Richard A. Ikeda

Production of N-terminal and C-terminal human serum transferrin in Escherichia coli

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.

Production of human serum transferrin in Escherichia coli

Transferrin (Tf) crystals diffract to only medium resolution. The mediocre quality of the crystals may be due to two factors: (1) the genetic variations naturally present in the primary sequence of Tf, and (2) the glycosylation of the protein. To control genetic variations and glycosylation of samples of Tf, it would be desirable to express the Tf gene from a recombinant clone. Additionally, expression of Tf from a clone would allow for manipulation of the structure of Tf. The cDNA encoding Tf has been cloned into the pL-based expression vector, pRE1, and the T7-based expression vectors, pRSETA and pET11A. The Tf expression plasmids, pTF-SSn and pTF-ESn, based on the T7 expression vectors, efficiently produce a 76-kDa protein that is approximately the same size as deglycosylated Tf, cross reacts with anti-Tf antibodies, and matches the deduced N-terminal amino acid sequence. Expression of Tf in Escherichia coli will allow the production of genetically pure, unglycosylated protein.