Children's Medical Center Corporation

Takeda Chemical Industries, Inc.

Yuen Shing

Moses J Folkman

Koichi Igarashi

A recombinant non-glycosylated mammalian growth factor (BTC-GF) stimulates proliferation of human smooth muscle cells. Especially, the amino acid sequence of the protein deduced from the nucleotide sequence coding for human BTC-GF is as that comprising the amino acids No. 1 to No. 147 of FIG. 10 (SEQ ID NO:18) or the amino acids No. 1 to 80 of FIG. 10, (SEQ ID NO:18) and the amino acid sequence of the protein deduced from the nucleotide sequence coding for mouse BTC-GF is as that comprising the amino acids No. 1 to No. 146 of FIG. 9 (SEQ ID NO:17).

Smooth muscle mitogen and isolated DNA coding therefor

A recombinant non-glycosylated mammalian growth factor (BTC-GF) stimulates proliferation of human smooth muscle cells. Especially, the amino acid sequence of the protein deduced from the nucleotide sequence coding for human BTC-GF is as that comprising the amino acids No. 1 to No. 147 of FIG. 10 (SEQ ID NO:5) or the amino acids No. 1 to 80 of FIG. 10 (SEQ ID NO:18), and the amino acid sequence of the protein deduced from the nucleotide sequence coding for mouse BTC-GF is as that comprising the amino acids No. 1 to No. 146 of FIG. 9 (SEQ ID NO:17).

Smooth muscle mitogen peptides and DNA coding therefor

A novel growth factor (BTC-GF) was purified from the conditioned medium of pancreatic beta tumor cells initially derived from transgenic mice (RIPI-Tag2). The purification scheme included BioRex 70 chromatography, phenyl-Sepharose chromatography, TSL-GEL heparin FPLC and C4 reverse phase HPLC. The peptide also stimulated proliferation of bovine smooth muscle cells. It was not inactivated by boiling, by 10 mM dithiothreitol or by exposure to 1M acetic acid. Biological activity of BTC-GF was recovered from a single band of protein which had a molecular weight of 32,000 on SDS-PAGE. The partial N-terminal amino acid sequence of this protein from BTC-3 and BTC-J10 cells was determined with an ABI 470A protein sequencer to be: ##STR1## An internal region amino acid sequence of BTC-GF was determined to be: ##STR2##

Smooth muscle mitogen

A novel growth factor (BTC-CG) was purified from the conditioned medium of pancreatic beta tumor cells initially derived from transgenic mice (RIPl-Tag2). The purification scheme included BioRex 70 chromatography, phenyl-Sepharose chromatography, TSL-GEL heparin FPLC and C4 reverse phase HPLC. The peptide also stimulated proliferation of bovine smooth muscle cells. It was not inactivated by boiling, by 10 mM dithiothreitol or by exposure to IM acetic acid. Biological activity of BTC-GF was recovered from a single band of protein which had a molecular weight of 2,000 on SDS-PAGE. The Partial N-terminal amino acid sequence of this protein was determined with an ABI 470A protein sequencer as: Asp-Gly-[X]-Thr-[X]-Arg-Thr-Pro-Glu-[X]-Asn-Gly.

Methods for stabilizing fibroblast growth factor (FGF) in heat, acid and/or proteolytic environments are provided. Such methods comprise combining or otherwise complexing FGF with a salt of sucrose octasulfate (SOS) and in particular with the aluminum (sucralfate) or potassium salts thereof. The present invention also provides FGF-stable compositions comprising SOS and FGF. Such compositions can be used in the treatment of FGF-responsive diseases including the treatment of wounds and ulcerative conditions of the gastrointestinal tract. The present invention also provides various diagnostic protocols employing SOS as well as diagnostic kits with SOS as a component thereof. Finally, the present invention provides methods of purifying FGF with SOS.

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Randolph, MA, United States of America

Yuen Shing