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.
Steinlein
L M
LM
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.
Graf
T N
TN
Ikeda
R A
RA
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