CHAPTER 2 Materials and methods
2.6 Molecular biology methods
2.6.11 Expression plasmids used in the study
The pET-23a plasmid (Figure 2.2) was the initial expression vector used for the recombinant expression of the amino terminal domain of the putative P. berghei copper transport protein (PbCtrNt). The 447bp gene fragment containing the amino terminal domain was ligated into the vector at the EcoRI and NotI restriction sites. The product was expected to have an addition of approximately 23 amino acid residues due the position of the insert relative to the start codon, which is situated at the NdeI restriction site (bp 238 on the plasmid). The host strain used for pET-23a expression was the E. coli BL21(DE3) pLysS strain. The recombinant construct is from here on referred to as pT-PbCtrNt.
Figure 2.2 Map of the pET-23a expression plasmid
The figure illustrates the restriction sites and important features of the pET-23a plasmid including the ampicillin resistant gene (Ap) and the multiple cloning region. The genes of interest were either inserted in the EcoRI and NotI (PbCtrNt; PyCox17) or BamHI and NotI (PfCox17) sites. Genes were inserted in frame with the C-terminal His6-tag and transcribed in the direction indicated by the solid black arrow ( ) housing the multiple cloning site (Adapted from Novagen manual).
In addition to the above, the pET23a construct was utilised for the recombinant expression of both the P. yoelii and P. falciparum putative Cox17 proteins. The P. yoelii gene (192bp) was
ligated into the vector at the EcoRI and NotI restriction sites, whilst the P. falciparum gene (195bp) was ligated into the BamHI and NotI restriction sites. As stated above, both products were expected to have an additional 23 amino acid residues. The host strain used for pET-23a expression of the Cox17 constructs was the E. coli BL21(DE3) strain. The recombinant constructs for P. yoelii and P. falciparum are referred to as pT23-PyCox17 and pT23-PfCox17, respectively.
pET-28a
In this study, pET-28a (Figure 2.3) was used for the recombinant expression of both the P.
yoelii and P. falciparum putative Cox17 copper chaperones. The 192bp P. yoelii gene was ligated into the vector at the EcoRI and NotI sites, whilst the 195bp P. falciparum gene was ligated into the BamHI and NotI sites. Both products were expected to have the addition of approximately 41 amino acid residues due the position of the insert relative to the start codon, which is situated at the NcoI restriction site (bp 296 on the plasmid). The host strain used for pET-28a expression in this study was the E. coli BL21(DE3) strain. As with the pET23a constructs, the P. yoelii and P. falciparum pET28a constructs are from here on referred to as pT28-PyCox17 and pT28-PfCox17, respectively.
Figure 2.3 Map of the pET-28a expression plasmid
The figure illustrates restriction sites and important features of the pET-28a plasmid including the kanamycin resistant gene (kan) and the multiple cloning region. The genes of interest were either inserted in the EcoRI and NotI (PyCox17) or BamHI and NotI (PfCox17) sites. Inserts were inserted in frame with the N-terminal His6-tag and transcribed in the direction indicated by the solid black arrow ( ) housing the multiple cloning site . (Adapted from Novagen manual).
pGex-4T-1
pGex-4T-1 (Figure 2.4) was initially used for the recombinant expression of PbCtrNt in an effort to overcome insoluble recombinant protein production. The 447bp gene fragment, coding for the putative P. berghei amino terminal domain, was ligated into the vector at the EcoRI and NotI sites in frame with the N-terminal GST protein. The host strain used for expression was E. coli BL21. The construct is referred to as pGx-PbCtrNt. In addition to this, the pGex-4T-1 vector was used for the recombinant expression of PfCox17, following unsuccessful attempts with the two pET vectors described above. The 195bp PfCox17 gene was ligated into the BamHI and NotI sites in frame with the N-terminal GST protein. The host strain used for expression was E.
coli BL21. The Cox17 construct is referred to as pGx-PfCox17.
Figure 2.4 Map of pGEX-4T-1 expression plasmid
The figure illustrates the restriction sites of the multiple cloning region and other important features of the plasmid.
These features include the glutathione S-transferase (GST) gene, the ampicillin resistant gene (Ampr) and the inducible Ptac promoter. The genes of interest were either inserted in the EcoRI and NotI sites (PbCtrNt) or the BamHI and NotI sites (PfCox17), in frame with the GST gene. (Adapted from GE Health care manual).
pMal-c2X and pMal-p2X
The pMal-2 series of expression vectors (Figure 2.5) were initially used for the same purpose as the pGex-4T-1 vector, to try to overcome the expression of insoluble PbCtrNt. Both pMal-2 vectors contain the malE gene, encoding the highly soluble maltose binding protein (MBP). The difference between pMal-c2X and -p2X lies in the removal of the malE signal sequence in the pMal-c2X vector, resulting in cytoplasmic expression of the recombinant protein. The retention
of the malE signal sequence in the pMal-p2X vector results in targeting of the expressed recombinant protein to the cell periplasm. The 447bp gene fragment, coding for the putative P.
berghei amino terminal domain, was ligated into both the pMal-c2X and -p2X vectors at the EcoRI and PstI sites, in frame with the N-terminal MBP. The host strain used for recombinant expression was E. coli JM103 or BL21. The pMal-c2X construct is referred to as pMc-PbCtrNt and the pMal-p2X construct as pMp-PbCtrNt
In addition to the above, the pMal-2 series of vectors were also used for the recombinant expression of the amino-terminal domains of the two putative P. falciparum copper transport proteins (PF14_0211 and PF14_0369). An identical approach was adopted for these two proteins where the respective gene fragments (132bp for PF14_0211 and 282bp for PF14_0369) were ligated into both the pMal vectors at the EcoRI and PstI sites, in frame with the N-terminal MBP. The host strain used for recombinant expression was E. coli JM103. The pMal-c2X constructs are referred to as pMc-PfCtr211Nt and pMc-PfCtr369Nt for PF14_0211 and PF14_0369, respectively. Similarly the pMal-p2X constructs are referred to as pMp- PfCtr211Nt and pMp-PfCtr369Nt for PF14_0211 and PF14_0369, respectively.
Figure 2.5 Map of the pMal-2x expression plasmids
The figure illustrates the restriction sites of the polylinker region and other important features of the plasmid. These features include the malE gene, the ampicillin resistant gene (Ampr) and the inducible Ptac promoter. The genes of interest were inserted in the EcoRI and PstI sites, in frame with the malE gene for fusion to a maltose binding carrier protein (Adapted from the NEB pMALTM protein fusion and purification manual).