CHAPTER 5 The Plasmodium spp. putative Cox17 copper metallochaperone
5.2 Results
5.2.3 Recombinant expression of P. yoelii and P. falciparum Cox17
Using recombinant P. yoelii and P. falciparum Cox17, the in vitro and in vivo copper binding ability of each protein was to be examined. The BCA release assay was again used to detect bound copper (Brenner and Harris, 1995). Preparation of the parasite Cox17 expression constructs followed the strategy outlined for expression of the amino terminal domain of the parasite copper transport proteins (Figure 4.9). Following PCR amplification of the P. yoelii and P. falciparum Cox17 coding domains (Figure 5.1), the PCR products were gel purified and ligated into a pGEM®-T cloning vector. These constructs were transformed into E. coli JM109 cells, positive colonies identified and the plasmid isolated. The P. yoelii Cox17 insert was excised by EcoRI and NotI digestion, whilst the P. falciparum insert was excised by BamHI and NotI digestion. Each insert was gel purified and ligated into a pET expression vector digested with the corresponding restriction enzymes. Initial attempts at recombinant expression of His6- PyCox17 and His6-PfCox17 made use of the pET23a and pET28a vectors transformed into E.
coli BL21(DE3) cells.
0 2 4 6 8 10 12
0 0.5 1 1.5 2 2.5 3
Elution volume
A280
0 2 4 6 8 10 12
0 0.5 1 1.5 2 2.5 3 3.5
Elution volume (ml)
A280
A. B.
Figure 5.5 Pilot expression and small-scale purification of recombinant His6-PyCox17
His6-PyCox17 expressed from the pET23a (A) and pET28a (B) vectors. All samples were analysed by 10% Tricine- SDS-PAGE. In A and B: lane 1, total cell protein; lane 2, soluble fraction; lane 3, unbound protein fraction; lane 4, resin wash eluate and lane 5, Ni-NTA bound protein. Molecular mass markers (M) are on the left of each gel (see materials and methods for details).
In initial screening, His6-PyCox17 was expressed as a soluble protein by both the pET23a and pET28a vectors (Figure 5.5), whilst His6-PfCox17 expression was not detected (data not shown). Small-scale experiments with the pT-PyCox17 constructs suggested higher expression levels for the pET28a construct (Figure 5.5b). This difference was not apparent in larger scale preparations, hence the pET23a construct was selected for further studies. The pET23a- expressed product was approximately 11.6kDa in size (Figure 5.5a), which is larger than its predicted size but is a similar finding to that reported for purified yeast Cox17 (Beers et al., 1997).
Table 5.1 Confirmation of PfCox17 amplicon ligation into expression vectors Insert presence confirmed by:
BamHI digestion PCR
pET primers PfCox17-specific
pET23a a a a
pET28a a a a
pGex-4T-1 a a a
Reasons for the apparent lack of His6-PfCox17 expression by the pET vectors were not understood. Ligation of the PfCox17 coding domain into the two pET vectors was confirmed by BamHI digestion, PCR using pET- and PfCox17-specific primers (Table 5.1), and sequencing the amplicon (Figure 5.2b). Altering the fermentation conditions made no difference to His6- PfCox17 expression (data not shown). A new PfCox17 expression construct was therefore prepared using the pGex-4T-1 vector, producing a GST-fusion protein.
kDa 66 42 34 27 20
6 3
M. 1. 2. 3. 4. 5. kDa
66 42 34 27 20
6 3
M. 1. 2. 3. 4. 5.
A. B.
Figure 5.6 Pilot expression and small-scale purification of recombinant GST-PfCox17
12.5% SDS-PAGE analysis of GST-PfCox17 expression. Lane 1, total cell protein; lane 2 soluble fraction; lane 3, unbound protein fraction; lanes 4 and 5, resin wash eluate; lane 6, glutathione agarose-bound protein. Molecular mass markers are on the left of each gel (see materials and methods for details).
GST-PfCox17 expression required that the PfCox17 coding sequence was excised from a pGEM®-T cloning vector by BamHI and NotI digestion, gel purified and ligated into a BamHI- NotI digested pGex-4T-1 expression vector. The construct was then transformed into E. coli BL21 host cells and screened for the expression of the GST-PfCox17 fusion protein. Successful ligation was confirmed by BamHI digestion and PCR amplification (Table 5.1). GST-PfCox17 was expressed as a soluble protein and purified by small-scale glutathione-agarose affinity chromatography (Figure 5.6). The two prominent expression products were approximately 33.5kDa and 30.3kDa in size, respectively, where the larger of the two corresponded to the predicted size for GST-PfCox17. The smaller protein may be a truncated product. The BL21 host cells used are protease deficient suggesting the smaller proteins are unlikely to be the result of proteolysis (Figure 5.6).
Figure 5.7 Recombinant expression and affinity purification of His6-PyCox17
10% Tricine-SDS-PAGE analysis of affinity purified His6-PyCox17 (A), detected with an anti-His6 antibody (B) and anti-KTC anti-peptide antibody (C) following blotting to nitrocellulose. Lane 1, soluble cell lysate; lane 2, unbound protein fraction; lanes 3 – 5, protein eluate from Ni-NTA affinity matrix. Molecular mass markers (M) are on the left of each gel (see materials and methods for details).
97 66
45 31
21
14 kDa
M. 1. 2. 3. 4. 5. 6.
66 4235
27 20 14 6
kDa M. 1. 2. 3. 4. 5.
66 4235
27 20 14 6
kDa 1. 2. 3. 4. 5.
66 4235
27 20 14 6
1. 2. 3. 4. 5.
kDa
A. B. C.
For the expression of His6-PyCox17 it was found that induction with 0.4 mM IPTG, for 4 hours, at 37°C was optimal, whilst for GST-PfCox17 induction with 0.5 mM IPTG, for 4 hours, at 37°C was optimal (data not shown). Each construct was then prepared for large-scale expression and purification of the recombinant protein. Following cell lysis, each recombinant protein was affinity purified using the Ni-NTA matrix for His6-PyCox17 (Figure 5.7) and glutathione-agarose for GST-PfCox17 (Figure 5.8). His6-PyCox17 yields were 3.95 mg and GST-PfCox17 6.87 mg protein per 500 ml of culture. The identity of each recombinant protein was confirmed by western blot. Purified His6-PyCox17 was detected by both an anti-His6 and anti-KTC anti- peptide antibody (Figure 5.7) and GST-PfCox17 was detected by an anti-GST and anti-NKG anti-peptide antibody (Figure 5.8). The His6-PyCox17 bands of a higher molecular weight than the monomeric species are likely to represent multimers. Although less evident, GST-PfCox17 also produced multimeric species (Figure 5.8).
Figure 5.8 Recombinant expression and affinity purification of GST-PfCox17
12.5% SDS-PAGE analysis of affinity purified GST-PfCox17 (A), detected by an anti-GST antibody (B) and anti-NKG (C) anti-peptide antibody following blotting to nitrocelulose. Lane 1, soluble cell lysate; lane 2, unbound protein fraction; lanes 3 – 5, protein eluate from glutathione-agarose affinity matrix. Molecular mass markers (M) are on the left of each gel (see materials and methods for details).