Cytosolic thioredoxin from Ruditapes philippinarum: Molecular cloning, characterization, expression and DNA protection activity of the recombinant protein

Thioredoxin (TRx) is a small redox protein that plays significant roles in protection against oxidative stress and in cell homeostasis by maintaining oxidized proteins in a reduced state. Here, we describe the isolation and characterization of a full-length TRx cDNA sequence from manila clam, Ruditapes philippinarum and named it as RpTRx. The full length sequence consists of 1416 bp with an open reading frame of 318 bp encoding for 106 amino acids. RpTRx protein harbors evolutionarily-conserved TRx active site 32WCGPC36. Phylogenetic analysis revealed a close proximity of RpTRx with the orthologue in Japanese scallop, Chlamys farreri. RpTRx was found to be constitutively expressed in hemocyte, gill, mantle, foot and siphon indicating a general role in physiological processes in various tissues. With regard to a potential role in immune responses, the RpTRx mRNA was found to be up-regulated in hemocytes after bacterial (Vibrio tapetis) and lipopolysaccharide (LPS) challenge at 3 h post-infection (p.i.); a wavering increase was observed up to 96 h p.i. for LPS challenge and 48 h p.i. for bacterial challenge. Thus, RpTRx may function as an intracellular antioxidant to protect the cells against ROS induced by LPS and bacterial challenges. Indeed, when recombinant RpTRx protein (rRpTRx) was over-expressed in Escherichiacoli Rosetta gamiTM (DE3) cells, it was able to scavenge free radicals and protect super-coiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. In summary, RpTRx plays an essential role in cellular defense and maintenance of homeostasis in the manila clam.

► Molecular cloning and functional characterization of thioredoxin from manila clam.
► Super-coiled DNA protection from oxidative damage induced by a metal-ion catalyzed oxidation.
► Tissue specific expression studies.
► Transcriptional profiling after LPS and bacterial challenges.