USRE37462EExpiredUtility

Protein sequence of the plant toxin gelonin

71
Assignee: RES DEV FOUNDATIONPriority: Aug 14, 1990Filed: May 20, 1999Granted: Dec 4, 2001
Est. expiryAug 14, 2010(expired)· nominal 20-yr term from priority
C07K 14/415A61P 35/00C12N 15/62A61P 31/00C07K 2319/55A61P 37/06C07K 14/00
71
PatentIndex Score
20
Cited by
42
References
18
Claims

Abstract

This invention relates to substantially purified gelonin, toxic fragments thereof, the DNA sequences encoding gelonin and use of the DNA for producing, by recombinant technology, gelonin, toxic fragments thereof and fusion proteins. More specifically, the invention relates to the primary amino acid sequence of gelonin, and of the DNA encoding said gelonin and the production of synthetic gelonin and toxic fragments thereof.

Claims

exact text as granted — not AI-modified
What is claimed as new and is desired to be covered under Letters Patent is:  
     
       1. Substantially pure gelonin toxin having the amino acid sequence: 
       
         
           
                 
                 
                 
               
                     
                   GlyLeuAspThrValSerPheSerThrLys 
                   10 
                 
                     
                     
                 
                     
                   GlyAlaThrTyrIleThrTyrValAsnPhe 
                   20 
                 
                     
                     
                 
                     
                   LeuAsnGluLeuArgValLysLeuLysPro 
                   30 
                 
                     
                     
                 
                     
                   GluGlyAsnSerHisGlyIleProLeuLeu 
                   40 
                 
                     
                     
                 
                     
                   ArgLysGlyAspAspProGlyLysCysPhe 
                   50 
                 
                     
                     
                 
                     
                   ValLeuValAlaLeuSerAsnAspAsnGly 
                   60 
                 
                     
                     
                 
                     
                   GlnLeuAlaGluIleAlaIleAspValThr 
                   70 
                 
                     
                     
                 
                     
                   SerValTyrValValGlyTyrGlnValArg 
                   80 
                 
                     
                     
                 
                     
                   AsnArgSerTyrPhePheLysAspAlaPro 
                   90 
                 
                     
                     
                 
                     
                   AspAlaAlaTyrGluGlyLeuPheLysAsn 
                   100 
                 
                     
                     
                 
                     
                   ThrIleLysAsnProLeuLeuPheGlyGly 
                   110 
                 
                     
                     
                 
                     
                   LysThrArgLeuHisPheGlyGlySerTyr 
                   120 
                 
                     
                     
                 
                     
                   ProSerLeuGluGlyGluLysAlaTyrArg 
                   130 
                 
                     
                     
                 
                     
                   GluThrThrAspLeuGlyIleGluProLeu 
                   140 
                 
                     
                     
                 
                     
                   ArgIleGlyIleLysLysLeuAspGluAsn 
                   150 
                 
                     
                     
                 
                     
                   AlaIleAspAsnTyrLysProThrGluIle 
                   160 
                 
                     
                     
                 
                     
                   AlaSerSerLeuLeuValValIleGlnMet 
                   170 
                 
                     
                     
                 
                     
                   ValSerGluAlaAlaArgPheThrPheIle 
                   180 
                 
                     
                     
                 
                     
                   GluAsnGlnIleArgAsnAsnPheGlnGln 
                   190 
                 
                     
                     
                 
                     
                   ArgIleArgProAlaAsnAsnThrIleSer 
                   200 
                 
                     
                     
                 
                     
                   LeuGlnAsnLysTrpGlyLysLeuSerPhe 
                   210 
                 
                     
                     
                 
                     
                   GlnIleArgThrSerGlyAlaAsnGlyMet 
                   220 
                 
                     
                     
                 
                     
                   PheSerGluAlaValGluLeuGluArgAla 
                   230 
                 
                     
                     
                 
                     
                   AsnGlyLysLysTyrTyrValThrAlaVal 
                   240 
                 
                     
                     
                 
                     
                   AspGlnValLysProLysIleAlaLeuLeu 
                   250 
                 
                     
                     
                 
                     
                   LysPheValAspLysAspProGlu 
                   260 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       or a fragment or derivative thereof, said fragment or derivative having an activity which inhibits cellular protein synthesis but does not bind to a cell surface receptor. 
     
