The thioredoxin system of Helicobacter pylori

This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Heli cobacter pylori, purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa prote in which possesses the conserved redox active motif CGPC, The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET exp ression vector and used to transform Escherichia coli, Trx was overexpresse d by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahist idine 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 dis ulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immu noglobulins (IgG/ IgA/sIgA), and soluble mucin. Subcellular fractionation a nalysis 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 har vested 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 homo dimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed t he 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 stres s response element as broth grown bacteria secreted Trx in response to chem ical, biological, and environmental stresses. These observations suggest th at Trx may conceivably assist H, pylori in the process of colonization by i nducing focal disruption of the oligomeric structure of mucin while renderi ng host antibody inactive through catalytic reduction.