4.2 Results
4.2.6 Assessing the recombinant Plasmodium proteins’ native structures
108
109
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Elution volume (ml)
250130 10070 55 35 25
15
Blue dextran Sheep IgG BSA Ovalbumin Myoglobin Blue dextran Sheep IgG BSA Ovalbumin Myoglobin
Calibration standardsMw
A
y = 442.39x-1.368 R² = 0.991 0
0.5 1 1.5 2 2.5 3 3.5
30 50 70
Log MW
Elution volume (ml) B
Figure 4.12 Calibration of the Sephacryl S-200 molecular exclusion chromatography column
The HiPrep 16/60 pre-packed Sephacryl S-200 High Resolution column (column volume of 120 ml) was calibrated using the following standards: 3 mg Blue Dextran (2000 kD); 5 mg Sheep IgG (150 kD); 5 mg BSA (68 kD); 5 mg Ovalbumin (45 kD); 5 mg Myoglobin (17 kD) , and the profile was recorded on the ÄKTAprime plus system (A), with the standards and their respective peaks analysed on a 12.5% reducing SDS-PAGE gel (insert in A). The column was run at 0.5 ml/min and 2 ml fractions were collected, with a total of 120 ml run over the column per run using a 50 mM NaH2PO4, 300 mM NaCl pH 8.0 buffer. Standard curve of molecular weights in (B).
110
11666.2 4535 25
Dimer Monomer
18.414.4
Mw 46 48 50 52 54 56 58 Eluent (ml)
224 165 125 97 77 62.5 51.5 ~ kD
0 10 20 30 40 50 60 70
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000 kD 150 kD 68 kD 45 kD 17 kD
145 kD
0 5 10 15 20 25 30 35 40
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000 kD 150 kD 68 kD 45 kD 17 kD
95 kD
66.2116 45 35 25 18.414.4
Mw 48 50 52 54 56 58 60 62 Eluent (ml)
165 125 97 77 63 52 43 37 ~ kD
Figure 4.13 rPfLDH elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPfLDH was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPfLDH elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis). The eluted fractions comprising the ~145 kDa peak were evaluated on a 12.5% reducing SDS-PAGE gel stained with Coomassie blue (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 46 to 58, with the dimeric and monomeric forms labelled alongside the gel.
Figure 4.14 rPvLDH elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPvLDH was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPvLDH elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis). The eluted fractions comprising the ~95 kDa peak were run on a 12.5% reducing SDS-PAGE gel and Coomassie stained (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 48 to 62.
111
0 2 4 6 8 10 12 14
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000 kD 150 kD 68 kD 45 kD 17 kD
112 kD
140260 9572 5242 3426
17
Mw 46 48 50 52 54 56 58 60 62 64 Eluent (ml)
224 165 125 97 77 63 52 43 37 31 ~ kD
0 0.05 0.1 0.15 0.2 0.25
0 50 100 150 200 250 300
A 340nm
Time (sec)
PfLDH PvLDH Figure 4.15 rPyLDH elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPyLDH was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPyLDH elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis). The eluted fractions comprising the ~112 kDa peak were run on a 12.5% reducing SDS-PAGE gel and Coomassie stained (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 46 to 64.
As further confirmation of the native conformation of the rPfLDH and rPvLDH, dehydrogenase activity of both proteins was demonstrated as shown in Figure 4.16. The oxidation of L-lactate by LDH producing NADH was measured at 340 nm, showing an increase of NADH over time.
Figure 4.16 Enzyme activity of both recombinant PfLDH and PvLDH assessed by measuring the increased formation of NADH due to the oxidation of L-lactate
Recombinant PfLDH and PvLDH both displayed dehydrogenase activity with the oxidation of L-lactate (1mM) resulting in the concomitant reduction of NAD (200 µM) to NADH. The increased NADH was measured as an increase in absorbance at 340 nm over a 5 min period. 35 µM of each of the recombinant proteins was used and the reactions were performed in a 100 mM Tris pH 9.0 buffer at 25°C. The control reaction without recombinant LDH or with 35 µM BSA instead remained at 0 even after 20 min.
