Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris.
The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter. Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter. As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system. Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues.
Mason
A B
AB
Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont, 05405, USA.
Woodworth
R C
RC
Oliver
R W
RW
Green
B N
BN
Lin
L N
LN
Brandts
J F
JF
Tam
B M
BM
Maxwell
A
A
MacGillivray
R T
RT
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007668:Kidney; D013058:Mass Spectrometry; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014168:Transferrin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014168:Transferrin; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase