Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein (original) (raw)

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a Hind111 site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-po1yacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, I Abbreviations: hTF, human serum transferrin; hTF/2N, aminoterminal half-molecule of hTF; hGH, human growth hormone; BHK, cultured baby hamster kidney cells; DHFR, dihydrofolate reductase; DMEM, Dulbecco's modified essential medium; FPLC, fast protein liquid chromatography; MTX, methotrexate; NMR, nuclear magnetic resonance; NTA, nitrilotriacetate; PAGE, polyacrylamide gel electrophoresis.