US4604234A — Protein having cell growth stimulating action, co…

Claim 1

Independent Claim 243 words

A protein having cell growth stimulating activity, said protein selected from those obtained from a culture of a microorganism selected from the group consisting of Staphylococcus epidermidis, Bacillus subtilis, Streptococcus faecalis, Serratia marcescens, Escherichia coli, Salmonella enteritidis, and Proteus vulgaris, the protein being characterized as follows: (a) Molecular weight in the range of 5,000-12,000, when said molecular weight is calculated on the basis of a calibration curve plotting the relationship between the molecular weight and an amount eluted by high performance liquid chromatography using as marker proteins, thioglobin (M.W. 670,000), bovine γ-globulin (M.W. 158,000), fowl ovalbumin (M.W. 44,000), equine myoglobulin (M.W. 17,000), and vitamin B-12 (M.W. 1,350); (b) Ultraviolet absorption at 278 nm; (c) Infrared absorption spectrum with dominant peaks at 1300, 1550, and 1630 cm -1 ; (d) Isoelectric point in the range of 4.0-6.6; (e) Soluble in water and insoluble in methanol, ethanol, acetone and ether; (f) Appears as a grayish white powder in the lyophilized state; (g) Positive for the following color reactions; Xanthoprotein, Pauli, Sakaguchi, Hunter, Sullivan, Hopkins-Cove, nitrosonaphthol, Millon, Neubauer-Road, and Libermann; and (h) ______________________________________ Amino Acid mol/M ______________________________________ Aspartic Acid 4.25-14.33 Glutamic Acid 4.76-21.35 Glycine 3.68-10.04 Valine 3.05-8.97 Alanine 3.51-18.09 Threonine 2.20-5.69 Serine 2.01-5.14 Cystine 0.00-0.40 Methionine 0.79-2.73 Isoleucine 2.34-7.97 Leucine 2.70-8.70 Tyrosine 0.39-3.17 Phenylalanine 0.79-3.20 Lysine 2.22-8.80 Histidine 0.54-1.35 Arginine 0.65-4.83 Proline 2.63-5.37 Tryptophan 0.25-0.70 ______________________________________ said protein exhibiting cell growth stimulating activity in a pH range between 4-10; and being substantially free of nucleoside phosphotransferase.

Claim 2

Dependent Claim 243 words

A protein having cell growth stimulating activity, said protein selected from those obtained from a culture of a microorganism selected from the group consisting of Staphylococcus epidermidis, Bacillus subtilis, Streptococcus faecalis, Serratia marcescens, Escherichia coli, Salmonella enteritidis, and Proteus vulgaris, the protein being characterized as follows: (a) Molecular weight in the range of 5,000-12,000, when said molecular weight is calculated on the basis of a calibration curve plotting the relationship between the molecular weight and an amount eluted by high performance liquid chromatography using as marker proteins, thioglobin (M.W. 670,000), bovine γ-globulin (M.W. 158,000), fowl ovalbumin (M.W. 44,000), equine myoglobulin (M.W. 17,000), and vitamin B-12 (M.W. 1,350); (b) Ultraviolet absorption at 278 nm; (c) Infrared absorption spectrum with dominant peaks at 1300, 1550, and 1630 cm -1 ; (d) Isoelectric point in the range of 4.0-6.6; (e) Soluble in water and insoluble in methanol, ethanol, acetone and ether; (f) Appears as a grayish white powder in the lyophilized state; (g) Positive for the following color reactions; Xanthoprotein, Pauli, Sakaguchi, Hunter, Sullivan, Hopkins-Cove, nitrosonaphthol, Millon, Neubauer-Road, and Libermann; and (h) ______________________________________ Amino Acid mol/M ______________________________________ Aspartic Acid 4.25-14.33 Glutamic Acid 4.76-21.35 Glycine 3.68-10.04 Valine 3.05-8.97 Alanine 3.51-18.09 Threonine 2.20-5.69 Serine 2.01-5.14 Cystine 0.00-0.40 Methionine 0.79-2.73 Isoleucine 2.34-7.97 Leucine 2.70-8.70 Tyrosine 0.39-3.17 Phenylalanine 0.79-3.20 Lysine 2.22-8.80 Histidine 0.54-1.35 Arginine 0.65-4.83 Proline 2.63-5.37 Tryptophan 0.25-0.70 ______________________________________ said protein exhibiting cell growth stimulating activity in a pH range between 4-10; and being substantially free of nucleoside phosphotransferase.

