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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 co-expression 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 PfAdoMetDC 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. PfAdoMetDC 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 .

plos one

Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli