                         SEQUENCE LISTING

<110>  EnzyPep B.V.
       Quaedflieg, Peter J.L.M.
       Nuijens, Timo
       Toplak, Ana
 
<120>  Enzymatic coupling of (oligo)peptides to the B-chain of an 
       insulin receptor  ligand

<130>  P112174PC00

<150>  EP 16175473.4
<151>  2016-06-21

<160>  7     

<170>  PatentIn version 3.5

<210>  1
<211>  1149
<212>  DNA
<213>  Bacillus amyloliquefaciens


<220>
<221>  CDS
<222>  (1)..(1146)

<220>
<221>  mat_peptide
<222>  (322)..(1146)

<400>  1
gtg aga ggc  aaa aaa gta tgg atc  agt ttg ctg ttt gct tta gcg tta       48
Val Arg Gly  Lys Lys Val Trp Ile  Ser Leu Leu Phe Ala Leu Ala Leu         
        -105                 -100                 -95                     

atc ttt acg atg gcg ttc ggc agc aca tcc tct gcc cag gcg gca ggg         96
Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly           
    -90                 -85                 -80                           

aaa tca aac ggg gaa aag aaa tat att gtc ggg ttt aaa cag aca atg        144
Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met           
-75                 -70                 -65                 -60           

agc acg atg agc gcc gct aag aag aaa gat gtc att tct gaa aaa ggc        192
Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly           
                -55                 -50                 -45               

ggg aaa gtg caa aag caa ttc aaa tat gta gac gca gct tca gct aca        240
Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr           
            -40                 -35                 -30                   

tta aac gaa aaa gct gta aaa gaa ttg aaa aaa gac ccg agc gtc gct        288
Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala           
        -25                 -20                 -15                       

tac gtt gaa gaa gat cac gta gca cat gcg tac gcg cag tcc gtg cct        336
Tyr Val Glu Glu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro           
    -10                 -5              -1  1               5             

tac ggc gta tca caa att aaa gcc cct gct ctg cac tct caa ggc tac        384
Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr           
                10                  15                  20                

act gga tca aat gtt aaa gta gcg gtt atc gac agc ggt atc gat tct        432
Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser           
            25                  30                  35                    

tct cat cct gat tta aag gta gca ggc gga gcc agc atg gtt cct tct        480
Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser           
        40                  45                  50                        

gaa aca aat cct ttc caa gac aac aac tct cac gga act cac gtt gcc        528
Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala           
    55                  60                  65                            

ggc aca gtt gcg gct ctt aat aac tca atc ggt gta tta ggc gtt gcg        576
Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala           
70                  75                  80                  85            

cca agc gca tca ctt tac gct gta aaa gtt ctc ggt gct gac ggt tcc        624
Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser           
                90                  95                  100               

ggc caa tac agc tgg atc att aac gga atc gag tgg gcg atc gca aac        672
Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn           
            105                 110                 115                   

aat atg gac gtt att aac atg agc ctc ggc gga cct tct ggt tct gct        720
Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala           
        120                 125                 130                       

gct tta aaa gcg gca gtt gat aaa gcc gtt gca tcc ggc gtc gta gtc        768
Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val           
    135                 140                 145                           

gtt gcg gca gcc ggt aac gaa ggc act tcc ggc agc tca agc aca gtg        816
Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val           
150                 155                 160                 165           

ggc tac cct ggt aaa tac cct tct gtc att gca gta ggc gct gtt gac        864
Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp           
                170                 175                 180               

agc agc aac caa aga gca tct ttc tca agc gta gga cct gag ctt gat        912
Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Pro Glu Leu Asp           
            185                 190                 195                   

gtc atg gca cct ggc gta tct atc caa agc acg ctt cct gga aac aaa        960
Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys           
        200                 205                 210                       

tac ggg gcg tac aac ggt acg tca atg gca tct ccg cac gtt gcc gga       1008
Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly           
    215                 220                 225                           

gcg gct gct ttg att ctt tct aag cac ccg aac tgg aca aac act caa       1056
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln           
230                 235                 240                 245           

gtc cgc agc agt tta gaa aac acc act aca aaa ctt ggt gat tct ttc       1104
Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp Ser Phe           
                250                 255                 260               

tac tat gga aaa ggg ctg atc aac gta cag gcg gca gct cag taa           1149
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln                   
            265                 270                 275                   


<210>  2
<211>  382
<212>  PRT
<213>  Bacillus amyloliquefaciens

<400>  2

Val Arg Gly  Lys Lys Val Trp Ile  Ser Leu Leu Phe Ala Leu Ala Leu 
        -105                 -100                 -95             


Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly 
    -90                 -85                 -80                 


Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met 
-75                 -70                 -65                 -60 


Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly 
                -55                 -50                 -45     


Gly Lys Val Gln Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr 
            -40                 -35                 -30         


Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala 
        -25                 -20                 -15             


Tyr Val Glu Glu Asp His Val Ala His Ala Tyr Ala Gln Ser Val Pro 
    -10                 -5              -1  1               5   


Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Tyr 
                10                  15                  20      


Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser 
            25                  30                  35          


Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser 
        40                  45                  50              


Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala 
    55                  60                  65                  


Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala 
70                  75                  80                  85  


Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser 
                90                  95                  100     


Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn 
            105                 110                 115         


Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala 
        120                 125                 130             


Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val 
    135                 140                 145                 


Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val 
150                 155                 160                 165 


Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp 
                170                 175                 180     


Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Pro Glu Leu Asp 
            185                 190                 195         


Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys 
        200                 205                 210             


Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly 
    215                 220                 225                 


Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln 
230                 235                 240                 245 


Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys Leu Gly Asp Ser Phe 
                250                 255                 260     


Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln 
            265                 270                 275 


<210>  3
<211>  266
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  subtilisin variant


<220>
<221>  misc_feature
<222>  (216)..(216)
<223>  Xaa can be any naturally occurring amino acid

<400>  3

Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 
1               5                   10                  15      


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


Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 
        35                  40                  45              


Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 
    50                  55                  60                  


Gly Thr His Val Ala Gly Thr Val Ala Ala Val Ala Pro Ser Ala Ser 
65                  70                  75                  80  


Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Gln Tyr Ser 
                85                  90                  95      


Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn Asn Met Asp Val 
            100                 105                 110         


Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala 
        115                 120                 125             


Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala 
    130                 135                 140                 


Gly Asn Glu Gly Thr Ser Gly Ser Ser Ser Thr Val Gly Tyr Pro Gly 
145                 150                 155                 160 


Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln 
                165                 170                 175     


Arg Ala Ser Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala Pro 
            180                 185                 190         


Gly Val Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys Tyr Gly Ala Tyr 
        195                 200                 205             


Asn Gly Thr Cys Met Ala Ser Xaa His Val Ala Gly Ala Ala Ala Leu 
    210                 215                 220                 


Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser 
225                 230                 235                 240 


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


Gly Leu Ile Asn Val Gln Ala Ala Ala Gln 
            260                 265     


<210>  4
<211>  266
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  subtilisin variant


<220>
<221>  misc_feature
<222>  (2)..(3)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (5)..(5)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (9)..(9)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (31)..(31)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (43)..(43)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (50)..(50)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (73)..(73)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (147)..(147)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (157)..(157)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (160)..(160)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (179)..(179)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (197)..(197)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (203)..(203)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (208)..(209)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (213)..(213)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (216)..(216)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (245)..(245)
<223>  Xaa can be any naturally occurring amino acid

<220>
<221>  misc_feature
<222>  (262)..(262)
<223>  Xaa can be any naturally occurring amino acid

<400>  4

Ala Xaa Xaa Val Xaa Tyr Gly Val Xaa Gln Ile Lys Ala Pro Ala Leu 
1               5                   10                  15      


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


Ser Gly Ile Asp Ser Ser His Pro Asp Leu Xaa Val Ala Gly Gly Ala 
        35                  40                  45              


Ser Xaa Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 
    50                  55                  60                  


Gly Thr His Val Ala Gly Thr Val Xaa Ala Val Ala Pro Ser Ala Ser 
65                  70                  75                  80  


Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gly Gln Tyr Ser 
                85                  90                  95      


Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn Asn Met Asp Val 
            100                 105                 110         


Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Ala Leu Lys Ala 
        115                 120                 125             


Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val Val Ala Ala Ala 
    130                 135                 140                 


Gly Asn Xaa Gly Thr Ser Gly Ser Ser Ser Thr Val Xaa Tyr Pro Xaa 
145                 150                 155                 160 


Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asp Ser Ser Asn Gln 
                165                 170                 175     


Arg Ala Xaa Phe Ser Ser Val Gly Pro Glu Leu Asp Val Met Ala Pro 
            180                 185                 190         


Gly Val Ser Ile Xaa Ser Thr Leu Pro Gly Xaa Lys Tyr Gly Ala Xaa 
        195                 200                 205             


Xaa Gly Thr Cys Xaa Ala Ser Xaa His Val Ala Gly Ala Ala Ala Leu 
    210                 215                 220                 


Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln Val Arg Ser Ser 
225                 230                 235                 240 


Leu Glu Asn Thr Xaa Thr Lys Leu Gly Asp Ser Phe Tyr Tyr Gly Lys 
                245                 250                 255     


Gly Leu Ile Asn Val Xaa Ala Ala Ala Gln 
            260                 265     


<210>  5
<211>  21
<212>  PRT
<213>  Homo sapiens

<400>  5

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 
1               5                   10                  15      


Glu Asn Tyr Cys Asn 
            20      


<210>  6
<211>  30
<212>  PRT
<213>  Homo sapiens

<400>  6

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 
1               5                   10                  15      


Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 
            20                  25                  30  


<210>  7
<211>  5
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  coupled pentapeptide


<220>
<221>  MOD_RES
<222>  (1)..(1)
<223>  ACETYLATION

<400>  7

Asp Phe Ser Lys Leu 
1               5   


