                         SEQUENCE LISTING

<110>  KINDRED BIOSCIENCES, INC.
 
<120>  VARIANT ASPARAGINASE POLYPEPTIDES FOR MEDICAL USE

<130>  01157-0013-00PCT

<150>  US 62/660,189
<151>  2018-04-19

<160>  15    

<170>  PatentIn version 3.5

<210>  1
<211>  348
<212>  PRT
<213>  Dickeya chrysanthemi

<400>  1

Met Glu Arg Trp Phe Lys Ser Leu Phe Val Leu Val Leu Phe Phe Val 
1               5                   10                  15      


Phe Thr Ala Ser Ala Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala 
            20                  25                  30          


Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr 
        35                  40                  45              


Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val 
    50                  55                  60                  


Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn 
65                  70                  75                  80  


Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln 
                85                  90                  95      


Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile 
            100                 105                 110         


Thr His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu 
        115                 120                 125             


Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro 
    130                 135                 140                 


Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val 
145                 150                 155                 160 


Arg Val Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val 
                165                 170                 175     


Ile Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala 
            180                 185                 190         


Ser Thr Leu Asp Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val 
        195                 200                 205             


Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His 
    210                 215                 220                 


Thr Thr Arg Ser Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys 
225                 230                 235                 240 


Val Asp Ile Leu Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp 
                245                 250                 255     


Ala Ala Ile Gln His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly 
            260                 265                 270         


Ala Gly Ser Val Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu 
        275                 280                 285             


Glu Lys Gly Val Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile 
    290                 295                 300                 


Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn 
305                 310                 315                 320 


Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser 
                325                 330                 335     


Asp Pro Lys Val Ile Gln Glu Tyr Phe His Thr Tyr 
            340                 345             


<210>  2
<211>  327
<212>  PRT
<213>  Erwinia chrysanthemi

<400>  2

Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr Ile 
1               5                   10                  15      


Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala Gly 
            20                  25                  30          


Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val Pro Glu Val Lys Lys 
        35                  40                  45              


Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu Asn 
    50                  55                  60                  


Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu Leu 
65                  70                  75                  80  


Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr Asp 
                85                  90                  95      


Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser Asp 
            100                 105                 110         


Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile Ser 
        115                 120                 125             


Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly Asp 
    130                 135                 140                 


Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg Ile 
145                 150                 155                 160 


Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp Thr 
                165                 170                 175     


Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn Arg 
            180                 185                 190         


Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser Val 
        195                 200                 205             


Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu Tyr 
    210                 215                 220                 


Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln His 
225                 230                 235                 240 


Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val Ser 
                245                 250                 255     


Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val Val 
            260                 265                 270         


Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp Glu 
        275                 280                 285             


Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala Arg 
    290                 295                 300                 


Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val Ile 
305                 310                 315                 320 


Gln Glu Tyr Phe His Thr Tyr 
                325         


<210>  3
<211>  328
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase with no leader sequence for 
       intracellular expression

<400>  3

Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 
1               5                   10                  15      


Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 
            20                  25                  30          


Gly Ala Leu Gly Val Asp Thr Leu Ile Asn Ala Val Pro Glu Val Lys 
        35                  40                  45              


Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 
    50                  55                  60                  


Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu 
65                  70                  75                  80  


Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 
                85                  90                  95      


Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 
            100                 105                 110         


Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 
        115                 120                 125             


Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 
    130                 135                 140                 


Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 
145                 150                 155                 160 


Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 
                165                 170                 175     


Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 
            180                 185                 190         


Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 
        195                 200                 205             


Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 
    210                 215                 220                 


Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 
225                 230                 235                 240 


His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 
                245                 250                 255     


Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val 
            260                 265                 270         


Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 
        275                 280                 285             


Glu Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 
    290                 295                 300                 


Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 
305                 310                 315                 320 


Ile Gln Glu Tyr Phe His Thr Tyr 
                325             


<210>  4
<211>  327
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase mature sequence with 3 
       cysteine substitutions

<400>  4

Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr Ile 
1               5                   10                  15      


Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala Gly 
            20                  25                  30          


Ala Leu Gly Val Asp Thr Leu Ile Cys Ala Val Pro Glu Val Lys Lys 
        35                  40                  45              


Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu Asn 
    50                  55                  60                  


Met Thr Gly Asp Val Val Leu Cys Leu Ser Gln Arg Val Asn Glu Leu 
65                  70                  75                  80  


Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr Asp 
                85                  90                  95      


Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser Asp 
            100                 105                 110         


Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile Ser 
        115                 120                 125             


Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly Asp 
    130                 135                 140                 


Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg Ile 
145                 150                 155                 160 


Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp Thr 
                165                 170                 175     


Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn Arg 
            180                 185                 190         


Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser Val 
        195                 200                 205             


Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu Tyr 
    210                 215                 220                 


Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln His 
225                 230                 235                 240 


Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val Ser 
                245                 250                 255     


Val Arg Gly Ile Ala Gly Met Arg Cys Ala Leu Glu Lys Gly Val Val 
            260                 265                 270         


Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp Glu 
        275                 280                 285             


Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala Arg 
    290                 295                 300                 


Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val Ile 
305                 310                 315                 320 


Gln Glu Tyr Phe His Thr Tyr 
                325         


<210>  5
<211>  336
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase mature sequence with 3 
       cysteine substitutions and N-terminal polyHis tag

<400>  5

Met Ala His His His His His His His Ala Asp Lys Leu Pro Asn Ile 
1               5                   10                  15      


Val Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly 
            20                  25                  30          


Thr Gln Thr Thr Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu 
        35                  40                  45              


Ile Cys Ala Val Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu 
    50                  55                  60                  


Gln Phe Ser Asn Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu 
65                  70                  75                  80  


Cys Leu Ser Gln Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val Asp 
                85                  90                  95      


Gly Val Val Ile Thr His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr 
            100                 105                 110         


Phe Leu His Leu Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala 
        115                 120                 125             


Ala Met Arg Pro Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu 
    130                 135                 140                 


Leu Glu Ala Val Arg Val Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly 
145                 150                 155                 160 


Val Met Val Val Ile Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr 
                165                 170                 175     


Lys Thr Asn Ala Ser Thr Leu Asp Thr Phe Arg Ala Asn Glu Glu Gly 
            180                 185                 190         


Tyr Leu Gly Val Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile 
        195                 200                 205             


Asp Lys Leu His Thr Thr Arg Ser Val Phe Asp Val Arg Gly Leu Thr 
    210                 215                 220                 


Ser Leu Pro Lys Val Asp Ile Leu Tyr Gly Tyr Gln Asp Asp Pro Glu 
225                 230                 235                 240 


Tyr Leu Tyr Asp Ala Ala Ile Gln His Gly Val Lys Gly Ile Val Tyr 
                245                 250                 255     


Ala Gly Met Gly Ala Gly Ser Val Ser Val Arg Gly Ile Ala Gly Met 
            260                 265                 270         


Arg Cys Ala Leu Glu Lys Gly Val Val Val Met Arg Ser Thr Arg Thr 
        275                 280                 285             


Gly Asn Gly Ile Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser 
    290                 295                 300                 


Asp Ser Leu Asn Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu 
305                 310                 315                 320 


Thr Arg Thr Ser Asp Pro Lys Val Ile Gln Glu Tyr Phe His Thr Tyr 
                325                 330                 335     


<210>  6
<211>  336
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase sequence with N-terminal 
       polyHis tag

<400>  6

Met Ala His His His His His His His Ala Asp Lys Leu Pro Asn Ile 
1               5                   10                  15      


Val Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Ser Ala Ala Thr Gly 
            20                  25                  30          


Thr Gln Thr Thr Gly Tyr Lys Ala Gly Ala Leu Gly Val Asp Thr Leu 
        35                  40                  45              


Ile Asn Ala Val Pro Glu Val Lys Lys Leu Ala Asn Val Lys Gly Glu 
    50                  55                  60                  


Gln Phe Ser Asn Met Ala Ser Glu Asn Met Thr Gly Asp Val Val Leu 
65                  70                  75                  80  


Lys Leu Ser Gln Arg Val Asn Glu Leu Leu Ala Arg Asp Asp Val Asp 
                85                  90                  95      


Gly Val Val Ile Thr His Gly Thr Asp Thr Val Glu Glu Ser Ala Tyr 
            100                 105                 110         


Phe Leu His Leu Thr Val Lys Ser Asp Lys Pro Val Val Phe Val Ala 
        115                 120                 125             


