Production and purification of N-terminal half-transferrin in Pichia pastoris.

Human serum transferrin, the major iron transport protein in humans, is a monomeric glycoprotein that is composed of two homologous domains; the N-terminal domain is formed by amino acids 1-331 and the C-terminal domain is formed by amino acids 338-679. Each domain is capable of binding one iron atom concomittantly with a carbonate anion; however, the two homologous iron binding sites are not chemically equivalent. The cDNA sequence coding for the N-terminal domain has been cloned and overexpressed in the methylotrophic yeast, Pichia pastoris. The transformants secrete a protein of approximately 38 kDa (the size expected for N-terminal half-transferrin), its N-terminal sequence agrees with the predicted sequence, and the protein reacts with anti-human serum transferrin antibodies. The purified protein appears to be properly folded and can bind iron as demonstrated by its spectral properties and urea-PAGE mobility. It is estimated that N-terminal half-transferrin represents approximately 90% of all protein secreted into the culture medium and that it is expressed at levels exceeding 50 mg/l. This study demonstrates that N-terminal half-transferrin can easily be expressed in the simple host system, Pichia pastoris, and that the purified protein is capable of reversibly binding iron.