RECOMBINANT HSP40 AND HSP70 CHAPERONES FROM Schistosoma mansoni: TOWARD THE STUDY OF THE PARASITE-HOST INTERFACE

Heat shock proteins (HSPs) are highly conserved molecular chaperones involved in protein folding, trafficking, and cellular proteostasis. In Schistosoma mansoni, members of the HSP70 and HSP40 families have been identified in secreted/extracellular fractions and may participate in processes occurring at the parasite-host interface and potentially contribute to parasite adaptation and interactions with the host. The characterization of these proteins may contribute to a better understanding of the molecular mechanisms underlying the parasite-host relationship. This study aims to obtain recombinant HSP40 and HSP70 proteins from S. mansoni for subsequent purification and biophysical characterization. Coding sequences for two HSP40 isoforms and one HSP70 were amplified by PCR using egg-derived cDNA as a template. The resulting amplicons were cloned into the pET-28a(+) expression vector using the NdeI and XhoI restriction sites. The resulting constructs were transformed into Escherichia coli DH5α, and positive clones were screened by restriction analysis and subsequently confirmed by DNA sequencing. Clones showing 100% sequence identity to the corresponding target sequences were selected for heterologous expression in E. coli BL21(DE3). Protein expression was induced with IPTG and analyzed by SDS-PAGE. For both HSP40 isoforms, recombinant proteins displaying electrophoretic mobilities consistent with their expected molecular masses were detected, and the corresponding constructs were subsequently used for large-scale expression. One HSP40 isoform was successfully purified by Ni-NTA affinity chromatography followed by size-exclusion chromatography using an ÄKTA system, while purification of the second isoform is currently being optimized. Circular dichroism analysis of the purified HSP40 revealed a predominantly α-helical secondary structure. For HSP70, cloning and screening of positive clones are currently underway. The proteins will be further investigated by isothermal titration calorimetry and interaction studies with mammalian serum, providing insights into their biophysical properties and potential role at the parasite-host interface.