Claim 3

Dependent Claim 21 words

A composition for stimulating cell growth comprising the protein of claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.

Claim 4

Dependent Claim 12 words

The composition according to claim 2, wherein the carrier is an ointment.

Claim 5

Dependent Claim 20 words

The composition according to claim 3, wherein the protein is present in an amount of about 0.0025-0.25 percent by weight.

Claim 6

Dependent Claim 12 words

The composition according to claim 2, and further comprising a zinc compound.

Claim 7

Dependent Claim 26 words

The composition according to claim 5, and wherein said zinc compound is selected from the group consisting of zinc oxide and zinc salts of fatty acids.

Claim 8

Dependent Claim 38 words

The composition according to claim 6, wherein the zinc compound is zinc oxide, and the ratio of protein to zinc oxide is in the range of from 1:2000 to 1:8000, calculated on the basis of percent by weight.

Claim 9

Dependent Claim 47 words

The composition according to claim 6, and wherein the zinc compound is a zinc salt of a fatty acid, and the ratio of protein to zinc salt of fatty acid, is in the range of from 1:400 to 1:4000, calculated on the basis of percent by weight.

Claim 10

Dependent Claim 13 words

The composition according to claim 2, and further comprising an adrenal cortical hormone.

Claim 11

Dependent Claim 29 words

The composition according to claim 9, and wherein the ratio of protein to adrenal cortical hormone is from 1:1 to 1:10, calculated on the basis of percent by weight.

Claim 12

Dependent Claim 228 words

A protein having cell growth stimulating activity, said protein selected from those obtained from a culture of a microorganism selected from the group consisting of Staphylococcus aureus, Sarcina lutea, Aerococcus viridians, Acinetobacter calcoaceticus, Corynebacterium fascians, and Clostridium perfringens; the protein being characterized as follows: (a) Molecular weight in the range 10,000-15,000, when said molecular weight is calculated on the basis of a calibration curve plotting the relationship between the molecular weight and an amount eluted by high performance liquid chromatography using as marker proteins, thioglobin (M.W. 670,000), bovine γ-globulin (M.W. 158,000), fowl ovalbumin (M.W. 44,000), equine myoglobin (M.W. 17,000), and vitamin B-12 (M.W. 1,350); (b) Ultraviolet absorption at 278 nm; (c) Infrared absorption spectrum with dominant peaks at 1300, 1550, and 1630 cm -1 ; (d) Soluble in water and insoluble in methanol, ethanol, acetone and ether; (e) Appears as a grayish white powder in the lyophilized state; (f) Positive for the following color reactions: Xanthoprotein, Pauli, Sakaguchi, Hunter, Sullivan; Hopkins-Cole, nitrosonaphthol, Millon, Neubauer-Road, and Liebermann; (g) Isoelectric point in the range of 4.0-5.5; and (h) ______________________________________ Amino Acid mol/M ______________________________________ Aspartic Acid 9.65-18.78 Glutamic Acid 12.36-43.08 Glycine 6.71-11.78 Valine 6.43-12.16 Alanine 8.07-14.68 Threonine 4.60-7.72 Serine 4.56-11.45 Cystine 0.00 Methionine 1.46-2.86 Isoleucine 3.98-6.77 Leucine 4.97-8.13 Tyrosine 0.62-2.63 Phenylalanine 1.75-3.44 Lysine 4.53-8.95 Histidine 1.04-1.78 Arginine 0.80-2.33 Proline 4.27-12.69 Tryptophan 0.32-0.63 ______________________________________ said protein being substantially free of nucleoside phosphotransferase.

