Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins

Abstract

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.

Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe–lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies

“…This mutation was introduced into the full-length non-glycosylated form of hTF [20], by exchanging appropriate DNA fragments from two plasmids : (1) the plasmid pNUT-HTF\2N-D63S contained the original D63S mutation in hTF\2N cDNA cloned into the expression vector pNUT, and (2) the plasmid pNUT-HTF-FL-NG contained the full-length cDNA for transferrin including the two mutations N413D and N611D that ensure an absence of N-linked glycosylation [20]. The plasmid pNUT-HTF\2N-D63S was cleaved with ClaI and BamHI and the resulting 1n3 kb fragment containing the D63S mutation was separated by PAGE and recovered from the gel with Geneclean (Bio101, La Jolla, CA) according to the manufacturer's instructions.…”

“…BHK cells were grown in DMEM-F12 medium with 5 % fetal bovine serum and transfected as previously described [20]. Selection of transfected cells with methotrexate, and expansion to roller bottles has also been described in detail [20,22].…”

“…Selection of transfected cells with methotrexate, and expansion to roller bottles has also been described in detail [20,22]. Better adhesion of the cells to the roller bottles was achieved by using DMEM-F12 containing 5 % fetal bovine serum through 2-3 medium changes prior to switching to DMEM-F12 containing 1 % UG for a single change of medium followed by DMEM-F12 containing 0n5 % UG and 2n5 % bovine embryonic fluid in all subsequent changes.…”

“…The Sprint system allows simultaneous monitoring of the pH, the conductivity and the absorbance at 280 nm. The homogeneity of the samples was assessed by gel electrophoresis as described [20].…”

“…The competitive solid-phase immunoassay used to determine the concentration of the recombinant hTF in the culture medium and at various stages of the purification has been described [20,22]. This same assay was used to derive binding constants for the antibody binding to the apo-and diferric forms of each protein by the method of Mu$ ller [24].…”