Ala Met Arg Pro Ala Thr Ala Ile Ser Ala Asp Gly Pro Met Asn Leu 
    130                 135                 140                 


Leu Glu Ala Val Arg Val Ala Gly Asp Lys Gln Ser Arg Gly Arg Gly 
145                 150                 155                 160 


Val Met Val Val Ile Asn Asp Arg Ile Gly Ser Ala Arg Tyr Ile Thr 
                165                 170                 175     


Lys Thr Asn Ala Ser Thr Leu Asp Thr Phe Arg Ala Asn Glu Glu Gly 
            180                 185                 190         


Tyr Leu Gly Val Ile Ile Gly Asn Arg Ile Tyr Tyr Gln Asn Arg Ile 
        195                 200                 205             


Asp Lys Leu His Thr Thr Arg Ser Val Phe Asp Val Arg Gly Leu Thr 
    210                 215                 220                 


Ser Leu Pro Lys Val Asp Ile Leu Tyr Gly Tyr Gln Asp Asp Pro Glu 
225                 230                 235                 240 


Tyr Leu Tyr Asp Ala Ala Ile Gln His Gly Val Lys Gly Ile Val Tyr 
                245                 250                 255     


Ala Gly Met Gly Ala Gly Ser Val Ser Val Arg Gly Ile Ala Gly Met 
            260                 265                 270         


Arg Lys Ala Leu Glu Lys Gly Val Val Val Met Arg Ser Thr Arg Thr 
        275                 280                 285             


Gly Asn Gly Ile Val Pro Pro Asp Glu Glu Leu Pro Gly Leu Val Ser 
    290                 295                 300                 


Asp Ser Leu Asn Pro Ala His Ala Arg Ile Leu Leu Met Leu Ala Leu 
305                 310                 315                 320 


Thr Arg Thr Ser Asp Pro Lys Val Ile Gln Glu Tyr Phe His Thr Tyr 
                325                 330                 335     


<210>  7
<211>  328
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase mature sequence with 4 
       cysteine substitutions

<400>  7

Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 
1               5                   10                  15      


Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 
            20                  25                  30          


Gly Ala Leu Gly Val Asp Thr Leu Ile Cys Ala Val Pro Glu Val Lys 
        35                  40                  45              


Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 
    50                  55                  60                  


Asn Met Thr Gly Asp Val Val Leu Cys Leu Ser Gln Arg Val Asn Glu 
65                  70                  75                  80  


Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 
                85                  90                  95      


Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 
            100                 105                 110         


Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 
        115                 120                 125             


Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 
    130                 135                 140                 


Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 
145                 150                 155                 160 


Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 
                165                 170                 175     


Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 
            180                 185                 190         


Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 
        195                 200                 205             


Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 
    210                 215                 220                 


Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 
225                 230                 235                 240 


His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 
                245                 250                 255     


Ser Val Arg Gly Ile Ala Gly Met Arg Cys Ala Leu Glu Lys Gly Val 
            260                 265                 270         


Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 
        275                 280                 285             


Cys Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 
    290                 295                 300                 


Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 
305                 310                 315                 320 


Ile Gln Glu Tyr Phe His Thr Tyr 
                325             


<210>  8
<211>  328
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase mature sequence with 1 
       lysine substitution

<400>  8

Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 
1               5                   10                  15      


Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 
            20                  25                  30          


Gly Ala Leu Gly Val Asp Thr Leu Ile Lys Ala Val Pro Glu Val Lys 
        35                  40                  45              


Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 
    50                  55                  60                  


Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu 
65                  70                  75                  80  


Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 
                85                  90                  95      


Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 
            100                 105                 110         


Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 
        115                 120                 125             


Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 
    130                 135                 140                 


Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 
145                 150                 155                 160 


Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 
                165                 170                 175     


Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 
            180                 185                 190         


Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 
        195                 200                 205             


Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 
    210                 215                 220                 


Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 
225                 230                 235                 240 


His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 
                245                 250                 255     


Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val 
            260                 265                 270         


Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 
        275                 280                 285             


Glu Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 
    290                 295                 300                 


Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 
305                 310                 315                 320 


Ile Gln Glu Tyr Phe His Thr Tyr 
                325             


<210>  9
<211>  328
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. chrysanthemi asparaginase mature sequence with 2 
       lysine substitutions

<400>  9

Met Ala Asp Lys Leu Pro Asn Ile Val Ile Leu Ala Thr Gly Gly Thr 
1               5                   10                  15      