Claim 13

Dependent Claim 228 words

A protein having cell growth stimulating activity, said protein selected from those obtained from a culture of a microorganism selected from the group consisting of Staphylococcus aureus, Sarcina lutea, Aerococcus viridians, Acinetobacter calcoaceticus, Corynebacterium fascians, and Clostridium perfringens; the protein being characterized as follows: (a) Molecular weight in the range 10,000-15,000, when said molecular weight is calculated on the basis of a calibration curve plotting the relationship between the molecular weight and an amount eluted by high performance liquid chromatography using as marker proteins, thioglobin (M.W. 670,000), bovine γ-globulin (M.W. 158,000), fowl ovalbumin (M.W. 44,000), equine myoglobin (M.W. 17,000), and vitamin B-12 (M.W. 1,350); (b) Ultraviolet absorption at 278 nm; (c) Infrared absorption spectrum with dominant peaks at 1300, 1550, and 1630 cm -1 ; (d) Soluble in water and insoluble in methanol, ethanol, acetone and ether; (e) Appears as a grayish white powder in the lyophilized state; (f) Positive for the following color reactions: Xanthoprotein, Pauli, Sakaguchi, Hunter, Sullivan; Hopkins-Cole, nitrosonaphthol, Millon, Neubauer-Road, and Liebermann; (g) Isoelectric point in the range of 4.0-5.5; and (h) ______________________________________ Amino Acid mol/M ______________________________________ Aspartic Acid 9.65-18.78 Glutamic Acid 12.36-43.08 Glycine 6.71-11.78 Valine 6.43-12.16 Alanine 8.07-14.68 Threonine 4.60-7.72 Serine 4.56-11.45 Cystine 0.00 Methionine 1.46-2.86 Isoleucine 3.98-6.77 Leucine 4.97-8.13 Tyrosine 0.62-2.63 Phenylalanine 1.75-3.44 Lysine 4.53-8.95 Histidine 1.04-1.78 Arginine 0.80-2.33 Proline 4.27-12.69 Tryptophan 0.32-0.63 ______________________________________ said protein being substantially free of nucleoside phosphotransferase.

Claim 14

Dependent Claim 21 words

A composition for stimulating cell growth comprising the protein of claim 11 as an active ingredient, and a pharmaceutically acceptable carrier.

Claim 15

Dependent Claim 12 words

The composition according to claim 12, wherein the carrier is an ointment.

Claim 16

Dependent Claim 20 words

The composition according to claims 13, wherein the protein is present in an amount of about 0.0025-0.25 percent by weight.

Claim 17

Dependent Claim 12 words

The composition according to claim 12, and further comprising a zinc compound.

Claim 18

Dependent Claim 26 words

The composition according to claim 15, and wherein said zinc compound is selected from the group consisting of zinc oxide and zinc salts of fatty acids.

Claim 19

Dependent Claim 38 words

The composition according to claim 16, wherein the zinc compound is zinc oxide and the ratio of protein to zinc oxide is in the range of from 1:2000 to 1:8000, calculated on the basis of percent by weight.

Claim 20

Dependent Claim 47 words

The composition according to claim 16, and wherein the zinc compound is a zinc salt of a fatty acid and the ratio of protein to zinc salt of fatty acid is in the range of from 1:400 to 1:4000, calculated on the basis of percent by weight.

Claim 21

Dependent Claim 13 words

The composition according to claim 12, and further comprising an adrenal cortical hormone.

Claim 22

Dependent Claim 29 words

The composition according to claim 19, and wherein the ratio of protein to adrenal cortical hormone is from 1:1 to 1:10, calculated on the basis of percent by weight.

Claim 23

Dependent Claim 214 words

A protein having cell growth stimulating activity, said protein selected from those obtained from a culture of the microorganism Clostridium perfringens, the protein being characterized as follows: (a) Molecular weight of 150,000-160,000, when said molecular weight is calculated on the basis of a calibration curve plotting the relationship between the molecular weight and an amount eluted by high performance liquid chromatography using as marker proteins, thioglobin (M.W. 670,000), bovine γ-globulin (M.W. 158,000), fowl ovalbumin (M.W. 44,000), equine myoglobulin (M.W. 17,000), and vitamin B-12 (M.W. 1,350); and a molecular weight of approximately 53,000, when determined by SDS disk electrophoresis; (b) Ultraviolet absorption at 278 nm; (c) Infrared absorption spectrum with dominant peaks at 1300, 1550, and 1630 cm -1 ; (d) Soluble in water and insoluble in methanol, ethanol, acetone and ether; (e) Appears as a grayish white powder in the lyophilized state; (f) Positive for the following color reactions: Xanthoprotein, Pauli, Sakaguchi, Hunter, Sullivan; Hopkins-Cole, nitrosonaphthol, millon, Neubauer-Road, and Liebermann; and (g) ______________________________________ Amino Acid mol/M ______________________________________ Aspartic Acid 109.80 Glutamic Acid 96.18 Glycine 71.66 Valine 73.01 Alanine 73.69 Threonine 40.39 Serine 45.59 Cystine 2.92 Methionine 16.56 Isoleucine 54.64 Leucine 65.42 Tyrosine 54.29 Phenylalanine 27.43 Lysine 75.66 Histidine 10.48 Arginine 21.37 Proline 32.55 Tryptophan 3.36 ______________________________________ said protein being substantially free of nucleoside phosphotransferase.

