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.
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.
L M
LM
Steinlein
T N
TN
Graf
R A
RA
Ikeda
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D011232:Chemical Precipitation; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018741:Replication Origin; D013104:Spheroplasts; D014168:Transferrin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase