EP0385060A2
Expired / Lapsed
EP
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Purified ciliary neurotrophic factor
Assignee: Syntex Synergen Neuroscience Joint Venture
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Filing: 1990-01-05
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Granted: —
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Expires: 2010-01-05
Abstract
Abstract A ciliary neurotrophic factor (CNTF), particularly sciatic nerve CNTF (SN-CNTF) is claimed. The SN-CNTF described herein is a single protein species and has a specific activity that increased to greater than 25,000-fold from crude extract. Amino acid data for this SN-CNTF is also provided. In addition, methods for using this data for providing SN-CNTF probes and for screening cDNA and genomic libraries are also provided. Recombinant-DNA methods for the production of SN-CNTF are described. Nucleic acid sequences encoding rabbit and human CNTF are provided. A recombinant expression system is provided for producing biologically active CNTF.
Inventors
- 👤 Franklin D. Collins
- 👤 Leu-Fen Lin
- 👤 Drzislav Mismer
- 👤 Christine Ko
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-08 03:57
CPC Classifications
A; A61; A61K; A61K38/00; A61K38/16; A61K38/17; A61K38/22; A61K38/27; C; C07; C07K; C07K14/00; C07K14/435; C07K14/475; A61P; A61P25/00; A61P25/28; C07K16/00; C07K16/18; C07K16/22; C12; C12N; C12N15/00; C12N15/09; C12N15/11
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
30 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) which has a specific activity more than 25,000 times greater than the specific activity of the natural sciatic nerve extract.
Claim 2
Dependent Claim
30 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) which has a specific activity more than 25,000 times greater than the specific activity of the natural sciatic nerve extract.
Claim 3
Dependent Claim
21 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) which has an activity of greater than 2 x 10⁸ TU/mg.
Claim 4
Dependent Claim
21 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) which has an activity of greater than 2 x 10⁸ TU/mg.
Claim 5
Dependent Claim
11 words
A substantially purified protein rabbit sciatic nerve ciliary neurotrophic factor (SN-CNTF).
Claim 6
Dependent Claim
11 words
A substantially purified protein rabbit sciatic nerve ciliary neurotrophic factor (SN-CNTF).
Claim 7
Dependent Claim
11 words
A substantially purified protein human sciatic nerve ciliary neurotrophic factor (SN-CNTF).
Claim 8
Dependent Claim
11 words
A substantially purified protein human sciatic nerve ciliary neurotrophic factor (SN-CNTF).
Claim 9
Dependent Claim
51 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) comprising a protein characterized by the following: (a) a molecular weight of about 25,000 on SDS-PAGE; (b) a specific activity of greater than 2 x 10⁸ Trophic Units per mg; (c) resistance of the biological activity to SDS and reducing agents.
Claim 10
Dependent Claim
51 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) comprising a protein characterized by the following: (a) a molecular weight of about 25,000 on SDS-PAGE; (b) a specific activity of greater than 2 x 10⁸ Trophic Units per mg; (c) resistance of the biological activity to SDS and reducing agents.
Claim 11
Dependent Claim
16 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) comprised of a single protein species.
Claim 12
Dependent Claim
16 words
A substantially purified protein sciatic nerve ciliary neurotrophic factor (SN-CNTF) comprised of a single protein species.
Claim 13
Independent Claim
48 words
A method for purifying sciatic nerve ciliary neurotrophic factor (SN-CNTF) from sciatic nerve extract comprising: subjecting the nerve extract preparation to acid treatment and ammonium sulfate fractionation; chromatofocussing the preparation subjected to acid treatment and ammonium sulfate fractionation; running the preparation on SDS-Page gel; and performing reverse phase-HPLC.
Claim 14
Independent Claim
48 words
A method for purifying sciatic nerve ciliary neurotrophic factor (SN-CNTF) from sciatic nerve extract comprising: subjecting the nerve extract preparation to acid treatment and ammonium sulfate fractionation; chromatofocussing the preparation subjected to acid treatment and ammonium sulfate fractionation; running the preparation on SDS-Page gel; and performing reverse phase-HPLC.
Claim 15
Dependent Claim
42 words
The method of claim 7 which includes, prior to said steps of running the preparation on an SDS-PAGE gel, additional purification steps comprising: hydrophobic interaction chromatography between ammonium sulfate fractionation and chromatofocussing; and FPLC hydrophobic interaction chromatography between chromatofocussing and preparative SDS-PAGE.
