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 protein. Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA. A soluble form of TlpA, which lacks its N-terminal membrane anchor, was overexpressed in E. coli and purified by a two-step procedure. Pure TlpA 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.
Loferer
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Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Hennecke
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010957:Plasmids; D012230:Rhizobiaceae; D012260:Ribonucleases / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; C084726:TlpA protein, Bradyrhizobium; D013879:Thioredoxins; D012260:Ribonucleases; D003545:Cysteine