Use of a chimeric Hsp70 to enhance the quality of recombinant Plasmodium falciparum S-adenosylmethionine decarboxylase protein produced in Escherichia coli

Show simple item record Makhoba, Xolani Henry Burger, Adelle Coertzen, Dina Zininga, Tawanda Birkholtz, Lyn-Marie Shonhai, Addmore 2016-06-01T08:15:42Z 2016-06-01T08:15:42Z 2016-03-31
dc.description 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. en_ZA
dc.description 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). en_ZA
dc.description S1 Table. E. coli strains and plasmids used in this study. en_ZA
dc.description S2 Table. Description of primers used towards generation of destination plasmids. en_ZA
dc.description.abstract 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. en_ZA
dc.description.department Biochemistry en_ZA
dc.description.department UP Centre for Sustainable Malaria Control (UP CSMC) en_ZA
dc.description.librarian am2016 en_ZA
dc.description.sponsorship The National Research Foundation for an equipment grant (UID, 75464) awarded to AS. AS is a recipient of a Georg Foster research fellowship awarded by the Alexander von Humboldt Foundation of Germany. XHM is a recipient of a National Research Foundation (South Africa) scarce skills scholarship and also received a grant from the University of Zululand Research Committee. AB is a recipient of a postdoctoral fellowship awarded by the NRF. en_ZA
dc.description.uri en_ZA
dc.identifier.citation Makhoba XH, Burger A, Coertzen D, Zininga T, Birkholtz L-M, Shonhai A (2016) Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum SAdenosylmethionine Decarboxylase Protein Produced in Escherichia coli. PLoS ONE 11(3): e0152626. DOI: 10.1371/journal.pone.0152626. en_ZA
dc.identifier.issn 1932-6203
dc.identifier.other 10.1371/journal.pone.0152626
dc.language.iso en en_ZA
dc.publisher Public Library of Science en_ZA
dc.rights © 2016 Makhoba et al. This is an open access article distributed under the terms of the Creative Commons Attribution License. en_ZA
dc.subject Plasmodium falciparum en_ZA
dc.subject Escherichia coli en_ZA
dc.subject Drug targets en_ZA
dc.subject S-adenosylmethionine decarboxylase en_ZA
dc.title Use of a chimeric Hsp70 to enhance the quality of recombinant Plasmodium falciparum S-adenosylmethionine decarboxylase protein produced in Escherichia coli en_ZA
dc.type Article en_ZA

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