Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins.
The coding sequence for human serum transferrin was assembled from restriction fragments derived from a full-length cDNA clone isolated from a human liver cDNA library. The assembled clone was inserted into the expression vector pNUT and stably transfected into transformed baby hamster kidney (BHK) cells, leading to secretion of up to 125 mg/L recombinant protein into the tissue culture medium. As judged by mobility on NaDodSO4-PAGE, immunoreactivity, spectral properties (indicative of correct folding and iron binding), and the ability to bind to receptors on a human cell line, initial studies showed that the recombinant transferrin, is identical to three commercial human serum transferrin samples. Electrospray mass spectrometry (ESMS), anion-exchange chromatography, and urea gel analysis showed that the recombinant protein has an extremely complex carbohydrate pattern with 16 separate masses ranging from 78,833 to 80,802 daltons. Mutation of the two asparagine carbohydrate linkage sites to aspartic acid residues led to the expression and secretion of up to 25 mg/L nonglycosylated transferrin. ESMS, anion-exchange chromatography, and urea gel analysis showed a single molecular species that was consistent with the expected theoretical mass of 75,143 daltons. In equilibrium binding experiments, the nonglycosylated mutant bound to HeLa S3 cells with the same avidity and to the same extent as the glycosylated protein and the three commercial samples. These studies demonstrate conclusively that carbohydrate has no role in this function.
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
A B
AB
Mason
M K
MK
Miller
W D
WD
Funk
D K
DK
Banfield
K J
KJ
Savage
R W
RW
Oliver
B N
BN
Green
R T
RT
MacGillivray
R C
RC
Woodworth
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002845:Chromatography; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007668:Kidney; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011990:Receptors, Transferrin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014162:Transfection; D014168:Transferrin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin