                                SEQUENCE LISTING

<110> Bachran, Christopher H.
      Leppla, Stephen H.
      Liu, Shihui
      Morley, Thomas
      The Government of the United States of America
         as represented by The Secretary of the
         Department of Health and Human Services

<120> Ubiquitin Fusions for Improving the
  Efficacy of Cytosolic Acting Targeted Toxins
  

<130> 77867-834975

<140> WO Not yet assigned 
<141> 2012-04-06  

<150> US 61/473,450       
<151> 2011-04-08  

<160> 22

<170> FastSEQ for Windows Version 4.0

<210> 1
<211> 76
<212> PRT
<213> Homo sapiens

<220> 
<223> human wild-type ubiquitin (Ub)

<400> 1
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
 1               5                  10                  15      
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
            20                  25                  30          
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
        35                  40                  45              
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
    50                  55                  60                  
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75      


<210> 2
<211> 76
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic modified ubiquitin Ub-K48

<400> 2
Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu
 1               5                  10                  15      
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp
            20                  25                  30          
Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
        35                  40                  45              
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu
    50                  55                  60                  
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75      


<210> 3
<211> 76
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic modified ubiquitin Ub-K63

<400> 3
Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu
 1               5                  10                  15      
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp
            20                  25                  30          
Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg
        35                  40                  45              
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
    50                  55                  60                  
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75      


<210> 4
<211> 76
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic modified ubiquitin Ub-Knull

<400> 4
Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu
 1               5                  10                  15      
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp
            20                  25                  30          
Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg
        35                  40                  45              
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu
    50                  55                  60                  
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75      


<210> 5
<211> 76
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic modified ubiquitin Ub-UN

<400> 5
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
 1               5                  10                  15      
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
            20                  25                  30          
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
        35                  40                  45              
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
    50                  55                  60                  
Ser Thr Leu His Leu Val Leu Arg Leu Arg Ala Ala
65                  70                  75      


<210> 6
<211> 9
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic variable portion of LFn-PEIII (FP59)
      fusion protein with linker

<400> 6
Ser Gly Gly Gly Ser Ala Glu Phe Leu
 1               5                  


<210> 7
<211> 86
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic variable portion of ubiquitin (Ub)
      fusion protein with linker

<400> 7
Gly Gly Gly Ser Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
 1               5                  10                  15      
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
            20                  25                  30          
Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
        35                  40                  45              
Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
    50                  55                  60                  
Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75                  80  
Gly Ser Ala Glu Phe Leu
                85      


<210> 8
<211> 86
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic variable portion of Ub-UN fusion protein
      with linker

<400> 8
Gly Gly Gly Ser Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
 1               5                  10                  15      
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
            20                  25                  30          
Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
        35                  40                  45              
Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
    50                  55                  60                  
Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Ala Ala
65                  70                  75                  80  
Ala Ser Ala Glu Phe Leu
                85      


<210> 9
<211> 86
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic variable portion of Ub-K48 fusion
      protein with linker

<400> 9
Gly Gly Gly Ser Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr
 1               5                  10                  15      
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala
            20                  25                  30          
Arg Ile Gln Asp Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
        35                  40                  45              
Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
    50                  55                  60                  
Ile Gln Arg Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75                  80  
Gly Ser Ala Glu Phe Leu
                85      


<210> 10
<211> 86
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic variable portion of Ub-K63 fusion
      protein with linker

<400> 10
Gly Gly Gly Ser Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr
 1               5                  10                  15      
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala
            20                  25                  30          
Arg Ile Gln Asp Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
        35                  40                  45              
Phe Ala Gly Arg Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
    50                  55                  60                  
Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75                  80  
Gly Ser Ala Glu Phe Leu
                85      


<210> 11
<211> 86
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic variable portion of Ub-Knull fusion
      protein with linker

<400> 11
Gly Gly Gly Ser Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr
 1               5                  10                  15      
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala
            20                  25                  30          
Arg Ile Gln Asp Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile
        35                  40                  45              
Phe Ala Gly Arg Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
    50                  55                  60                  
Ile Gln Arg Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly
65                  70                  75                  80  
Gly Ser Ala Glu Phe Leu
                85      


<210> 12
<211> 255
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic cytosol-targeting moiety, N-terminal section of
      Bacillus anthracis anthrax toxin lethal factor (LFn)

<400> 12
Ala Gly Gly His Gly Asp Val Gly Met His Val Lys Glu Lys Glu Lys
 1               5                  10                  15      
Asn Lys Asp Glu Asn Lys Arg Lys Asp Glu Glu Arg Asn Lys Thr Gln
            20                  25                  30          
Glu Glu His Leu Lys Glu Ile Met Lys His Ile Val Lys Ile Glu Val
        35                  40                  45              
Lys Gly Glu Glu Ala Val Lys Lys Glu Ala Ala Glu Lys Leu Leu Glu
    50                  55                  60                  
Lys Val Pro Ser Asp Val Leu Glu Met Tyr Lys Ala Ile Gly Gly Lys
65                  70                  75                  80  
Ile Tyr Ile Val Asp Gly Asp Ile Thr Lys His Ile Ser Leu Glu Ala
                85                  90                  95      
Leu Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile Tyr Gly Lys Asp Ala
            100                 105                 110         
Leu Leu His Glu His Tyr Val Tyr Ala Lys Glu Gly Tyr Glu Pro Val
        115                 120                 125             
Leu Val Ile Gln Ser Ser Glu Asp Tyr Val Glu Asn Thr Glu Lys Ala
    130                 135                 140                 
Leu Asn Val Tyr Tyr Glu Ile Gly Lys Ile Leu Ser Arg Asp Ile Leu
145                 150                 155                 160 
Ser Lys Ile Asn Gln Pro Tyr Gln Lys Phe Leu Asp Val Leu Asn Thr
                165                 170                 175     
Ile Lys Asn Ala Ser Asp Ser Asp Gly Gln Asp Leu Leu Phe Thr Asn
            180                 185                 190         
Gln Leu Lys Glu His Pro Thr Asp Phe Ser Val Glu Phe Leu Glu Gln
        195                 200                 205             
Asn Ser Asn Glu Val Gln Glu Val Phe Ala Lys Ala Phe Ala Tyr Tyr
    210                 215                 220                 
Ile Glu Pro Gln His Arg Asp Val Leu Gln Leu Tyr Ala Pro Glu Ala
225                 230                 235                 240 
Phe Asn Tyr Met Asp Arg Phe Asn Glu Gln Glu Ile Asn Leu Ser
                245                 250                 255 