Claim 24

Dependent Claim 214 words

A protein having cell growth stimulating activity, said protein selected from those obtained from a culture of the microorganism Clostridium perfringens, the protein being characterized as follows: (a) Molecular weight of 150,000-160,000, when said molecular weight is calculated on the basis of a calibration curve plotting the relationship between the molecular weight and an amount eluted by high performance liquid chromatography using as marker proteins, thioglobin (M.W. 670,000), bovine γ-globulin (M.W. 158,000), fowl ovalbumin (M.W. 44,000), equine myoglobulin (M.W. 17,000), and vitamin B-12 (M.W. 1,350); and a molecular weight of approximately 53,000, when determined by SDS disk electrophoresis; (b) Ultraviolet absorption at 278 nm; (c) Infrared absorption spectrum with dominant peaks at 1300, 1550, and 1630 cm -1 ; (d) Soluble in water and insoluble in methanol, ethanol, acetone and ether; (e) Appears as a grayish white powder in the lyophilized state; (f) Positive for the following color reactions: Xanthoprotein, Pauli, Sakaguchi, Hunter, Sullivan; Hopkins-Cole, nitrosonaphthol, millon, Neubauer-Road, and Liebermann; and (g) ______________________________________ Amino Acid mol/M ______________________________________ Aspartic Acid 109.80 Glutamic Acid 96.18 Glycine 71.66 Valine 73.01 Alanine 73.69 Threonine 40.39 Serine 45.59 Cystine 2.92 Methionine 16.56 Isoleucine 54.64 Leucine 65.42 Tyrosine 54.29 Phenylalanine 27.43 Lysine 75.66 Histidine 10.48 Arginine 21.37 Proline 32.55 Tryptophan 3.36 ______________________________________ said protein being substantially free of nucleoside phosphotransferase.

Claim 25

Dependent Claim 21 words

A composition for stimulating cell growth comprising the protein of claim 21 as an active ingredient, and a pharmaceutically acceptable carrier.

Claim 26

Dependent Claim 12 words

The composition according to claim 22, wherein the carrier is an ointment.

Claim 27

Dependent Claim 20 words

The composition according to claim 23, wherein the protein is present in an amount of about 0.0025-0.25 percent by weight.

Claim 28

Dependent Claim 12 words

The composition according to claim 22, and further comprising a zinc compound.

Claim 29

Dependent Claim 26 words

The composition according to claim 25, and wherein said zinc compound is selected from the group consisting of zinc oxide and zinc salts of fatty acids.

Claim 30

Dependent Claim 38 words

The composition according to claim 26, wherein the zinc compound is zinc oxide and the ratio of protein to zinc oxide is in the range of from 1:2000 to 1:8000, calculated on the basis of percent by weight.

Claim 31

Dependent Claim 47 words

The composition according to claim 26, and wherein the zinc compound is a zinc salt of a fatty acid and the ratio of protein to zinc salt of fatty acid is in the range of from 1:400 to 1:4000, calculated on the basis of percent by weight.

Claim 32

Dependent Claim 13 words

The composition according to claim 22, and further comprising an adrenal cortical hormone.

Claim 33

Dependent Claim 29 words

The composition according to claim 29, and wherein the ratio of protein to adrenal cortical hormone is from 1:1 to 1:10, calculated on the basis of percent by weight.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1989-08-04	1990-02-03	1990-08-05	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1993-08-04	1994-02-03	1994-08-05	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1997-08-04	1998-02-03	1998-08-05	$7700.00	Window Closed / Lapsed

Protein having cell growth stimulating action, composition thereof and method for producing the same

Abstract

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