Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-11-12
2002-12-10
Kemmerer, Elizabeth C. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S387100, C530S387900
Reexamination Certificate
active
06492493
ABSTRACT:
The present invention relates to a novel family of purified proteins designated as Bone Morphogenetic Proteins (BMP)-17 and BMP-18, related proteins, DNA encoding them, and processes for obtaining them. These proteins may be used to induce bone and/or cartilage or other connective tissue formation, and in wound healing and tissue repair. These proteins may also be used for augmenting the activity of other bone morphogenetic proteins.
BACKGROUND OF THE INVENTION
The search for the molecule or molecules responsible for the bone-, cartilage-, and other connective tissue-inductive activity present in bone and other tissue extracts has led to the discovery of a novel set of molecules called the Bone Morphogenetic Proteins (BMPs). The structures of several proteins, designated BMP-1 through BMP-16 have previously been elucidated. The unique inductive activities of these proteins, along with their presence in bone, suggests that they are important regulators of bone repair processes, and may be involved in the normal maintenance of bone tissue. There is a need to identify whether additional proteins, particularly human proteins, exist which play a role in these processes. The present invention relates to the identification of such a novel human protein, which the inventors have designated human BMP-17 and BMP-18.
Human BMP-17 and BMP-18 appear to be human homologs of a murine protein called Lefty. The nucleotide and amino acid sequences of murine Lefty are described in Zhou et al.,
Nature,
361:543-547 (1993). The murine Lefty gene has been described as being expressed in the mouse node during gastrulation. A related human protein, designated endometrial bleeding associated factor [EBAF] was published in Kothapelli et al.,
J. Clin. Invest.,
99:2342-2350 (1997).
SUMMARY OF THE INVENTION
As used herein, the term BMP-17 and BMP-18 proteins refer to the human BMP-17 and BMP-18 proteins, having the amino acid sequences specified in SEQUENCE (SEQ) ID NO:2 and SEQ ID NO: 4, as well as DNA sequences encoding the BMP-17 and BMP-18 proteins, such as the native human sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3. Also included are naturally occurring allelic sequences of SEQ ID NO:1 and 3, and equivalent degenerative codon sequences of the above.
The BMP-17 (SEQ ID NO: 1) and BMP-18 (SEQ ID NO: 3) DNA sequences and amino acid sequences (SEQ ID NO: 2 and 4, respectively) are set forth in the Sequence Listings. BMP-17 and BMP-18 proteins may be capable of inducing the formation of cartilage, bone, or other connective tissue, or combinations thereof. The cartilage and/or bone and/or other connective tissue formation activity in the rat bone formation assay described below. BMP-17 and BMP-18 proteins may be further characterized by the ability to demonstrate effects upon the growth and/or differentiation of embryonic cells and/or stem cells. Thus, the proteins or compositions of the present invention may also be useful for treating cell populations, such as embryonic cells or stem cell populations, to enhance or enrich the growth and/or differentiation of the cells. Alternatively, the proteins or compositions of the present invention may also be useful for maintenance of a cell population, including differentiated cell populations, for example, neuronal cells, epithelial cells, dendritic cells, chondrocytes, osteocytes, muscle cells or cells of other differentiated phenotypes.
Human BMP-17 proteins may be produced by culturing a cell transformed with a DNA sequence comprising nucleotide a DNA sequence encoding the mature BMP-17 polypeptide, comprising nucleotide #427 to nucleotide #1098 as shown in SEQ ID NO: 1, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #224 as shown in SEQ ID NO:2 substantially free from other proteinaceous materials with which it is co-produced. For production in mammalian cells, the DNA sequence further comprises a DNA sequence encoding a suitable propeptide 5′ to and linked in frame to the nucleotide sequence encoding the mature BMP-17-related polypeptide.
Human BMP-18 proteins may be produced by culturing a cell transformed with a DNA sequence comprising nucleotide a DNA sequence encoding the mature BMP-18 polypeptide, comprising nucleotide #406 to nucleotide #1098 as shown in SEQ ID NO: 3, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #231 as shown in SEQ ID NO:4 substantially free from other proteinaceous materials with which it is co-produced. For production in mammalian cells, the DNA sequence further comprises a DNA sequence encoding a suitable propeptide 5′ to and linked in frame to the nucleotide sequence encoding the mature BMP-18-related polypeptide.
The propeptide may be the native BMP-17 or BMP-18-related propeptide, or may be a propeptide from another protein of the TGF-&bgr; superfamily. Where the native BMP-17 and BMP-18 propeptide is used, human BMP-17 and BMP-18 may be produced by culturing a cell transformed with a DNA sequence comprising a DNA sequence encoding the full BMP-17 and BMP-18 polypeptide, comprising nucleotides #1 to #1098 as shown in SEQ ID NO: 1, or nucleotides #1 to #1098 as shown in SEQ ID NO: 3, producing a protein characterized by the amino acid sequence comprising amino acids #−142 to #224 as shown in SEQ ID NO:2, of which amino acids #−142 to −1 comprise the native propeptide of human BMP-17; or #−135 to #231 of SEQ ID NO: 4 of which amino acids #−135 to −1 comprise the native propeptide of human BMP-18, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #224 as shown in SEQ ID NO:2, or #1 to #231 of SEQ ID NO:4, respectively, substantially free from other proteinaceous materials with which it is co-produced.
Based in part upon the Von Heginje signal peptide prediction algorithm, approximately the first 17 to 23 amino acids of SEQ ID NO: 2 and 4 appear to be involved in signalling for the secretion of the mature peptide. Accordingly, in one embodiment of the invention, DNA encoding a signal peptide,such as the native BMP-17 or BMP-18 signal peptide, or another recognized signal peptide, may be linked directly to the sequence encoding the mature BMP-17 or BMP-18 peptide.
It is expected that other species, particularly human, have DNA sequences homologous to human BMP-17 and BMP-18 protein. The invention, therefore, includes methods for obtaining the DNA sequences encoding human BMP-17 and BMP-18 proteins, the DNA sequences obtained by those methods, and the human proteins encoded by those DNA sequences. This method entails utilizing the human BMP-17 and BMP-18 nucleotide sequences or portions thereof to design probes to screen libraries for the corresponding gene from other species or coding sequences or fragments thereof from using standard techniques. Thus, the present invention may include DNA sequences from other species, which are homologous to human BMP-17 and BMP-18 proteins and can be obtained using the human BMP-17 and/or BMP-18 sequences. The present invention may also include functional fragments of the human BMP-17 and BMP-18 proteins, and DNA sequences encoding such functional fragments, as well as functional fragments of other related proteins. The ability of such a fragment to function is determinable by assay of the protein in the biological assays described for the assay of the BMP-17 and BMP-18 proteins. DNA sequences encoding the complete mature human BMP-17 (SEQ ID NO: 1 and BMP-18 protein (SEQ ID NO:3) and the corresponding amino acid sequences (SEQ ID NO:2 and 4, respectively) are set forth herein. The BMP-17 and BMP-18 proteins of the present invention, such as human BMP-17 and BMP-18, may be produced by culturing a cell transformed with the correlating DNA sequence, such as the human BMP-17 and BMP-18 DNA sequen
Celeste Anthony J.
Murray Beth L.
Genetics Institute LLC
Gyure Barbara A.
Kemmerer Elizabeth C.
LandOfFree
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