Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-10-19
2004-06-29
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S252300, C435S254200, C435S325000, C530S300000, C530S350000
Reexamination Certificate
active
06756215
ABSTRACT:
FIELD
The disclosure is in the field of protein labeling and detection. More specifically, the disclosure relates to the use of molecular tags to identify and track recombinant molecules, such as small proteins, in a subject.
BACKGROUND
TGF-&bgr; Protein Family
Transforming growth factor-&bgr;1 (TGF-&bgr;1) is a growth factor and immunomodulatory cytokine that is secreted from cells and acts through specific binding interactions with a collection of different cell-surface localized receptors. TGF-&bgr;1 is the prototype for a large family of secreted polypeptides that includes the three mammalian TGF-&bgr; isoforms (TGF-&bgr;1, TGF-&bgr;2, and TGF-&bgr;3), bone morphogenesis proteins (BMPs), activins, and Müllerian inhibitory substance (MIS). More distantly related members of this protein family include murine nodal gene products, Drosophila decapentaplegic complex gene products, and Vg1 from Xenopus.
In general, TGF-&bgr; family proteins are homodimers, wherein each functional protein complex includes two identical, associated monomer subunits. The crystal structure of the TGF-&bgr;1 homodimer is known (Hinck el al.,
Biochem
., 35:8517-8534, 1996; Qian et al.,
J. Biol. Chem
., 271:30656-30662, 1996). TGF-&bgr; is a very compact protein, having four intramolecular disulfide bridges within each subunit, as well as one intermolecular disulfide bridge.
Each monomer of the protein is synthesized as a large (~55 kDa) precursor molecule with a long (about 278 residue) N-terminal pro-region and a much shorter (112 residue, 12.5 kDa) C-terminal active domain (the mature region). During the maturation process, two precursor molecules associate with each other; the pro-region is important for proper folding of and proper association between the two active domain monomers. The pro-region of each monomer is proteolytically cleaved from the associated active domain; in most instances however, the pro-region remains associated with the mature TGF-&bgr; fragment. The severed pro-region is referred to as the “latency-associated peptide” (LAP). LAP is responsible for blocking the correctly folded TGF-&bgr; homodimer so that it does not interact with its receptor. For an excellent discussion of TGF-&bgr; synthesis, see Khalil,
Micro. Infect
., 1:1255-1263, 1999.
TGF-&bgr;s and their receptors are expressed in essentially all tissues, and have been found to be important in many cellular processes. These include cell growth and differentiation, immunosupression, inflammation, and the expression of extracellular matrix proteins. By way of example, in animal models TGF-&bgr; has been shown to attenuate the symptoms associated with various diseases and disorders, including rheumatoid arthritis, multiple sclerosis, wound healing, bronchial asthma, and inflammatory bowel disease, and has been used in the clinical setting to enhance wound healing.
TGF-&bgr;1 was the first identified member of the TGF-&bgr; family, and has been intensely studied for over 20 years. There are some TGF-&bgr;1 antibodies available, but their usefulness in a clinical setting is limited at least in part because they often display some degree of cross-reactivity to other TGF-&bgr; family proteins (see, e.g., U.S. Pat. No. 5,571,714). In most experiments, TGF-&bgr; is iodinated with
125
I to enable researchers to track the protein. Radioactive iodination is an expensive and hazardous process, and it usually would be inappropriate to use
125
I labeled proteins for in vivo experimentation, for instance in clinical trials.
The ability to track the distribution of any exogenously administered, recombinant forms of TGF-&bgr; family proteins has been restricted by the inability to distinguish between the endogenous forms of the protein produced in treated cells or tissues. In addition, available antibodies to these proteins exhibit some degree of cross-reactivity with related family members.
There have been a few reports of TGF-&bgr; fusions in the literature, but the described molecules have been essentially biologically non-functional. In an effort to produce large quantities of easily purified TGF-&bgr; that retained activity, Nimni and co-workers expressed 6x His-tagged TGF-&bgr; fusion proteins in
Escherichia coli
(Tuan et al.,
Conn. Tiss. Res
., 34:1-9, 1996; Han et al.,
Prot. Expr. Purif
., 11:169-178, 1997). Serious difficulties were encountered in refolding the denatured fusion protein, and full biological activity was not retained using this system. In addition, the Nimni constructs cannot be used to express a tagged TGF-&bgr; in a mammalian host, since the constructs lack a part the TGF-&bgr; pro-protein (the LAP), which is essential for secretion and proper folding of the TGF-&bgr; protein. In an earlier effort, Wakefield et al. (
Growth Factors
, 5:243-253, 1991) reported attaching an endoplasmic reticulum retention signal to the C-terminus of full-length TGF-&bgr;1, in an attempt to maintain the protein in the cell (rather than secreting it to the extracellular matrix). This construct had no biological activity.
It is believed that all prior efforts to fuse a TGF-&bgr; family protein to a peptide or protein have resulted in biologically non-functional molecules. Therefore a need still exists for TGF-&bgr; family protein fusions that maintain substantial biological activity.
SUMMARY OF THE DISCLOSURE
This disclosure provides functionalized TGF-&bgr; fusion proteins that maintain substantial TGF-&bgr; biological activity. These fusion proteins are achieved by placing a functionalizing peptide between the pro- and active (mature) portions of a TGF-&bgr; protein, or at a relatively non-conserved site within the mature region of a TGF-&bgr; protein.
Encompassed herein are functional TGF-&bgr; family fusion proteins that contain a functionalizing peptide portion for detecting, quantifying or providing a specific additional function to the fusion protein and a mature TGF-&bgr; family protein, both as a monomer and in the form of a dimer (e.g., a homodimer). Also encompassed are nucleic acid molecules encoding such fusion proteins, and conservative substitutions of such molecules. This disclosure also provides methods for making and using the fusion proteins described, as well as kits.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.
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Letterio John J.
Wolfraim Lawrence A.
Kemmerer Elizabeth
Klarquist & Sparkman, LLP
Nichols Christopher James
The United States of America as represented by the Secretary of
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