112 Under native conditions the purified rPfGAPDH eluted in two peaks estimated at 148 kD and 36 kD from the Sephacryl column. Comparing this to the SDS-PAGE result in Figure 4.10 where rPfGAPDH was detected at ~ 38 kD, the eluted peak of 148 kD was close to the expected tetrameric form and that of 35 kD was close to the monomeric form. Interestingly the monomer in the SDS-PAGE gel (B) ran slightly lower at ~ 35 kD than it did in Figure 4.10, although a different molecular weight marker was also used in this case.
Figure 4.17 rPfGAPDH elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPfGAPDH was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPfGAPDH elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis). The eluted fractions comprising the ~148 and ~ 36 kDa peaks were run on a 12.5% reducing SDS-PAGE gel and Coomassie stained (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 42 to 52 and 64 to 68.
In comparison rPyGAPDH was estimated to be around 41 kD on the SDS-PAGE gel (Figure 4.10), and eluted as an estimated 110 kD protein from the Sephacryl column (Figure 4.17), which suggested trimer formation.
Finally analysis of the PMT proteins revealed that both proteins formed monomers in solution, with rPfPMT eluting as an estimated 29 kD protein and rPvPMT as a ~ 27 kD protein.
250130 100 7055
35 25
Mw 42 44 46 48 50 52 64 66 68 Eluent (ml)
459 315 224 165 125 97 31 27 24 ~ kD
B A
250 130 100 70 55 35 25 0
5 10 15 20 25 30
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000kD 150kD 68kD 45kD 17kD
148kD 36kD
113
0 10 20 30 40 50 60 70 80 90
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000 kD 150 kD 68 kD 45 kD 17 kD
110 kD
260140 7295 52 42 34 26
17
Mw 44 46 48 50 52 54 56 58 Eluent (ml)
314 224 165 125 97 77 63 52 ~ kD
0 10 20 30 40 50 60 70 80
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000kD 150kD 68kD 45kD 17kD
29kD
116 66.2 45 35 25 18.4
Mw 60 62 64 66 68 70 72 Eluent (ml)
43 37 31 27 24 21 19 ~ kD
B A
Figure 4.18 rPyGAPDH elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPyGAPDH was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPyGAPDH elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis).
The eluted fractions comprising the ~110 kDa peak were run on a 12.5% reducing SDS-PAGE gel and Coomassie stained (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 44 to 58.
Figure 4.19 rPfPMT elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPfPMT was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPfPMT elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis). The eluted fractions comprising the ~29 kDa peak were run on a 12.5% reducing SDS-PAGE gel and Coomassie stained (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 60 to 72.
114
0 20 40 60 80 100 120 140 160
0 50 100 150 200 250 300 350 400 450
0 20 40 60 80 100 120
Absorbance (mAu)
Absorbance (mAu)
Elution volume (ml)
2000kD 150kD 68kD 45kD 17kD
27kD
A
66.2116 45 35 25 18.4
Mw
37 31 27 24 21 19 17 15 14 ~ kD
62 64 66 68 70 72 74 76 78 Eluent (ml)
B
Figure 4.20 rPvPMT elution profile over a Sephacryl S-200 MEC column
Four milligrams of affinity purified rPvPMT was passed over a HiPrep 16/60 pre-packed Sephacryl S-200 high resolution column with a column volume of 120 ml, using a 50 mM NaH2PO4; 300 mM NaCl pH 8.0 running buffer. The profile was recorded on the ÄKTAprime plus system using milli Absorbance units (mAu) labelled on the primary and secondary axes (A). The solid line depicts the rPvPMT elution profile (plotted on the secondary axis), with the calibration standards included as the dashed line (plotted on the primary axis). The eluted fractions comprising the ~27 kDa peak were run on a 12.5% reducing SDS-PAGE gel and Coomassie stained (B). The lanes were labelled as follows: molecular weight marker (Mw), followed by the respective 2 ml eluents 62 to 78, with their respective estimated molecular weights indicated above the lanes.
115