     
       2. A DNA sequence of the formula: 
       
         
           
                 
                 
                 
               
                   GGNYTNGAYA CNGTNWSNTT YWSNACNAAR GGNGCNACNT AYATHACNTA YGTNAAYTTY 
                   60 
                     
                 
                     
                 
                   YTNAAYGARY TNMGNGTNAA RYTNAARCCN GARGGNAAYW SNCAYGGNAT HCCNYTNYTN 
                   120 
                 
                     
                 
                   MGNAARGGNG AYGAYCCNGG NAARTGYTTY GTNYTNGTNG CNYTNWSNAA YGAYAAYGGN 
                   180 
                 
                     
                 
                   CARYTNGCNG ARATHGCNAT HGAYGTNACN WSNGTNTAYG TNGTNGGNTA YCARGTNMGN 
                   240 
                 
                     
                 
                   AAYMGNWSNT AYTTYTTYAA RGAYGCNCCN GAYGCNGCNT AYGARGGNYT NTTYAATAAY 
                   300 
                 
                     
                 
                   ACNATHAARA AYCCNYTNYT NTTYGGNGGN AARACNMGNY TNCAYTTYGG NGGNWSNTAY 
                   360 
                 
                     
                 
                   CCNWSNYTNG ARGGNGARAA RGCNTAYMGN GARACNACNG AYYTNGGNAT HGARCCNYTN 
                   420 
                 
                     
                 
                   MGNATHGGNA THAATAARYT NGAYGARAAY GCNATHGAYA AYTAYAARCC NACNGARATH 
                   480 
                 
                     
                 
                   GCNWSNWSNY TNYTNGTNGT NATHCARATG GTNWSNGARG CNGCNMGNTT YACNTTYATH 
                   540 
                 
                     
                 
                   GARAAYCARA THMGNAAYAA YTTYCARCAR MGNATHMGNC CNGCNAAYAA YACNATHWSN 
                   600 
                 
                     
                 
                   YTNGARAAYA ARTGGGGNAA RYTNWSNTTY CARATHMGNA CNWSNGGNGC NAAYGGNATG 
                   660 
                 
                     
                 
                   TTYWSNGARG CNGTNGARYT NGARMGNGCN AAYGGNAARA ARTAYTAYGT NACNGCNGTN 
                   720 
                 
                     
                 
                   GAYCARGTNA ARCCNAARAT HGCNYTNYTN AARTTYGTNG AYAARGAYCC NGAR 
                   774 
                 
                 
                 
                 
                 
               
                   wherein 
                   R =  A or G 
                   K =  G or T 
                   N =  any 
                 
                     
                   Y =  C or T 
                   M =  A or C 
                   S =  C or G 
                 
                     
                   B =  C, G, or T 
                   V =  A, C, or G 
                   W =  A or T 
                 
                     
                   D =  A, G, or T 
                   H =  A, C, or T 
                   X =  unknown 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
             
                
                
                
                
               
            
           
         
       
       or a fragment or derivative thereof, said fragment or derivative coding for gelonin or for a polypeptide having an activity which inhibits cellular protein synthesis but does not bind to a cell surface receptor. 
     