Ile Ala Gly Ser Ala Ala Thr Gly Thr Gln Thr Thr Gly Tyr Lys Ala 
            20                  25                  30          


Gly Ala Leu Gly Val Asp Thr Leu Ile Lys Ala Val Pro Glu Val Lys 
        35                  40                  45              


Lys Leu Ala Asn Val Lys Gly Glu Gln Phe Ser Asn Met Ala Ser Glu 
    50                  55                  60                  


Asn Met Thr Gly Asp Val Val Leu Lys Leu Ser Gln Arg Val Asn Glu 
65                  70                  75                  80  


Leu Leu Ala Arg Asp Asp Val Asp Gly Val Val Ile Thr His Gly Thr 
                85                  90                  95      


Asp Thr Val Glu Glu Ser Ala Tyr Phe Leu His Leu Thr Val Lys Ser 
            100                 105                 110         


Asp Lys Pro Val Val Phe Val Ala Ala Met Arg Pro Ala Thr Ala Ile 
        115                 120                 125             


Ser Ala Asp Gly Pro Met Asn Leu Leu Glu Ala Val Arg Val Ala Gly 
    130                 135                 140                 


Asp Lys Gln Ser Arg Gly Arg Gly Val Met Val Val Ile Asn Asp Arg 
145                 150                 155                 160 


Ile Gly Ser Ala Arg Tyr Ile Thr Lys Thr Asn Ala Ser Thr Leu Asp 
                165                 170                 175     


Thr Phe Arg Ala Asn Glu Glu Gly Tyr Leu Gly Val Ile Ile Gly Asn 
            180                 185                 190         


Arg Ile Tyr Tyr Gln Asn Arg Ile Asp Lys Leu His Thr Thr Arg Ser 
        195                 200                 205             


Val Phe Asp Val Arg Gly Leu Thr Ser Leu Pro Lys Val Asp Ile Leu 
    210                 215                 220                 


Tyr Gly Tyr Gln Asp Asp Pro Glu Tyr Leu Tyr Asp Ala Ala Ile Gln 
225                 230                 235                 240 


His Gly Val Lys Gly Ile Val Tyr Ala Gly Met Gly Ala Gly Ser Val 
                245                 250                 255     


Ser Val Arg Gly Ile Ala Gly Met Arg Lys Ala Leu Glu Lys Gly Val 
            260                 265                 270         


Val Val Met Arg Ser Thr Arg Thr Gly Asn Gly Ile Val Pro Pro Asp 
        275                 280                 285             


Lys Glu Leu Pro Gly Leu Val Ser Asp Ser Leu Asn Pro Ala His Ala 
    290                 295                 300                 


Arg Ile Leu Leu Met Leu Ala Leu Thr Arg Thr Ser Asp Pro Lys Val 
305                 310                 315                 320 


Ile Gln Glu Tyr Phe His Thr Tyr 
                325             


<210>  10
<211>  348
<212>  PRT
<213>  Escherichia coli

<400>  10

Met Glu Phe Phe Lys Lys Thr Ala Leu Ala Ala Leu Val Met Gly Phe 
1               5                   10                  15      


Ser Gly Ala Ala Leu Ala Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly 
            20                  25                  30          


Gly Thr Ile Ala Gly Gly Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr 
        35                  40                  45              


Val Gly Lys Val Gly Val Glu Asn Leu Val Asn Ala Val Pro Gln Leu 
    50                  55                  60                  


Lys Asp Ile Ala Asn Val Lys Gly Glu Gln Val Val Asn Ile Gly Ser 
65                  70                  75                  80  


Gln Asp Met Asn Asp Asn Val Trp Leu Thr Leu Ala Lys Lys Ile Asn 
                85                  90                  95      


Thr Asp Cys Asp Lys Thr Asp Gly Phe Val Ile Thr His Gly Thr Asp 
            100                 105                 110         


Thr Met Glu Glu Thr Ala Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp 
        115                 120                 125             


Lys Pro Val Val Met Val Gly Ala Met Arg Pro Ser Thr Ser Met Ser 
    130                 135                 140                 


Ala Asp Gly Pro Phe Asn Leu Tyr Asn Ala Val Val Thr Ala Ala Asp 
145                 150                 155                 160 


