EXPRESSION, PURIFICATION AND FUNCTIONAL-PROPERTIES OF A SOLUBLE FORM OF BRADYRHIZOBIUM-JAPONICUM TLPA, A THIOREDOXIN-LIKE PROTEIN

The TlpA protein of Bradyrhizobium japonicum was previously identified genetically as a membrane-anchored, periplasmic thioredoxin-like prot ein. Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA. A s oluble form of TlpA, which lacks its N-terminal membrane anchor, was o verexpressed in E. coli and purified by a two-step procedure. Pure Tlp A was shown to be a monomer in solution and was active in reducing the disulfides of insulin and in reactivating reduced, denatured RNaseA. Evidence is presented that two non-active-site cysteine residues form an intramolecular disulfide bond, a feature that is not normally found in other prokaryotic thioredoxins.