Claim 16
Dependent Claim
13 words
The nucleic acid sequence encoding rabbit SN-CNTF as set forth in Fig. 11.
Claim 17
Dependent Claim
13 words
The nucleic acid sequence encoding rabbit SN-CNTF as set forth in Fig. 11.
Claim 18
Dependent Claim
13 words
The amino acid sequence of rabbit SN-CNTF as set forth in Fig. 11.
Claim 19
Dependent Claim
13 words
The amino acid sequence of rabbit SN-CNTF as set forth in Fig. 11.
Claim 20
Dependent Claim
13 words
The nucleic acid sequence encoding human CNTF as set forth in Fig. 12.
Claim 21
Dependent Claim
13 words
The nucleic acid sequence encoding human CNTF as set forth in Fig. 12.
Claim 22
Dependent Claim
13 words
The amino acid sequence encoding human CNTF as set forth in Fig. 12.
Claim 23
Dependent Claim
13 words
The amino acid sequence encoding human CNTF as set forth in Fig. 12.
Claim 24
Dependent Claim
11 words
A recombinant animal cell expression system for producing biologically active CNTF.
Claim 25
Dependent Claim
11 words
A recombinant animal cell expression system for producing biologically active CNTF.
Claim 26
Dependent Claim
10 words
A bacterial cell expression system for producing biologically active CNTF.
Claim 27
Dependent Claim
10 words
A bacterial cell expression system for producing biologically active CNTF.
Claim 28
Dependent Claim
14 words
The CNTF of claim 4 wherein said CNTF is produced by recombinant DNA methods.
Claim 29
Dependent Claim
118 words
A recombinant DNA method for the production of CNTF comprising: (a) Preparation of a DNA sequence capable of directing a host cell to produce a protein having CNTF activity; (b) Cloning the DNA sequence into a vector capable of being transferred into and replicated in a host cell, such vector containing operational elements needed to express the DNA sequence; (c) Transferring the vector containing the synthetic DNA sequence and operational elements into a host cell capable of expressing the DNA encoding CNTF; (d) Culturing the host cells under conditions appropriate to amplification of the vector and expression of CNTF; and (e) Harvesting CNTF; and (f) Permitting CNTF to assume an active tertiary structure whereby it possess CNTF activity.
Claim 30
Dependent Claim
118 words
A recombinant DNA method for the production of CNTF comprising: (a) Preparation of a DNA sequence capable of directing a host cell to produce a protein having CNTF activity; (b) Cloning the DNA sequence into a vector capable of being transferred into and replicated in a host cell, such vector containing operational elements needed to express the DNA sequence; (c) Transferring the vector containing the synthetic DNA sequence and operational elements into a host cell capable of expressing the DNA encoding CNTF; (d) Culturing the host cells under conditions appropriate to amplification of the vector and expression of CNTF; and (e) Harvesting CNTF; and (f) Permitting CNTF to assume an active tertiary structure whereby it possess CNTF activity.
Claim 31
Dependent Claim
16 words
The method of claim 16 wherein said DNA encodes the amino acid sequence of human CNTF.
Claim 32
Dependent Claim
16 words
The method of claim 17 wherein said DNA sequence includes the nucleic acids present in CNTF-Syn1/3.
Claim 33
Dependent Claim
16 words
The method of claim 17 wherein said DNA sequence includes the nucleic acids present in CNTF-Syn2/3.
Claim 34
Dependent Claim
41 words
The method of claim 16 wherein said DNA sequence encodes a physiologically functional CNTF comprising a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently similar to CNTF to allow possession of at least one of CNTF's biological properties.
Claim 35
Dependent Claim
12 words
The method of claim 16 wherein said host cell is a microorganism.
Claim 36
Dependent Claim
12 words
The method of claim 21 wherein said microorganism is E. coli .
Claim 37
Dependent Claim
12 words
The method of claim 16 wherein said host cells are mammalian cells.
Claim 38
Dependent Claim
14 words
The method of claim 23 wherein said mammalian cells are Chinese Hampster Ovary cells.
Claim 39
Dependent Claim
5 words
A substantially purified recombinant CNTF.
Claim 40
Dependent Claim
5 words
A substantially purified recombinant CNTF.
References Cited (Backward Citations)
No citation records indexed.
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