Lys Ala Ser Ala Asn Arg Gly Val Leu Val Val Met Asn Asp Thr Val 
                165                 170                 175     


Leu Asp Gly Arg Asp Val Thr Lys Thr Asn Thr Thr Asp Val Ala Thr 
            180                 185                 190         


Phe Lys Ser Val Asn Tyr Gly Pro Leu Gly Tyr Ile His Asn Gly Lys 
        195                 200                 205             


Ile Asp Tyr Gln Arg Thr Pro Ala Arg Lys His Thr Ser Asp Thr Pro 
    210                 215                 220                 


Phe Asp Val Ser Lys Leu Asn Glu Leu Pro Lys Val Gly Ile Val Tyr 
225                 230                 235                 240 


Asn Tyr Ala Asn Ala Ser Asp Leu Pro Ala Lys Ala Leu Val Asp Ala 
                245                 250                 255     


Gly Tyr Asp Gly Ile Val Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr 
            260                 265                 270         


Lys Ser Val Phe Asp Thr Leu Ala Thr Ala Ala Lys Thr Gly Thr Ala 
        275                 280                 285             


Val Val Arg Ser Ser Arg Val Pro Thr Gly Ala Thr Thr Gln Asp Ala 
    290                 295                 300                 


Glu Val Asp Asp Ala Lys Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn 
305                 310                 315                 320 


Pro Gln Lys Ala Arg Val Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys 
                325                 330                 335     


Asp Pro Gln Gln Ile Gln Gln Ile Phe Asn Gln Tyr 
            340                 345             


<210>  11
<211>  326
<212>  PRT
<213>  Escherichia coli

<400>  11

Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly 
1               5                   10                  15      


Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Val Gly Lys Val Gly Val 
            20                  25                  30          


Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 
        35                  40                  45              


Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Asn 
    50                  55                  60                  


Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 
65                  70                  75                  80  


Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 
                85                  90                  95      


Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 
            100                 105                 110         


Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 
        115                 120                 125             


Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg 
    130                 135                 140                 


Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 
145                 150                 155                 160 


Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 
                165                 170                 175     


Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 
            180                 185                 190         


Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 
        195                 200                 205             


Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 
    210                 215                 220                 


Asp Leu Pro Ala Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val 
225                 230                 235                 240 


Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Ser Val Phe Asp Thr 
                245                 250                 255     


Leu Ala Thr Ala Ala Lys Thr Gly Thr Ala Val Val Arg Ser Ser Arg 
            260                 265                 270         


Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 
        275                 280                 285             


Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 
    290                 295                 300                 


Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys Asp Pro Gln Gln Ile Gln 
305                 310                 315                 320 


Gln Ile Phe Asn Gln Tyr 
                325     


<210>  12
<211>  326
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. coli asparaginase mature sequence with one additional 
       cysteine pair

<400>  12

Leu Pro Asn Ile Thr Ile Leu Cys Thr Gly Gly Thr Ile Ala Gly Gly 
1               5                   10                  15      


Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Val Gly Lys Val Gly Cys 
            20                  25                  30          


Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 
        35                  40                  45              


Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Asn 
    50                  55                  60                  


Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 
65                  70                  75                  80  


Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 
                85                  90                  95      


Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 
            100                 105                 110         


Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 
        115                 120                 125             


Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg 
    130                 135                 140                 


Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 
145                 150                 155                 160 


Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 
                165                 170                 175     


Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 
            180                 185                 190         


Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 
        195                 200                 205             


Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 
    210                 215                 220                 


Asp Leu Pro Ala Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val 
225                 230                 235                 240 


Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Ser Val Phe Asp Thr 
                245                 250                 255     


Leu Ala Thr Ala Ala Lys Thr Gly Thr Ala Val Val Arg Ser Ser Arg 
            260                 265                 270         


Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 
        275                 280                 285             


Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 
    290                 295                 300                 


Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys Asp Pro Gln Gln Ile Gln 
305                 310                 315                 320 


Gln Ile Phe Asn Gln Tyr 
                325     


<210>  13
<211>  321
<212>  PRT
<213>  Escherichia coli

<400>  13

Met Gly Lys Ala Val Ile Ala Ile His Gly Gly Ala Gly Ala Ile Ser 
1               5                   10                  15      


Arg Ala Gln Met Ser Leu Gln Gln Glu Leu Arg Tyr Ile Glu Ala Leu 
            20                  25                  30          


