US2003004319A1PendingUtilityA1

Mammalian growth factor

Assignee: UNIV NEW YORKPriority: Jun 16, 1987Filed: Aug 27, 2001Published: Jan 2, 2003
Est. expiryJun 16, 2007(expired)· nominal 20-yr term from priority
C07K 14/50
47
PatentIndex Score
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Claims

Abstract

A mammalian growth factor, displaying homology to both basic and acidic fibroblast growth factor in a single polypeptide chain, is disclosed herein. The growth factor was isolated from cells transfected with the DNA extracted from Kaposi's Sarcoma cells.

Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . A polypeptide having substantial homology to each of basic and acidic fibroblast growth factor said polypeptide having growth factor activity and having an amino acid sequence consisting essentially of the amino acid sequence of the expression product of a fragment of an oncogene isolated from Kaposi's sarcoma cells.  
     
     
         2 . A polypeptide comprising the amino acid sequence:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu 
                 
                     
                     
                 
                     
                   Pro Ala Val Leu Leu Ala Leu Leu Ala Pro Trp Ala 
                 
                     
                     
                 
                     
                   Gly Arg Gly Gly Ala Ala Ala Pro Thr Ala Pro Asn 
                 
                     
                     
                 
                     
                   Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 
                 
                     
                     
                 
                     
                   Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val 
                 
                     
                     
                 
                     
                   Ala Ala Gln Pro Lys Glu Ala Ala Val Gln Ser Gly 
                 
                     
                     
                 
                     
                   Ala Gly Asp Tyr Leu Leu Gly Ile Lys Arg Leu Arg 
                 
                     
                     
                 
                     
                   Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu 
                 
                     
                     
                 
                     
                   Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His 
                 
                     
                     
                 
                     
                   Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu Ser Pro 
                 
                     
                     
                 
                     
                   Val Glu Arg Gly Val Val Ser Ile Phe Gly Val Ala 
                 
                     
                     
                 
                     
                   Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 
                 
                     
                     
                 
                     
                   Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr 
                 
                     
                     
                 
                     
                   Phe Lys Glu Ile Leu Leu Pro Asn Asn Tyr Asn Ala 
                 
                     
                     
                 
                     
                   Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala 
                 
                     
                     
                 
                     
                   Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 
                 
                     
                     
                 
                     
                   Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro 
                 
                     
                     
                 
                     
                   Arg Leu. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         3 . A gene coding for the polypeptide of  claim 1  or 2.  
     
     
         4 . A polypeptide comprising the amino acid sequence  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile Lys Arg 
                 
                     
                     
                 
                     
                   Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe 
                 
                     
                     
                 
                     
                   His Leu Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly 
                 
                     
                     
                 
                     
                   Ala His Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu 
                 
                     
                     
                 
                     
                   Ser Pro Val Glu Arg Gly Val Val Ser Ile Phe Gly 
                 
                     
                     
                 
                     
                   Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys 
                 
                     
                     
                 
                     
                   Gly Lys Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu 
                 
                     
                     
                 
                     
                   Cys Thr Phe Lys Glu Ile Leu Leu Pro Asn Asn Tyr 
                 
                     
                     
                 
                     
                   Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe 
                 
                     
                     
                 
                     
                   Ile Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly 
                 
                     
                     
                 
                     
                   Asn Arg Val Ser Pro Thr Met Lys Val Thr His Phe 
                 
                     
                     
                 
                     
                   Leu Pro Arg Leu. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         5 . The polypeptide of  claim 1  comprising the amino acid sequence:  
       
         
           
                 
                 
               
                     
                 
                   Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val 
                     
                 
                     
                 
                   Leu Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala 
                 
                     
                 
                   Ala Pro Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg 
                 
                     
                 
                   Arg Trp Glu Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val 
                 
                     
                 
                   Ala Ala Gln Pro Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp 
                 
                     
                 
                   Tyr Leu Leu Gly Ile Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val 
                 
                     
                 
                   Gly Ile Gly Phe His Leu Gln Ala Leu Pro Asp Gly Arg Ile Gly 
                 
                     
                 
                   Gly Ala His Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu Ser Pro 
                 
                     
                 
                   Val Glu Arg Gly Val Val Ser Ile Phe Gly Val Ala Ser Arg Phe 
                 
                     
                 
                   Phe Val Ala Met Ser Ser Lys Gly Lys Leu Tyr Gly Ser Pro Phe 
                 
                     
                 
                   Phe Thr Asp Glu Cys Thr Phe Lys Glu Ile Leu Leu Pro Asn Asn 
                 
                     
                 
                   Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala 
                 
                     
                 
                   Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg Val Ser Pro 
                 
                     
                 
                   Thr Met Lys Val Thr His Phe Leu Pro Arg Leu. 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         6 . The polypeptide of  claim 1  wherein said homology to each of said acidic and basic growth factors is at least about 35%.  
     
