S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective
antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical
validation as a drug target is important. The production of PfAdoMetDC in Escherichia
coli has been reported to result in unsatisfactory yields and poor quality product. The coexpression
of recombinant proteins with molecular chaperones has been proposed as one
way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK,
GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant
proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf,
which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain
of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function
when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired.
We proposed that because of its domain constitution, KPf would most likely be recognised
by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding
domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC
expressed in E. coli. First, using site-directed mutagenesis, followed by complementation
assays, we established that KPf with a mutation in the hydrophobic residue located in its
substrate binding cavity was functionally compromised. We further co-expressed PfAdo-
MetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced
PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdo-
MetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to
protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial
proteins in E. coli.
S1 Fig. KPf and PfHsp70 do not co-purify with PfAdoMetDC. Western blot representing the
purification of PfAdoMetDC expressed in E. coli BL21 (DE3) Star cells rehosted with various chaperone
combinations. Lanes: U–PfAdoMetDC expressed in the absence of supplemented chaperones;
K–PfAdoMetDC co-expressed with supplemented DnaK; KPf–PfAdoMetDC expressed in
cells supplemented with KPf; Pf70 –PfAdoMetDC expressed in cells supplemented with PfHsp70;
K-EL–PfAdoMetDC expressed in cells supplemented with DnaK and GroEL-GroES; KP-EL–PfAdoMetDC
expressed in cells supplemented with KPf and GroEL-GroES; Pf70-EL–PfAdoMetDC
expressed in cells supplemented with PfHsp70 and GroEL-GroES; +C–positive consisting of purified
PfHsp70 protein.Western blot analysis of PfHsp70 (70 kDa) detected using α-PfHsp70 antibody.
Numbers to the left represent protein markers (Fermentas) in kDa.
S2 Fig. Sequence alignment of PfHsp70 and E. coli DnaK. Sequence alignment of E. coli
DnaK (accession number: BAA01595.1) and PfHsp70 (accession number: PF08_0054) were
conducted using ClustalW and Boxshade. The following structural features are highlighted: the
highly conserved linker segment (black horizontal line) which separates the ATPase domain
from the peptide binding domain. Residues Y145, N147, D148, N170 and T173 in the ATPase
domain that interact with DnaJ as reviewed by Shonhai et al (8) are shown with black arrows.
Residues G400, D526 and G539 in the peptide binding domain of DnaK that are important for
interaction with DnaJ, and the aligned residues in PfHsp70 are shown as black arrows. Identical
residues are presented in white against a black background and similar residues are shown in
black against a grey background).
S1 Table. E. coli strains and plasmids used in this study.
S2 Table. Description of primers used towards generation of destination plasmids.