     
       3. The toxin of claim  1  further defined as having the amino acid sequence: 
       
         
           
                 
                 
               
                     
                   GlyLeuAspThrValSerPheSerThrLys 
                 
                     
                     
                 
                     
                   
                     GlyAlaThrTyrIleThrTyrValAsnPhe 
                   
                 
                     
                     
                 
                     
                   
                     LeuAsnGluLeuArgValLysLeuLysPro 
                   
                 
                     
                     
                 
                     
                   
                     GluGlyAsnSerHisGlyIleProLeuLeu 
                   
                 
                     
                     
                 
                     
                   
                     ArgLysGlyAspAspProGlyLysCysPhe 
                   
                 
                     
                     
                 
                     
                   
                     ValLeuValAlaLeuSerAsnAspAsnGly 
                   
                 
                     
                     
                 
                     
                   
                     GlnLeuAlaGluIleAlaIleAspValThr 
                   
                 
                     
                     
                 
                     
                   
                     SerValTyrValValGlyTyrGlnValArg 
                   
                 
                     
                     
                 
                     
                   
                     AsnArgSerTyrPhePheLysAspAlaPro 
                   
                 
                     
                     
                 
                     
                   
                     AspAlaAlaTyrGluGlyLeuPheLysAsn 
                   
                 
                     
                     
                 
                     
                   
                     ThrIleLysAsnProLeuLeuPheGlyGly 
                   
                 
                     
                     
                 
                     
                   
                     LysThrArgLeuHisPheGlyGlySerTyr 
                   
                 
                     
                     
                 
                     
                   
                     ProSerLeuGluGlyGluLysAlaTyrArg 
                   
                 
                     
                     
                 
                     
                   
                     GluThrThrAspLeuGlyIleGluProLeu 
                   
                 
                     
                     
                 
                     
                   
                     ArgIleGlyIleLysLysLeuAspGluAsn 
                   
                 
                     
                     
                 
                     
                   
                     AlaIleAspAsnTyrLysProThrGluIle 
                   
                 
                     
                     
                 
                     
                   
                     AlaSerSerLeuLeuValValIleGlnMet 
                   
                 
                     
                     
                 
                     
                   
                     ValSerGluAlaAlaArgPheThrPheIle 
                   
                 
                     
                     
                 
                     
                   
                     GluAsnGlnIleArgAsnAsnPheGlnGln 
                   
                 
                     
                     
                 
                     
                   
                     ArgIleArgProAlaAsnAsnThrIleSer 
                   
                 
                     
                     
                 
                     
                   
                     LeuGluAsnLysTrpGlyLysLeuSerPhe 
                   
                 
                     
                     
                 
                     
                   
                     GlnIleArgThrSerGlyAlaAsnGlyMet 
                   
                 
                     
                     
                 
                     
                   
                     PheSerGluAlaValGluLeuGluArgAla 
                   
                 
                     
                     
                 
                     
                   
                     AsnGlyLysLysTyrTyrValThrAlaVal 
                   
                 
                     
                     
                 
                     
                   
                     AspGlnValLysProLysIleAlaLeuLeu 
                   
                 
                     
                     
                 
                     
                   
                     LysPheValAspLysAspProGlu. 
                   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
       4. The DNA sequence of claim  2 , further defined as having the nucleotide sequence: 
       
         
           
                 
                 
                 
               
                   GGNYTNGAYA CNGTNWSNTT YWSNACNAAR GGNGCNACNT AYATHACNTA YGTNAAYTTY 
                   
                     60 
                   
                     
                 
                     
                 
                   
                     YTNAAYGARY TNMGNGTNAA RYTNAARCCN GARGGNAAYW SNCAYGGNAT HCCNYTNYTN 
                   
                   
                     120 
                   
                 
                     
                 
                   
                     MGNAARGGNG AYGAYCCNGG NAARTGYTTY GTNYTNGTNG CNYTNWSNAA YGAYAAYGGN 
                   
                   
                     180 
                   
                 
                     
                 
                   
                     CARYTNGCNG ARATHGCNAT HGAYGTNACN WSNGTNTAYG TNGTNGGNTA YCARGTNMGN 
                   
                   
                     240 
                   
                 
                     
                 
                   
                     AAYMGNWSNT AYTTYTTYAA RGAYGCNCCN GAYGCNGCNT AYGARGGNYT NTTYAATAAY 
                   
                   
                     300 
                   
                 
                     