Ser Ala Ile Val Glu Thr Gly Gln Lys Met Leu Glu Ala Gly Glu Ser 
        35                  40                  45              


Ala Leu Asp Val Val Thr Glu Ala Val Arg Leu Leu Glu Glu Cys Pro 
    50                  55                  60                  


Leu Phe Asn Ala Gly Ile Gly Ala Val Phe Thr Arg Asp Glu Thr His 
65                  70                  75                  80  


Glu Leu Asp Ala Cys Val Met Asp Gly Asn Thr Leu Lys Ala Gly Ala 
                85                  90                  95      


Val Ala Gly Val Ser His Leu Arg Asn Pro Val Leu Ala Ala Arg Leu 
            100                 105                 110         


Val Met Glu Gln Ser Pro His Val Met Met Ile Gly Glu Gly Ala Glu 
        115                 120                 125             


Asn Phe Ala Phe Ala Arg Gly Met Glu Arg Val Ser Pro Glu Ile Phe 
    130                 135                 140                 


Ser Thr Ser Leu Arg Tyr Glu Gln Leu Leu Ala Ala Arg Lys Glu Gly 
145                 150                 155                 160 


Ala Thr Val Leu Asp His Ser Gly Ala Pro Leu Asp Glu Lys Gln Lys 
                165                 170                 175     


Met Gly Thr Val Gly Ala Val Ala Leu Asp Leu Asp Gly Asn Leu Ala 
            180                 185                 190         


Ala Ala Thr Ser Thr Gly Gly Met Thr Asn Lys Leu Pro Gly Arg Val 
        195                 200                 205             


Gly Asp Ser Pro Leu Val Gly Ala Gly Cys Tyr Ala Asn Asn Ala Ser 
    210                 215                 220                 


Val Ala Val Ser Cys Thr Gly Thr Gly Glu Val Phe Ile Arg Ala Leu 
225                 230                 235                 240 


Ala Ala Tyr Asp Ile Ala Ala Leu Met Asp Tyr Gly Gly Leu Ser Leu 
                245                 250                 255     


Ala Glu Ala Cys Glu Arg Val Val Met Glu Lys Leu Pro Ala Leu Gly 
            260                 265                 270         


Gly Ser Gly Gly Leu Ile Ala Ile Asp His Glu Gly Asn Val Ala Leu 
        275                 280                 285             


Pro Phe Asn Thr Glu Gly Met Tyr Arg Ala Trp Gly Tyr Ala Gly Asp 
    290                 295                 300                 


Thr Pro Thr Thr Gly Ile Tyr Arg Glu Lys Gly Asp Thr Val Ala Thr 
305                 310                 315                 320 


Gln 
    


<210>  14
<211>  303
<212>  PRT
<213>  Escherichia coli

<400>  14

Gln Met Ser Leu Gln Gln Glu Leu Arg Tyr Ile Glu Ala Leu Ser Ala 
1               5                   10                  15      


Ile Val Glu Thr Gly Gln Lys Met Leu Glu Ala Gly Glu Ser Ala Leu 
            20                  25                  30          


Asp Val Val Thr Glu Ala Val Arg Leu Leu Glu Glu Cys Pro Leu Phe 
        35                  40                  45              


Asn Ala Gly Ile Gly Ala Val Phe Thr Arg Asp Glu Thr His Glu Leu 
    50                  55                  60                  


Asp Ala Cys Val Met Asp Gly Asn Thr Leu Lys Ala Gly Ala Val Ala 
65                  70                  75                  80  


Gly Val Ser His Leu Arg Asn Pro Val Leu Ala Ala Arg Leu Val Met 
                85                  90                  95      


Glu Gln Ser Pro His Val Met Met Ile Gly Glu Gly Ala Glu Asn Phe 
            100                 105                 110         


Ala Phe Ala Arg Gly Met Glu Arg Val Ser Pro Glu Ile Phe Ser Thr 
        115                 120                 125             


Ser Leu Arg Tyr Glu Gln Leu Leu Ala Ala Arg Lys Glu Gly Ala Thr 
    130                 135                 140                 


Val Leu Asp His Ser Gly Ala Pro Leu Asp Glu Lys Gln Lys Met Gly 
145                 150                 155                 160 


