                         SEQUENCE LISTING

<110>  CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)
       UNIVERSITE CLAUDE BERNARD LYON 1
       HULMES, David
       MOALI, Catherine
       BOURHIS, Jean-Marie
       MARIANO, Natacha
       VADON-LE GOFF, Sandrine
       AGHAJARI-HASER, Nushin
 
<120>  CRYSTAL STRUCTURE OF THE PROCOLLAGEN III C-PROPEPTIDE TRIMER AND 
       APPLICATIONS THEREOF

<130>  361678D30391

<150>  US 61/560,494
<151>  2011-11-16

<150>  US 61/677,882
<151>  2012-07-31

<160>  9     

<170>  PatentIn version 3.5

<210>  1
<211>  245
<212>  PRT
<213>  artificial

<220>
<223>  procollagen III C-propeptide (CPIII)

<400>  1

Asp Glu Pro Met Asp Phe Lys Ile Asn Thr Asp Glu Ile Met Thr Ser 
1               5                   10                  15      


Leu Lys Ser Val Asn Gly Gln Ile Glu Ser Leu Ile Ser Pro Asp Gly 
            20                  25                  30          


Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys His 
        35                  40                  45              


Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly Cys 
    50                  55                  60                  


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


Cys Ile Ser Ala Asn Pro Leu Asn Val Pro Arg Lys His Trp Trp Thr 
                85                  90                  95      


Asp Ser Ser Ala Glu Lys Lys His Val Trp Phe Gly Glu Ser Met Asp 
            100                 105                 110         


Gly Gly Phe Gln Phe Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val 
        115                 120                 125             


Leu Asp Val Gln Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser 
    130                 135                 140                 


Gln Gln Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln 
145                 150                 155                 160 


Ala Ser Gly Asn Val Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu 
                165                 170                 175     


Gly Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu 
            180                 185                 190         


Glu Asp Gly Cys Thr Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe 
        195                 200                 205             


Glu Tyr Arg Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp Ile Ala 
    210                 215                 220                 


Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly 
225                 230                 235                 240 


Pro Val Cys Phe Leu 
                245 


<210>  2
<211>  256
<212>  PRT
<213>  artificial

<220>
<223>  CPIII with a Histidine Tag ETGHHHHHHSA in N-terminal

<400>  2

Glu Thr Gly His His His His His His Ser Ala Asp Glu Pro Met Asp 
1               5                   10                  15      


Phe Lys Ile Asn Thr Asp Glu Ile Met Thr Ser Leu Lys Ser Val Asn 
            20                  25                  30          


Gly Gln Ile Glu Ser Leu Ile Ser Pro Asp Gly Ser Arg Lys Asn Pro 
        35                  40                  45              


Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys His Pro Glu Leu Lys Ser 
    50                  55                  60                  


Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile 
65                  70                  75                  80  


Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Ile Ser Ala Asn 
                85                  90                  95      


Pro Leu Asn Val Pro Arg Lys His Trp Trp Thr Asp Ser Ser Ala Glu 
            100                 105                 110         


Lys Lys His Val Trp Phe Gly Glu Ser Met Asp Gly Gly Phe Gln Phe 
        115                 120                 125             


Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val Leu Asp Val Gln Leu 
    130                 135                 140                 


Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser Gln Gln Ile Thr Tyr 
145                 150                 155                 160 


His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln Ala Ser Gly Asn Val 
                165                 170                 175     


Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu Gly Glu Phe Lys Ala 
            180                 185                 190         


Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu Glu Asp Gly Cys Thr 
        195                 200                 205             


Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe Glu Tyr Arg Thr Arg 
    210                 215                 220                 


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


Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly Pro Val Cys Phe Leu 
                245                 250                 255     


<210>  3
<211>  252
<212>  PRT
<213>  artificial

<220>
<223>  CUB1-CUB2 domains of PCPE1

<400>  3

Gln Thr Pro Asn Tyr Ala Arg Pro Val Phe Leu Cys Gly Gly Asp Val 
1               5                   10                  15      


Lys Gly Glu Ser Gly Tyr Val Ala Ser Glu Gly Phe Pro Asn Leu Tyr 
            20                  25                  30          


Pro Pro Asn Lys Glu Cys Ile Trp Thr Ile Thr Val Pro Glu Gly Gln 
        35                  40                  45              