                 
                   
                     ACNATHAARA AYCCNYTNYT NTTYGGNGGN AARACNMGNY TNCAYTTYGG NGGNWANTAY 
                   
                   
                     360 
                   
                 
                     
                 
                   
                     CCNWSNYTNG ARGGNGARAA RGCNTAYMGN GARACNACNG AYYTNGGNAT HGARCCNYTN 
                   
                   
                     420 
                   
                 
                     
                 
                   
                     MGNATHGGNA THAARAARYT NGAYGARAAY GCNATHGAYA AYTAYAARCC NACNGARATH 
                   
                   
                     480 
                   
                 
                     
                 
                   
                     GCNWSNWSNY TNYTNGTNGT NATHCARATG GTNWSNGARG CNGCNMGNTT YACNTTYATH 
                   
                   
                     540 
                   
                 
                     
                 
                   
                     GARAAYCARA THMGNAAYAA YTTYCARCAR MGNATHMGNC CNGCNAAYAA YACNATHWSN 
                   
                   
                     600 
                   
                 
                     
                 
                   
                     YTNGARAAYA ARTGGGGNAA RYTNWSNTTY CARATHMGNA CNWSNGGNGC NAAYGGNATG 
                   
                   
                     660 
                   
                 
                     
                 
                   
                     TTYWSNGARG CNGTNGARYT NGARMGNGCN AAYGGNAARA ARTAYTAYGT NACNGCNGTN 
                   
                   
                     720 
                   
                 
                     
                 
                   
                     GAYCARGTNA ARCCNAARAT HGCNYTNYTN AARTTYGTNG AYAARGAYCC NGAR 
                   
                   
                     774 
                   
                 
                 
                 
                 
                 
               
                   
                     wherein 
                   
                   
                     R =  or G 
                   
                   
                     K =  G or T 
                   
                   
                     N =  any 
                   
                 
                     
                   
                     Y =  C or T 
                   
                   
                     M =  A or C 
                   
                   
                     S =  C or G 
                   
                 
                     
                   
                     B =  C, G, or T 
                   
                   
                     V =  A, C, or G 
                   
                   
                     W =  A or T 
                   
                 
                     
                   
                     D =  A, G, or T 
                   
                   
                     H =  A, C, or T 
                   
                   
                     X =  unknown. 
                   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
             
                
                
                
                
               
            
           
         
       
     
     
       5. The gelonin toxin of claim  1  or claim  3 , further defined as a fusion protein that includes a functional modality in addition to said toxin. 
     
     
       6. The gelonin toxin of claim  5  wherein the fusion protein is prepared by conjugation of the functional modality to the toxin. 
     
     
       7. The gelonin toxin of claim  5 , wherein the fusion protein is prepared by recombinant technology. 
     
     
       8. The gelonin toxin of claim  5 , wherein said functional modality is an antibody. 
     
     
       9. The gelonin toxin of claim  5 , wherein the functional modality is a targeting ligand. 
     
     
       10. The gelonin toxin of claim  9 , wherein the targeting ligand is IL-   2 .   
     
     
       11. The gelonin toxin of claim  9 , wherein the targeting ligand is EGF. 
     
     
       12. The gelonin toxin of claim  9 , wherein the targeting ligand is IFN. 
     
     
       13. The gelonin toxin of claim  5 , wherein the functional modality is a second toxic activity. 
     
     
       14. The gelonin toxin of claim  13 , wherein said second toxic activity is diptheria toxin action. 
     
     
       15. The gelonin toxin of claim  5  wherein the functional modality is TNF. 
     
     
       16. The gelonin toxin of claim  5 , wherein the functional modality is a chemotherapeutic agent. 
     
     
       17. The gelonin toxin of claim  5 , wherein the functional modality is a radioisotope. 
     
     
       18. The gelonin toxin of claim  1  or claim  3 , further defined as lacking carbohydrate groups.

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