The Salk Institute for Biological Studies

Frederick S Esch

Nicholas C Ling

Roger C Guillemin

Peter Bohlen

Shao-Yao Ying

Andrew J Baird

Denis Gospodarowicz

Paul E Brazeau, Jr

Naoto Ueno

Shunichi Shimasaki

Jean E Rivier

Wylie W Vale, Jr

Joachim Spiess

Joan Vaughan

William B Wehrenberg

Paul E Brazeau

Anne Corrigan

Richard A McClintock

Substantially pure mammalian basic fibroblast growth factors are produced. The amino acid residue sequences of bovine and human bFGF are disclosed as well as a DNA chain encoding the polypeptide of the bovine species. By appropriately inserting a synthesized DNA chain into a cloning vector and using the cloning vector to transform cells, synthetic bovin bFGF can be obtained from transformed cell lines, both prokaryotic and eukaryotic.

Bovine basic fibroblast growth factor

Two follistatin proteins with inhibin-like activity were isolated from porcine follicular fluid using heparin-Sepharose affinity chromatography, followed by gel filtration on Sephacryl S-200 and then six steps of high-performance liquid chromatography. The larger protein has 315 residues and is believed to be glycosylated. The smaller protein is a 288-residue, C-terminally shortened version thereof. These proteins specifically inhibit basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system. The half-maximal effective dose for both is 2.5-6.0 ng/ml. Human and rat follistatins exhibit very high homology with the porcine protein, with the human differing from porcine in only 4 residues out of 315 and with the rat differing from porcine in only 8 residues out of 315. Using the porcine amino acid sequence information, cDNA clones encoding these proteins were identified from a porcine ovarian cDNA library. Then, using the porcine cDNA as a probe, the cloning and sequencing of the corresponding human and rat proteins were accomplished.

DNA encoding follistatin

Substantially pure mammalian basic fibroblast growth factors are produced. The amino acid residue sequences of bovine and human bFGF are disclosed as well as a DNA chain encoding the polypeptide of the bovine species. By appropriately inserting a synthesized DNA chain into a cloning vector and using the cloning vector to transform cells, synthetic bFGF can be obtained from transformed cell lines, both prokaryotic and eukaryotic.

Basic fibroblast growth factor

Two follistatin proteins with inhibin-like activity were isolated from porcine follicular fluid using heparin-Sepharose affinity chromatography, followed by gel filtration on Sephacryl S-200 and then six steps of high-performance liquid chromatography. Each isolated molecule is a monomer having a molecular weight of at least about 32,000 daltons. Microsequencing revealed the NH.sub.2 -terminal portions both to be Gly-Asn-Cys-Trp-Leu-Arg-Gln-Ala-Lys-Asn-Gly-Arg-Cys-Gln-Val-Leu. The larger protein has 315 residues and is believed to be glycosylated. The smaller protein is a 288-residue, C-terminally shortened version thereof. These proteins specifically inhibit basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system. The Half-maximal effective dose for both is 2.5-6.0 ng/ml. Human and rat follistatins exhibit very high homology with the porcine protein, with the human differing from porcine in only 4 residues out of 315 and with the rat differing from porcine in only 8 residues out of 315.

Mammalian follistatin

A 28,000-dalton protein with FSH-releasing activity is isolated from porcine follicular fluid in a multi-step procedure including gel filtration, ion exchange chromatography and several reverse-phase high-performance liquid chromatography steps. The 28 kD protein promotes secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system, exhibiting an EC.sub.50 of 0.5 ng/ml. These peptides are dimers of inhibin .beta.-chains of mammals, and homodimers or heterodimers of the .beta..sub.A and .beta..sub.B chains are biologically active to regulate fertility of various mammalian species, including humans.

FSH-releasing peptides

Substantially pure bovine pituitary fibroblast growth factor, a 146 amino acid residue polypeptide, is produced. The amino acid residue sequence of bpFGF is disclosed as well as a DNA chain encoding the polypeptide. By appropriately inserting a synthesized DNA chain into a cloning vector and using the cloning vector to transform cells, synthetic bpFGF can be obtained from transformed cell lines, both prokaryotic and eukaryotic.

Bovine fibroblast growth factor

The invention provides synthetic peptides which are extremely potent in stimulating the release of pituitary GH in mammals and which have the formula: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser- Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu -Gln-Gly-Ala-Arg-Val-Arg-Leu-Y wherein Y is OH or NH.sub.2. These peptides or biologically active fragments thereof, or analogs thereof having well-known substitutions and, or additions, as well as nontoxic salts of any of the foregoing, may be administered therapeutically to mammals, including humans, and may be used diagnostically. The peptides are useful in stimulating the release of GH and accelerating growth in warm-blooded non-human animals, particularly pigs, and in improving aquiculture.

Porcine GRF

A synthetic peptide is extremely potent in stimulating the release of pituitary GH in mammals, particularly in sheep, since it is the replicate of the native (hormone) releasing factor of the sheep hypothalamus. It contains the following sequence: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Al a-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-G ln-Gly-Ala-Lys-Val-Arg-Leu. This peptide or a biologically active fragment thereof, or analogs thereof having well-known substitutions and/or additions, as well as nontoxic salts of any of the foregoing, may be administered therapeutically to mammals and may be used diagnostically. The peptide is particularly useful in stimulating the release of GH so as to accelerate growth in warm-blooded non-human animals, particularly sheep, and/or to improve aquiculture.

Ovine GRF

The invention provides synthetic peptides which are extremely potent in stimulating the release of pituitary GH in mammals and which have the formula: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser- Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu -Gln-Gly-Ala-Lys-Val-Arg-Leu-Y wherein Y is OH or NH.sub.2. These peptides or biologically active fragments thereof, or analogs thereof having well-known substitutions and/or additions, as well as nontoxic salts of any of the foregoing, may be administered therapeutically to mammals, including humans, and may be used diagnostically. The peptides are useful in stimulating the release of GH so as to accelerate growth in warm-blooded non-human animals, particularly cattle, and/or to increase the production of milk in lactating cows, and also in improving aquiculture.

Bovine GRF

PGRF has been synthesized. The invention provides synthetic peptides which are extremely potent in stimulating the release of pituitary GH in mammals and which have the formula: ##STR1## wherein R is OH or NH.sub.2, R.sub.34 is Ser or Ala, R.sub.38 is Arg or Ser, R.sub.40 is Ala or Arg and R.sub.41 is Arg, Arg-Ala, Arg-Ala-Arg, Arg-Ala-Arg-Leu or des-R.sub.41. These peptides or biologically active fragments thereof, or analogs thereof having well-known substitutions and/or additions, as well as nontoxic salts of any of the foregoing, may be administered therapeutically to mammals, including humans, and may be used diagnostically.

Mammalian PGRF

A substantially purified substance has been isolated from bovine gonadal secretions which mediates pituitary hormone secretion, using an extraction and purification sequence that includes gel filtration, partition chromatography and HPLC in that order. The substance, designated gonadostatin, is a moderate-size peptide which below a threshold level selectively inhibits LRF-stimulated pituitary secretion of LH and FSH. Above a threshold level, gonadostatin generally stimulates pituitary hormone secretion.

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Foster City, CA, United States of America

Frederick S Esch