Human prothymosin alpha (ProT alpha) is a small acidic protein (12.1 kDa; pI similar to 3.5) ubiquitously expressed in a wide variety of tissues. The amino acid composition of this protein is highly unusual. While close to half of its sequence is composed of acidic amino acids. the protein does not contain any aromatic residues. ProTa has been shown to play crucial roles in different biological processes including cell proliferation, transcriptional regulation and apoptosis. Despite the multiple functions this protein has, it does not adopt a stable tertiary fold under physiological conditions. In order to understand how ProTot functions, detailed structural characterization of this protein is essential. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for elucidating the protein structure and dynamics at the atomic level. However, milligrams of isotopically labeled protein with high purity are usually required for the studies. In this work, we developed a high-yield protocol for purifying recombinant ProTot expressed in Escherichia coli by exploiting the intrinsically disordered and acidic natures of this protein. By combining the heat-cooling extraction, ammonium sulfate precipitation, and anion exchange chromatography, we were able to obtain over 20 mg of ProTa with > 97% purity from 1 L of M9 minimal media culture. The new purification protocol provides a cost effective and an efficient way to produce large quantities of high purity recombinant human ProTot in various isotopically labeled forms, which will greatly facilitate the structural studies of this protein by NMR and other biophysical methods. (c) 2007 Elsevier Inc. All rights reserved.