title: Production of N-terminal and C-terminal human serum transferrin in Escherichia coli.
abstract: The elucidation of the relationship of the structure of human serum transferrin to its iron-binding activity and the delineation of the interactions between transferrin and its receptor will require the construction and production of site-specific mutants of human serum transferrin to test the importance of specific structural motifs to the functions of transferrin. The N-terminal domain of transferrin has been previously produced in BHK cells, but the production of the C-terminal domain of transferrin has never been reported. The amino-terminal and carboxyl-terminal half-molecules of human serum transferrin have been cloned into the T7 expression vector pET11a. Contrary to previous reports, nTf and cTf can be easily produced in E. coli. The plasmids produce 38-kDa proteins that are approximately the sizes predicted for N-terminal and C-terminal half-molecules of transferrin, and both proteins react with anti-human serum transferrin antibodies. It is estimated that nTf represents 30-40% of total cellular protein after induction, while cTf represents less than 5% of total cellular protein. This demonstrates that recombinant forms of human serum transferrin can be produced in E. coli and suggests that it will be possible to use a bacterial system to produce other structural variants of transferrin.
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
authors.forename: L M
authors.initials: LM
authors.lastname: Steinlein
authors.forename: R A
authors.initials: RA
authors.lastname: Ikeda
mesh_terms: D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014168:Transferrin / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D014168:Transferrin