<210> 13
<211> 216
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic effector moiety, catalytic domain of
      Pseudomonas exotoxin A (PEIII)

<400> 13
Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly
 1               5                  10                  15      
Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu
            20                  25                  30          
Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu
        35                  40                  45              
Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp
    50                  55                  60                  
Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu
65                  70                  75                  80  
Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile
                85                  90                  95      
Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro
            100                 105                 110         
Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly
        115                 120                 125             
Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala
    130                 135                 140                 
Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly
145                 150                 155                 160 
Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr
                165                 170                 175     
Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp
            180                 185                 190         
Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly
        195                 200                 205             
Lys Pro Pro Arg Glu Asp Leu Lys
    210                 215     


<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic HIV protease cleavage sequence,
      recognition sequence

<400> 14
Gly Ser Gly Ile Phe Leu Glu Thr Ser Leu
 1               5                  10  


<210> 15
<211> 5
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic native Psudomonas exotoxin A C-terminus

<400> 15
Arg Glu Asp Leu Lys
 1               5  


<210> 16
<211> 5
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic ubiquitin fusion protein construct
      N-terminal sequence

<400> 16
His Met Ala Gly Gly
 1               5  


<210> 17
<211> 4
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic cell-surface furin-like protease
      cleavage sequence

<400> 17
Arg Lys Lys Arg
 1              


<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic linker with Cys positioned C-terminal of
      di-glycine motif at C-terminus of ubiquitin to provide
      spacer between ubiquitin and effector moiety

<400> 18
Ala Gly Gly Gly Gly Ser Cys
 1               5          


<210> 19
<211> 10
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic construct N-terminal 10 X histidine tag

<400> 19
His His His His His His His His His His
 1               5                  10  


<210> 20
<211> 5
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic enterokinase cleavage site

<400> 20
Asp Asp Asp Asp Lys
 1               5  


<210> 21
<211> 4
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic linker at C-terminus of LFn

<400> 21
Gly Gly Gly Ser
 1              


<210> 22
<211> 263
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic effector domain of Haemophilus ducreyi
      cytolethal distending toxin B (CdtB)

<400> 22
Ser Gly Asn Leu Ser Asp Phe Lys Val Ala Thr Trp Asn Leu Gln Gly
 1               5                  10                  15      
Ser Ser Ala Val Asn Glu Ser Lys Trp Asn Ile Asn Val Arg Gln Leu
            20                  25                  30          
Leu Ser Gly Glu Gln Gly Ala Asp Ile Leu Met Val Gln Glu Ala Gly
        35                  40                  45              
Ser Leu Pro Ser Ser Ala Val Arg Thr Ser Arg Val Ile Gln His Gly
    50                  55                  60                  
Gly Thr Pro Ile Glu Glu Tyr Thr Trp Asn Leu Gly Thr Arg Ser Arg
65                  70                  75                  80  
Pro Asn Met Val Tyr Ile Tyr Tyr Ser Arg Leu Asp Val Gly Ala Asn
                85                  90                  95      
Arg Val Asn Leu Ala Ile Val Ser Arg Arg Gln Ala Asp Glu Ala Phe
            100                 105                 110         
Ile Val His Ser Asp Ser Ser Val Leu Gln Ser Arg Pro Ala Val Gly
        115                 120                 125             
Ile Arg Ile Gly Thr Asp Val Phe Phe Thr Val His Ala Leu Ala Thr
    130                 135                 140                 
Gly Gly Ser Asp Ala Val Ser Leu Ile Arg Asn Ile Phe Thr Thr Phe
145                 150                 155                 160 
Asn Ser Ser Ser Ser Pro Pro Glu Arg Arg Val Tyr Ser Trp Met Val
                165                 170                 175     
Val Gly Asp Phe Asn Arg Ala Pro Ala Asn Leu Glu Val Ala Leu Arg
            180                 185                 190         
Gln Glu Pro Ala Val Ser Glu Asn Thr Ile Ile Ile Ala Pro Thr Glu
        195                 200                 205             
Pro Thr His Arg Ser Gly Asn Ile Leu Asp Tyr Ala Ile Leu His Asp
    210                 215                 220                 
Ala His Leu Pro Arg Arg Glu Gln Ala Arg Glu Arg Ile Gly Ala Ser
225                 230                 235                 240 
Leu Met Leu Asn Gln Leu Arg Ser Gln Ile Thr Ser Asp His Phe Pro
                245                 250                 255     
Val Ser Phe Val Arg Asp Arg
            260             



