This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-␤-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/ IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2,5-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.Redox control of a broad range of biochemical and immunological processes is now well documented to exercise a pivotal role in the cellular activity of both eukaryotes and prokaryotes. To date the redox properties of Helicobacter pylori have received little attention. Accumulating evidence indicates that the redox status of cells controls various cellular functions including cellular activation and proliferation in addition to growth inhibition and apoptosis (for reviews, see Refs. 1-5). Cellular redox status is maintained by intracellular redoxregulating molecules, including thioredoxin (Trx), 1 glutaredoxin, and protein disulfide isomerase, which catalyze the formation and reduction of disulfide bonds in proteins.The redox protein Trx and the associated enzyme thioredoxin reductase (TR) constitute a thiol-dependent reduction-oxidation system that can catalyze the reduction of certain proteins by NADPH, usually with high selectivity (1). In anaerobic bacteria, the generation of ...