     
         7 . The polypeptide of  claim 6  herein said homology is at least about 60% taking conservative substitutions into account.  
     
     
         8 . The polypeptide of  claim 5  consisting essentially of said amino acid sequence.  
     
     
         9 . A purified, isolated DNA fragment comprising the nucleotide sequence:  
       
         
           
                 
                 
               
                     
                 
                   ATG TCG GGG CCC GGG ACG GCC GCG GTA GCG CTG CTC CCG GCG GTC 
                     
                 
                     
                 
                   CTG CTG GCC TTG CTG GCG CCC TGG GCG GGC CGA GGG GGC GCC GCC 
                 
                     
                 
                   GCA CCC ACT GCA CCC AAC GGC ACG CTG GAG GCC GAG CTG GAG CGC 
                 
                     
                 
                   CGC TGG GAG AGC CTG GTG GCG CTC TCG TTG GCG CGC CTG CCG GTG 
                 
                     
                 
                   GCA GCG CAG CCC AAG GAG GCG GCC GTC CAG AGC GGC GCC GGC GAC 
                 
                     
                 
                   TAC CTG CTG GGC ATC AAG CGG CTG CGG CGG CTC TAC TGC AAC GTG 
                 
                     
                 
                   GGC ATC GGC TTC CAC CTC CAG GCG CTC CCC GAC GGC CGC ATC GGC 
                 
                     
                 
                   GGC GCG CAC GCG GAC ACC CGC GAC AGC CTG CTG GAG CTC TCG CCC 
                 
                     
                 
                   GTG GAG CGG GGC GTG GTG AGC ATC TTC GGC GTG GCC AGC CGG TTC 
                 
                     
                 
                   TTC GTG GCC ATG AGC AGC AAG GGC AAG CTC TAT GGC TCG CCC TTC 
                 
                     
                 
                   TTC ACC GAT GAG TGC ACG TTC AAG GAG ATT CTC CTT CCC AAC AAC 
                 
                     
                 
                   TAC AAC GCC TAC GAG TCC TAC AAG TAC CCC GGC ATG TTC ATC GCC 
                 
                     
                 
                   CTG AGC AAG AAT GGG AAG ACC AAG AAG GGG AAC CGA GTG TCG CCC 
                 
                     
                 
                   ACC ATG AAG GTC ACC CAC TTC CTC CCC AGG CTG 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and coding for a mammalian growth factor.  
     
     
         10 . A method for healing wounds or burns in mammals comprising administering to mammals in need of such treatment a cell-growth promoting effective amount of the polypeptide of  claim 1 .  
     
     
         11 . The method of  claim 10  wherein said polypeptide comprises the amino acid sequence:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu 
                 
                     
                     
                 
                     
                   Pro Ala Val Leu Leu Ala Leu Leu Ala Pro Trp Ala 
                 
                     
                     
                 
                     
                   Gly Arg Gly Gly Ala Ala Ala Pro Thr Ala Pro Asn 
                 
                     
                     
                 
                     
                   Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 
                 
                     
                     
                 
                     
                   Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val 
                 
                     
                     
                 
                     
                   Ala Ala Gln Pro Lys Glu Ala Ala Val Gln Ser Gly 
                 
                     
                     
                 
                     
                   Ala Gly Asp Tyr Leu Leu Gly Ile Lys Arg Leu Arg 
                 
                     
                     
                 
                     
                   Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu 
                 
                     
                     
                 
                     
                   Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly Ala His 
                 
                     
                     
                 
                     
                   Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu Ser Pro 
                 
                     
                     
                 
                     
                   Val Glu Arg Gly Val Val Ser Ile Phe Gly Val Ala 
                 
                     
                     
                 
                     
                   Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 
                 
                     
                     
                 
                     
                   Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr 
                 
                     
                     
                 
                     
                   Phe Lys Glu Ile Leu Leu Pro Asn Asn Tyr Asn Ala 
                 
                     
                     
                 
                     
                   Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala 
                 
                     
                     
                 
                     
                   Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 
                 
                     
                     
                 
                     
                   Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro 
                 
                     
                     
                 
                     
                   Arg Leu. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         12 . The method of  claim 10  comprising topically administering said polypeptide.  
     
     
         13 . The method of  claim 10  comprising an angiogenesis-promoting effective amount of said polypeptide.  
     
     
         14 . A pharmaceutical formulation for promoting healing of wounds or burns in mammals comprising as an active ingredient a cell-growth promoting effective amount of a member selected from the group consisting of the polypeptide of  claim 1  and physiologically acceptable salts thereof.  
     
