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

The coding sequence for human serum transferrin was assembled from res triction 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 h amster kidney (BHK) cells, leading to secretion of up to 125 mg/L reco mbinant 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 recepto rs on a human cell line, initial studies showed that the recombinant t ransferrin, is identical to three commercial human serum transferrin s amples. Electrospray mass spectrometry (ESMS), anion-exchange chromato graphy, and urea gel analysis showed that the recombinant protein has an extremely complex carbohydrate pattern with 16 separate masses rang ing from 78 833 to 80 802 daltons. Mutation of the two asparagine carb ohydrate linkage sites to aspartic acid residues led to the expression and secretion of up to 25 mg/L nonglycosylated transferrin. ESMS, ani on-exchange chromatography, and urea gel analysis showed a single mole cular species that was consistent with the expected theoretical mass o f 75 143 daltons. In equilibrium binding experiments, the nonglycosyla ted mutant bound to HeLa S3 cells with the same avidity and to the sam e extent as the glycosylated protein and the three commercial samples. These studies demonstrate conclusively that carbohydrate has no role in this function.