EXPRESSION OF GLYCOSYLATED AND NONGLYCOSYLATED HUMAN TRANSFERRIN IN MAMMALIAN-CELLS - CHARACTERIZATION OF THE RECOMBINANT PROTEINS WITH COMPARISON TO 3 COMMERCIALLY AVAILABLE TRANSFERRINS

被引:96
作者
MASON, AB
MILLER, MK
FUNK, WD
BANFIELD, DK
SAVAGE, KJ
OLIVER, RWA
GREEN, BN
MACGILLIVRAY, RTA
WOODWORTH, RC
机构
[1] UNIV BRITISH COLUMBIA,DEPT BIOCHEM,VANCOUVER V6T 1Z3,BC,CANADA
[2] UNIV SALFORD,DEPT BIOL SCI,BIOL RES UNIT,SALFORD M5 4WT,LANCS,ENGLAND
[3] VG BIOTECH,ALTRINCHAM WA14 5R2,CHESHIRE,ENGLAND
关键词
D O I
10.1021/bi00071a025
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:5472 / 5479
页数:8
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