CHAPTER 4 Recombinant expression of the Plasmodium falciparum protein PFC0760C
4.3 Results
4.3.8 Diethylaminoethyl (DEAE)-Sepharose TM ion exchange chromatography to
been excess Pf33-MBP cleavage mixture. The second peak was observed after the NaCl gradient was started. The protein eluted from the column at a NaCl concentration of approximately 100 mM. This should correspond to Pf33 as it was expected to elute first due to a lower nett negative charge. The second peak of protein was observed to elute at a concentration of approximately 370 mM NaCl, which is expected to be MBP. The last absorbance peak was observed at a NaCl concentration of approximately 400 mM. This peak could be due to small amounts of un-cleaved Pf33-MBP. This peak could also be due to Factor Xa as it elutes at a NaCl concentration in the region of 400 mM (New England Biolabs pMALTM Protein Fusion and Purification System Manual). Cleaved MBP is reported to elute as a sharp peak at 100-150 mM NaCl (New England Biolabs pMALTM Protein Fusion and Purification System Manual), however this doesn‟t appear to be the case here.
MBP expressed from non-recombinant pMAL-c2X was passed over the DEAE Sepharose column as a control (Figure 4.10 B). MBP eluted at a NaCl concentration of approximately 275 mM. MBP expressed from pMAL-c2X has a different pI (5.28) and molecular mass (54 kDa), in comparison to cleaved-off MBP (pI/of 5.09; molecular mass of 45.8) from Pf33-MBP and would therefore be expected to elute at different NaCl concentrations. MBP expressed from pMAL-c2X would be expected to elute at a lower salt concentration in comparison to cleaved-off MBP and therefore MBP expressed from pMAL- c2X was not an adequate control.
The eluted fractions (filled diamonds, Figure 4.10 A) from the DEAE purification of cleaved Pf33-MBP was analysed on an SDS-PAGE gel (Figure 4.11) to determine which fractions contained Pf33 and which contained MBP. Protein bands of approximately 43 kDa (Figure 4.11 A lanes 5-8 and lane 13; B lanes 9-10), 45 kDa and 50 kDa (Figure 4.11 lane 11) were observed. The protein bands of 43 kDa correspond to the same molecular mass observed for the predominant band in the cleaved Pf33-MBP sample (Figure 4.9, A, lane 3).
Unfortunately antibodies were not used to confirm the identity of the proteins.
kDa
14.4 21.5 31.0 45.0 97.4 66.2
1 2 3 4 5 6 7 8
kDa 97.4 66.2 45.0 31.0
14.4 21.5
1 2 3 4 5 6 7 8 Factor Xa (μg/ml) 0 10 5 2.5 0.5 0.25 0.05 0 10 5 2.5 0.5 0.25 0.05
RT 4˚C
A B
Figure 4.9. Analysis of protease cleavage of amylose-purified Pf33-MBP with Factor Xa on a 12.5% acrylamide gel. (A) Samples from incubation with factor Xa at room temperature for 46 h, (B) Samples from incubation with factor Xa at 4˚C for 46 h.
Factor Xa was used at a final concentration of 10 μg/ml (lane 3), 5 μg/ml (lane 4), 2.5 μg/ml (lane 5), 500 ng/ml (lane 6), 250 ng/ml (lane 7) and 50 ng/ml (lane 8). Pf33-MBP incubated with buffer, as a non-cleaved control (lane 2) and a protein molecular weight marker (lane 1) were included.
Figure 4.10. DEAE Sepharose purification of cleaved Pf33 and MBP. Factor Xa-treated Pf33-MBP (A) was separated with ion exchange chromatography. Amylose-purified MBP expressed from pMAL- c2X was used as a control (B). The absorbance of each fraction (1 ml) was read at 280 nm. A NaCl gradient of 25 mM – 500 mM NaCl was used (dashed line) to elute bound protein. Filled diamonds () indicate the fractions analysed by SDS-PAGE (Figure 4.11).
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0 10 20 30 40 50 60 70
Fraction number
Absorbance at 280 nm
0 50 100 150 200 250 300 350 400 450 500
NaCl concentration (mM)
0 0.1 0.2 0.3 0.4 0.5
0 10 20 30 40 50 60 70 80
Fraction number
Absorbance at 280 nm
0 100 200 300 400 500
NaCl concentration (mM)
A
B
Figure 4.11. Analysis of fractions obtained from DEAE Sepharose purification of cleaved Pf33-MBP. (A) Fractions from DEAE Sepharose purification of cleaved Pf33-MBP. Lane 1, Bio- Rad low molecular weight marker; lane 2 fraction 1; lane 3, fraction 6; lane 4, fraction 7; lane 5, fraction 8; lane 6, fraction 29; lane 7, fraction 30; lane 8, fraction 31; lane 9, fraction 32; lane 10, fraction 64; lane 11, fraction 67; lane 12, fraction 68; lane 13, amylose-purified factor Xa-cleaved Pf33-MBP; lane 14, amylose-purified, non-cleaved Pf33-MBP. (B) Fractions from DEAE Sepharose purification of cleaved Pf33-MBP. Lane 1, Bio-Rad low molecular weight marker;
lane 2 fraction 5; lane 3, pooled fractions 14-26; lane 4, fraction 36; lane 5, fraction 41; lane 6, fraction 42; lane 7, fraction 43; lane 8, fraction 45; lane 9, fraction 53; lane 10, fraction 54; lane 11, fraction 59; lane 12, fraction 64; lane 13, fraction 67; lane 14, amylose-purified MBP.
Fractions were run on a 12.5% SDS-PAGE gel and silver-stained.
B kDa 97.4 66.2 45.0 31.0
21.5 14.4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14
14.4 31.0 45.0 66.2 kDa 97.4 A
21.5
4.3.9 ‘On-column’ Factor Xa cleavage of Pf33-MBP
„On-column‟ cleavage of Pf33-MBP was attempted as an alternative method for separating MBP and Pf33. Pf33-MBP was cycled over an amylose resin column, to allow binding of the MBP fusion to amylose. The Pf33-MBP bound to the amylose resin was then incubated with Factor Xa to cleave off Pf33, leaving MBP bound to the amylose. The MBP was later eluted with 10 mM maltose. The absorbances of the fractions collected were determined at 280 nm to determine the presence of protein (Figure 4.12). An SDS-PAGE gel and western blot of the „on-column‟ cleavage fractions was run (Figure 4.13). The bound MBP fraction (Figure 4.13, lane 8) has a banding pattern almost identical to the cleaved off Pf33 (Figure 4.13, lane 7), except that there is more of the 43 kDa band in lane 7 than in lane 8.
However, when probed with anti-MBP antiserum (Figure 4.13 B) the banding pattern is identical, suggesting that all of the 3 bands observed in each lane are MBP or have MBP as part of their sequence.
It was thought possible that Pf33 was being recognised by anti-MBP antibodies as perhaps MBP and Pf33 had some similar epitopes. To ensure that there were no linear epitopes, an alignment of their amino acid sequences was done using the ClutalW program (Section 3.2.1). No sequence similarity exists between the two sequences (Result not shown) and therefore the anti-MBP antibodies are detecting MBP in Figure 4.13 B. The possibility also existed that there was perhaps a second Factor Xa recognition site that perhaps resulted in some of the MBP still being attached to Pf33. Factor Xa cleaves at the sequence IEGR and sometimes IDGR. Only one IEGR was observed in the MBP-fusion amino acid sequence, at a position corresponding to the plasmid derived Factor Xa site (result not shown).
A further possibility to explain the results could be that after cleavage, some of the MBP protein was not tightly bound and therefore eluted with Pf33 before elution with maltose.
Figure 4.12. Absorbance readings of fractions collected from ‘on-column’ cleavage of Pf33-MBP.
Fractions (1 ml) were read at 280 nm.
0 0.5 1 1.5 2 2.5 3 3.5
0 5 10 15 20 25 30
Volume (ml)
Absorbance at 280 nm
10 mM maltose
MBP Pf33
1 2 3 4 5 6 7 8
66.2
Figure 4.13. Separation of Pf33 from MBP using ‘On-column’ cleavage. (A) 10 % (m/v) acrylamide gel, (B) western blot analysis with rabbit anti-MBP serum. Lane1, Bio-Rad low molecular weight marker; lane 2, uninduced pTS822 lysate; lane 3, IPTG-induced pTS822 lysate;
lane 4, pellet from centrifugation of sonicated, IPTG-induced pTS822 lysate; lane 5, supernatant from centrifugation of sonicated, IPTG- induced pTS822 lysate; lane 6, Pf33-MBP purified from IPTG-induced pTS822 supernatant on amylose resin; lane 7, product from „on- column‟ cleavage of Pf33-MBP with Factor Xa; lane 8, bound fraction from „on-column‟ cleavage of Pf33-MBP, eluted with 10 mM maltose.
A B
31.0 45.0 97.4 66.2
21.5 kDa
1 2 3 4 5 6 7 8
31.0
21.5 45.0 kDa 97.4
4.4 Expression of Pf33 as a glutathione S-transferase fusion protein