PRODUCTION AND PURIFICATION OF N-TERMINAL HALF-TRANSFERRIN IN PICHIA-PASTORIS

Human serum transferrin, the major iron transport protein in humans, i s a monomeric glycoprotein that is composed of two homologous domains; the N-terminal domain is formed by amino acids 1-331 and the C-termin al 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, T he cDNA sequence coding for the N-terminal domain has been cloned and overexpressed in the methylotrophic yeast, Pichia pastoris. The transf ormants secrete a protein of approximately 38 kDa (the size expected f or N-terminal half-transferrin), its N-terminal sequence agrees with t he predicted sequence, and the protein reacts with anti-human serum tr ansferrin antibodies. The purified protein appears to be properly fold ed and can bind iron as demonstrated by its spectral properties and ur ea-PAGE mobility, It is estimated that N-terminal half-transferrin rep resents approximately 90% of all protein secreted into the culture med ium and that it is expressed at levels exceeding 50 mg/l, This study d emonstrates that N-terminal half-transferrin can easily be expressed i n the simple host system, Pichia pastoris, and that the purified prote in is capable of reversibly binding iron, (C) 1995 Academic Press, Inc .