Production of His-BbST [hexahistidine-tagged BbST (bubaline somatotropin)] by auto-induction in high-density shake-flask cultures coupled with a single-step, on-column purification and refolding strategy is described here. To optimize expression of BbST, different media and expression conditions were tested. The highest expression levels of BbST, exceeding 30% of the total Escherichia coli cellular proteins, were achieved when YNG and M9NG media were used. Using these auto-inducing media, the final concentration of BbST increased up to 455 mg/l and was severalfold higher than that obtained by isopropyl beta-D-thiogalactoside induction. Most of the target protein, however, was in the form of inclusion bodies, which were solubilized in 8 M urea solution (pH 8.5). Using immobilized-metal-ion-affinity chromatography, His-BbST could be purified from solubilized sample to >97% homogeneity in a single step in a biologically active state as judged by its efficient growth-promoting activity in Nb2 rat lymphoma cell proliferation assays. The expression and purification scheme, presented here, has a potential of scaling up to obtain pure and biologically active His-BbST relatively inexpensively for further studies and applications.