Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1999-11-15
2004-11-30
Bugaisky, Gabriele (Department: 1653)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S320100, C435S325000, C435S069600, C435S069100, C530S394000
Reexamination Certificate
active
06825037
ABSTRACT:
GOVERNMENT SUPPORT
The work leading to this invention was supported by one or more grants from the United States Government.
BACKGROUND OF THE INVENTION
The iron-binding pseudoglobulins collectively called transferrins or siderophilins comprise a class of proteins with strikingly similar features. X-ray crystallographic analyses of human lactoferrin (Anderson, B. F. et al. (1987)
Proc. Natl. Acad. Sci. USA
84:1769-1773) and rabbit serum transferrin (Bailey, S. et al. (1988)
Biochemistry
27:5804-5812) reveal that these proteins consist of two similar lobes connected by a short bridging peptide and that each lobe contains two domains defining a deep cleft containing the binding site for a metal ion and a synergistic anion.
The chicken ovotransferrin gene has been expressed in transgenic mice (McKnight, G. S. et al. (1983)
Cell
(Cambridge, Mass.) 34:335-341) and a fusion protein of part of rat transferrin with galactosidase has been expressed in
E. coli
(Aldred, A. et al. (1984)
Biochem. Biophys. Res. Commun
. 122:960-965). Except for this fusion protein, attempts to express transferrin or portions of the molecule in prokaryotic systems have been unsuccessful (Aldred, A. et al. (1984)
Biochem. Biophys. Res. Commun
. 122:960-965). The highly convoluted structure of the protein and large number of disulfide bridges in the molecule are probably the major impediments to expression in bacterial hosts. Attempts to mimic partially the natural protein folding environment by targeting the protein for bacterial membrane transport via an attached alkaline phosphatase signal sequence have been unsuccessful.
SUMMARY OF THE INVENTION
This invention pertains to recombinant transferrin, to recombinant transferrins that bind to the transferrin receptor, to recombinant transferrin half-molecules comprising at least the metal-binding domains of a single lobe (amino-terminal or carboxy-terminal) of transferrin and to stable cell culture system for expression of the transferrin. The recombinant transferrin can be expressed in stable, transformed eukaryotic cells, such as baby hamster kidney cells, to yield essentially homogeneous (monodisperse) preparations of the full or half-molecule forms. The invention also pertains to mutant transferring, non-glycosylated transferrins and transferrin half-molecules which have metal-binding or other properties which are different from the natural (wild-type) form of the transferrin. These include mutant transferrins and transferrin half-molecules which bind iron or other metals more or less avidly than natural transferrin.
Transferrin half-molecules can be used in metal chelation therapy to treat individuals affected with abnormalities of metal regulation or with metal poisoning. For example, transferrin half-molecules, especially mutant forms which bind iron with a higher avidity than natural transferrin, can be administered to iron-overloaded individuals, e.g., thalassemics, in order to clear excess toxic iron from their bodies. In addition, half-molecules, or mutants thereof having altered metal ion selectivities, could be used to clear other toxic metals, e.g., lead, mercury, cadmium, copper and zinc from the body.
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Funk Walter D.
MacGillivray Ross T.A.
Mason Anne B.
Woodworth Robert C.
Bugaisky Gabriele
DeConti, Jr. Giulio A.
Lahive & Cockfield LLP
Milasincic Debra J.
University of Vermont
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