     
         15 . The formulation of  claim 14  further comprising a physiologically acceptable carrier.  
     
     
         16 . The pharmaceutical formulation of  claim 14  further comprising a water-miscible antibacterial agent.  
     
     
         17 . The pharmaceutical formulation of  claim 14  comprising between about 0.01 micrograms and about 10 micrograms of said polypeptide.  
     
     
         18 . A method for promoting cell growth in a cell culture comprising introducing into said culture a cell growth-promoting effective amount of the polypeptide of  claim 1 .  
     
     
         19 . A polypeptide comprising the amino acid sequence:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   X - Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu 
                 
                     
                     
                 
                     
                   Glu Arg Arg Trp Glu Ser Leu Val Ala Leu Ser Leu 
                 
                     
                     
                 
                     
                   Ala Arg Leu Pro Val Ala Ala Gln Pro Lys Glu Ala 
                 
                     
                     
                 
                     
                   Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly 
                 
                     
                     
                 
                     
                   Ile Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly 
                 
                     
                     
                 
                     
                   Ile Gly Phe His Leu Gln Ala Leu Pro Asp Gly Arg 
                 
                     
                     
                 
                     
                   Ile Gly Gly Ala His Ala Asp Thr Arg Asp Ser Leu 
                 
                     
                     
                 
                     
                   Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser 
                 
                     
                     
                 
                     
                   Ile Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met 
                 
                     
                     
                 
                     
                   Ser Ser Lys Gly Lys Leu Tyr Gly Ser Pro Phe Phe 
                 
                     
                     
                 
                     
                   Thr Asp Glu Cys Thr Phe Lys Glu Ile Leu Leu Pro 
                 
                     
                     
                 
                     
                   Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro 
                 
                     
                     
                 
                     
                   Gly Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr 
                 
                     
                     
                 
                     
                   Lys Lys Gly Asn Arg Val Ser Pro Thr Met Lys Val 
                 
                     
                     
                 
                     
                   Thr His Phe Leu Pro Arg Leu; 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       wherein X is selected from the group consisting of Ala-Pro or Pro.  
     
     
         20 . A method for healing wounds or burns in mammals comprising administering to mammals in need of such treatment a cell-growth promoting effective amount of the polypeptide of  claim 19 .  
     
     
         21 . The method of  claim 20  comprising topically administering said growth factor.  
     
     
         22 . A pharmaceutical formulation for promoting healing of wounds or burns in mammals comprising as an active ingredient a cell-growth promoting effective amount of a member selected from the group consisting of the polypeptide of  claim 19  and physiologically acceptable salts thereof.  
     
     
         23 . The formulation of  claim 22  further comprising a physiologically acceptable carrier.  
     
     
         24 . The pharmaceutical formulation of  claim 22  further comprising a water-miscible antibacterial agent.  
     
     
         25 . The pharmaceutical formulation of  claim 22  comprising between about 0.01 micrograms and about 10 micrograms of said polypeptide.  
     
     
         26 . A method for promoting cell growth in a cell culture comprising introducing into said culture a cell growth-promoting effective amount of the polypeptide of  claim 19 .  
     
     
         27 . The method of  claim 20  further comprising administering an amount of heparin effective to stabilize said polypeptide.  
     
     
         28 . The formulation of  claim 24  further comprising an amount of heparin effective to stabilize said polypeptide.  
     
     
         29 . The polypeptide of  claim 19  wherein said polypeptide is further characterized by having the ability to induce the production of plasminogen activator in endothelial cells in culture.  
     
     
         30 . A procaryotic or eukaryotic host cell transformed or transfected with a DNA sequence according to  claim 9  in a manner allowing the host cell to express a polypeptide having substantial homology to each of acidic and basic fibroblast growth factor activity and having an amino acid sequence consisting essentially of the amino acid sequence of the expression product of a fragment of an oncogene isolated from Kaposi's Sarcoma cells.  
     
     
         31 . A procaryotic or eukaryotic host cell stably transformed with a DNA vector according to  claim 30 .  
     
     
         32 . A purified and isolated DNA sequence consisting essentially of a DNA sequence encoding a polypeptide having substantial homology to each of acidic and basic fibroblast growth factor activity and having an amino acid sequence consisting essentially of the amino acid sequence of the expression product of a fragment of an oncogene isolated from Kaposi's Sarcoma cells.  
     
     
         33 . A transformed or transfected COS cell according to  claim 31 .  
     
     
         34 . A transformed or transfected N3H3T3 cell according to  claim 31 .  
     
     
         35 . Transformed or transfected  E. coli  cell according to claim  31 .

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