IPTG 80 25

TB 30°C 25 80

LB 37°C 25 50

LB 37°C + IPTG 25 80

TB 37°C 25 25

Both of the recombinant Plasmodium PMT proteins eluted off the TALON^® (Co²⁺) resin between 25 to 80 mM imidazole, dependant on the conditions used for expression.

4.2.5 Expression and affinity purification of the recombinant Plasmodium proteins

104 the LB with IPTG induction method. The second change was that rPfPMT was expressed overnight using the LB with IPTG induction method. All proteins were expressed at greater than 5 mg from 50 ml cultures, except for rPvLDH which yielded approximately 3 mg per 50 ml culture. Another change to the methodology used in the optimisation studies was that 10 mM imidazole was included in the lysis and wash buffers, with 250 mM imidazole used in the elution buffer.

Examples of the resulting purifications were shown in Figures 4.9 to 4.11 to follow. Purity of the eluted proteins was assessed on 12.5% reducing SDS-PAGE and the recombinant proteins were detected in each case with an anti-His tag mouse monoclonal antibody in western blots shown alongside the respective gels.

105

Figure 4.9 Recombinant expression and affinity purification of Pf, Pv and PyLDH analysed by SDS- PAGE and western blot

All three recombinant proteins were expressed in E. coli BL21(DE3) cells. The His-tagged proteins were affinity purified using a TALON^® (Co²⁺) resin and the purification steps were analysed on 12.5% reducing SDS- PAGE gels (left panel) and probed with an anti-His tag mouse monoclonal antibody at 1/10000 dilution and a secondary goat anti-mouse-HRPO antibody at 1/6000 dilution (right panel). The elution profiles measuring absorbance at 280 nm were shown as inserts in the respective western blots. The lanes were loaded with molecular weight marker (Mw); BL21(DE3) untransformed cell lysate (lane 1); supernatant sample loaded onto the TALON^® (Co²⁺) resin (lane 2); unbound lysate (lane 3); 10 mM imidazole wash sample (lane 4); eluents 1 to 5 using 250 mM imidazole (lanes 5 to 9).

116 66.2

45 35

25 18.4

rPfLDH Anti-His tag blot

0 0.5 1 1.5

A 280nm

Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

rPvLDH Anti-His tag blot

0 0.2 0.4 0.6 0.8

A 280nm

260 140 95 72

17 52

42 34

Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

0 1 2 3

A 280nm

260140 7295 52 42 34 26

rPyLDH Anti-His tag blot

Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

rPfLDH Anti-His tag blot

rPvLDH Anti-His tag blot

106 The recombinant PfLDH had an estimated molecular weight of ~ 37 kD in the anti-His blot, with an additional protein band at ~ 84 kD, suggesting the presence of a dimeric form of the protein. Two additional minor bands of ~ 35 kD and ~ 63 kD were also detected (Figure 4.9).

None of the untransformed E. coli BL21(DE3) lysate proteins (lane 2, Figures 4.9) were detected in any of the western blots probed with anti-His tag antibodies. Recombinant PvLDH was detected as a single band of approximately 39 kD, where rPyLDH was similarly detected at ~ 40 kD, with low concentrations of a dimer at around 90 kD. The overall yield of the rPfLDH may have been improved further since the protein was still detected in the unbound fraction, suggesting the TALON^® (Co²⁺) resin may have been saturated.

Figure 4.10 Recombinant expression and affinity purification of Pf and PyGAPDH analysed by SDS- PAGE and western blot

Both recombinant proteins were expressed in E. coli BL21(DE3) cells. The His-tagged proteins were affinity purified using a TALON^® (Co²⁺) resin and the purification steps were analysed on 12.5% reducing SDS-PAGE gels (left panel) and probed with an anti-His tag mouse monoclonal antibody at 1/10000 dilution and a secondary goat anti-mouse-HRPO antibody at 1/6000 dilution (right panel). The elution profiles measuring absorbance at 280 nm were shown as inserts in the respective western blots. The lanes were loaded with molecular weight marker (Mw); BL21(DE3) untransformed cell lysate (lane 1); supernatant sample loaded onto the TALON^® (Co²⁺) resin (lane 2); unbound lysate (lane 3); 10 mM imidazole wash sample (lane 4); eluents 1 to 5 using 250 mM imidazole (lanes 5 to 9).

rPfGAPDH Anti-His tag blot

66.2 45 35

25 116

18.4

Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

0 0.5 1 1.5 2 2.5

A 280nm

Mw 1 2 3 4 5 6 7 8 9 260140

9572 52 42 34 26

0 1 2 3

A 280nm

rPyGAPDH Anti-His tag blot

Mw 1 2 3 4 5 6 7 8 9

rPfGAPDH Anti-His tag blot

rPyGAPDH Anti-His tag blot

107

rPfPMT Anti-His tag blot

140 95 72 52 42 34 26 260

0 1 2 3

A280 nm

Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

rPvPMT Anti-His tag blot

260140 9572

52 42 34 26

0 1 2 3

A 280nm

Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

rPfPMT Anti-His tag blot

rPvPMT Anti-His tag blot

The recombinant PfGAPDH protein had an estimated molecular mass of 38 kD, where rPyGAPDH was detected at ~ 40 kD. Similarly to the rPfLDH result, some rPfGAPDH was detected in the unbound sample run in lane three (Figure 4.10), suggesting some of the protein was unable to bind the TALON^® (Co²⁺) resin. This was also the case for both rPfPMT and rPvPMT as shown in Figure 4.11. The rPfPMT was detected at ~ 29 kD and the rPvPMT at ~30 kD in their respective blots.

Figure 4.11 Recombinant expression and affinity purification of Pf and PvPMT analysed by SDS- PAGE and western blot

108

Dalam dokumen Developing antibodies against Plasmodium lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and phosphoethanolamine-N-methyltransferase (Halaman 123-128)