Thr Val Gly Ala Val Ala Leu Asp Leu Asp Gly Asn Leu Ala Ala Ala 
                165                 170                 175     


Thr Ser Thr Gly Gly Met Thr Asn Lys Leu Pro Gly Arg Val Gly Asp 
            180                 185                 190         


Ser Pro Leu Val Gly Ala Gly Cys Tyr Ala Asn Asn Ala Ser Val Ala 
        195                 200                 205             


Val Ser Cys Thr Gly Thr Gly Glu Val Phe Ile Arg Ala Leu Ala Ala 
    210                 215                 220                 


Tyr Asp Ile Ala Ala Leu Met Asp Tyr Gly Gly Leu Ser Leu Ala Glu 
225                 230                 235                 240 


Ala Cys Glu Arg Val Val Met Glu Lys Leu Pro Ala Leu Gly Gly Ser 
                245                 250                 255     


Gly Gly Leu Ile Ala Ile Asp His Glu Gly Asn Val Ala Leu Pro Phe 
            260                 265                 270         


Asn Thr Glu Gly Met Tyr Arg Ala Trp Gly Tyr Ala Gly Asp Thr Pro 
        275                 280                 285             


Thr Thr Gly Ile Tyr Arg Glu Lys Gly Asp Thr Val Ala Thr Gln 
    290                 295                 300             


<210>  15
<211>  303
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Variant E. coli asparaginase mature sequence with no unpaired 
       cysteines

<400>  15

Gln Met Ser Leu Gln Gln Glu Leu Arg Tyr Ile Glu Ala Leu Ser Ala 
1               5                   10                  15      


Ile Val Glu Thr Gly Gln Lys Met Leu Glu Ala Gly Glu Ser Ala Leu 
            20                  25                  30          


Asp Val Val Thr Glu Ala Val Arg Leu Leu Glu Glu Ser Pro Leu Phe 
        35                  40                  45              


Asn Ala Gly Ile Gly Ala Val Phe Thr Arg Asp Glu Thr His Glu Leu 
    50                  55                  60                  


Asp Ala Cys Val Met Asp Gly Asn Thr Leu Lys Ala Gly Ala Val Ala 
65                  70                  75                  80  


Gly Val Ser His Leu Arg Asn Pro Val Leu Ala Ala Arg Leu Val Met 
                85                  90                  95      


Glu Gln Ser Pro His Val Met Met Ile Gly Glu Gly Ala Glu Asn Phe 
            100                 105                 110         


Ala Phe Ala Arg Gly Met Glu Arg Val Ser Pro Glu Ile Phe Ser Thr 
        115                 120                 125             


Ser Leu Arg Tyr Glu Gln Leu Leu Ala Ala Arg Lys Glu Gly Ala Thr 
    130                 135                 140                 


Val Leu Asp His Ser Gly Ala Pro Leu Asp Glu Lys Gln Lys Met Gly 
145                 150                 155                 160 


Thr Val Gly Ala Val Ala Leu Asp Leu Asp Gly Asn Leu Ala Ala Ala 
                165                 170                 175     


Thr Ser Thr Gly Gly Met Thr Asn Lys Leu Pro Gly Arg Val Gly Asp 
            180                 185                 190         


Ser Pro Leu Val Gly Ala Gly Cys Tyr Ala Asn Asn Ala Ser Val Ala 
        195                 200                 205             


Val Ser Ser Thr Gly Thr Gly Glu Val Phe Ile Arg Ala Leu Ala Ala 
    210                 215                 220                 


Tyr Asp Ile Ala Ala Leu Met Asp Tyr Gly Gly Leu Ser Leu Ala Glu 
225                 230                 235                 240 


Ala Ser Glu Arg Val Val Met Glu Lys Leu Pro Ala Leu Gly Gly Ser 
                245                 250                 255     


Gly Gly Leu Ile Ala Ile Asp His Glu Gly Asn Val Ala Leu Pro Phe 
            260                 265                 270         


Asn Thr Glu Gly Met Tyr Arg Ala Trp Gly Tyr Ala Gly Asp Thr Pro 
        275                 280                 285             


Thr Thr Gly Ile Tyr Arg Glu Lys Gly Asp Thr Val Ala Thr Gln 
    290                 295                 300             


