Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein.
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 HindIII 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-polyacrylamide 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, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences. Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium. Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI. The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)
Funk
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Department of Biochemistry, University of British Columbia, Vancouver, Canada.
MacGillivray
R T
RT
Mason
A B
AB
Brown
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SA
Woodworth
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection; D014168:Transferrin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014168:Transferrin; D004247:DNA