Loferer H, Hennecke H
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Eur J Biochem. 1994 Jul 15;223(2):339-44. doi: 10.1111/j.1432-1033.1994.tb18999.x.
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.
日本慢生根瘤菌的TlpA蛋白先前通过遗传学方法鉴定为一种膜锚定的周质硫氧还蛋白样蛋白。在此，我们描述了TlpA在大肠杆菌中的异源表达、随后的纯化及其生化特性。一种缺少N端膜锚定序列的可溶性TlpA在大肠杆菌中过表达，并通过两步法进行纯化。纯TlpA在溶液中显示为单体，并且在还原胰岛素二硫键以及使还原的、变性的核糖核酸酶A重新激活方面具有活性。有证据表明，两个非活性位点的半胱氨酸残基形成了分子内二硫键，这一特征在其他原核硫氧还蛋白中通常不存在。