Thr Val Ser Leu Ser Phe Arg Val Phe Asp Leu Glu Leu His Pro Ala 
    50                  55                  60                  


Cys Arg Tyr Asp Ala Leu Glu Val Phe Ala Gly Ser Gly Thr Ser Gly 
65                  70                  75                  80  


Gln Arg Leu Gly Arg Phe Cys Gly Thr Phe Arg Pro Ala Pro Leu Val 
                85                  90                  95      


Ala Pro Gly Asn Gln Val Thr Leu Arg Met Thr Thr Asp Glu Gly Thr 
            100                 105                 110         


Gly Gly Arg Gly Phe Leu Leu Trp Tyr Ser Gly Arg Ala Thr Ser Gly 
        115                 120                 125             


Thr Glu His Gln Phe Cys Gly Gly Arg Leu Glu Lys Ala Gln Gly Thr 
    130                 135                 140                 


Leu Thr Thr Pro Asn Trp Pro Glu Ser Asp Tyr Pro Pro Gly Ile Ser 
145                 150                 155                 160 


Cys Ser Trp His Ile Ile Ala Pro Pro Asp Gln Val Ile Ala Leu Thr 
                165                 170                 175     


Phe Glu Lys Phe Asp Leu Glu Pro Asp Thr Tyr Cys Arg Tyr Asp Ser 
            180                 185                 190         


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


Lys Phe Cys Gly Asp Ala Val Pro Gly Ser Ile Ser Ser Glu Gly Asn 
    210                 215                 220                 


Glu Leu Leu Val Gln Phe Val Ser Asp Leu Ser Val Thr Ala Asp Gly 
225                 230                 235                 240 


Phe Ser Ala Ser Tyr Lys Thr Leu Pro Arg Gly Thr 
                245                 250         


<210>  4
<211>  260
<212>  PRT
<213>  artificial

<220>
<223>  CUB1-CUB2 domains of PCPE1 with a Histine Tag in C-terminal

<400>  4

Gln Thr Pro Asn Tyr Ala Arg Pro Val Phe Leu Cys Gly Gly Asp Val 
1               5                   10                  15      


Lys Gly Glu Ser Gly Tyr Val Ala Ser Glu Gly Phe Pro Asn Leu Tyr 
            20                  25                  30          


Pro Pro Asn Lys Glu Cys Ile Trp Thr Ile Thr Val Pro Glu Gly Gln 
        35                  40                  45              


Thr Val Ser Leu Ser Phe Arg Val Phe Asp Leu Glu Leu His Pro Ala 
    50                  55                  60                  


Cys Arg Tyr Asp Ala Leu Glu Val Phe Ala Gly Ser Gly Thr Ser Gly 
65                  70                  75                  80  


Gln Arg Leu Gly Arg Phe Cys Gly Thr Phe Arg Pro Ala Pro Leu Val 
                85                  90                  95      


Ala Pro Gly Asn Gln Val Thr Leu Arg Met Thr Thr Asp Glu Gly Thr 
            100                 105                 110         


Gly Gly Arg Gly Phe Leu Leu Trp Tyr Ser Gly Arg Ala Thr Ser Gly 
        115                 120                 125             


Thr Glu His Gln Phe Cys Gly Gly Arg Leu Glu Lys Ala Gln Gly Thr 
    130                 135                 140                 


Leu Thr Thr Pro Asn Trp Pro Glu Ser Asp Tyr Pro Pro Gly Ile Ser 
145                 150                 155                 160 


Cys Ser Trp His Ile Ile Ala Pro Pro Asp Gln Val Ile Ala Leu Thr 
                165                 170                 175     


Phe Glu Lys Phe Asp Leu Glu Pro Asp Thr Tyr Cys Arg Tyr Asp Ser 
            180                 185                 190         


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


Lys Phe Cys Gly Asp Ala Val Pro Gly Ser Ile Ser Ser Glu Gly Asn 
    210                 215                 220                 


Glu Leu Leu Val Gln Phe Val Ser Asp Leu Ser Val Thr Ala Asp Gly 
225                 230                 235                 240 


Phe Ser Ala Ser Tyr Lys Thr Leu Pro Arg Gly Thr Leu Glu His His 
                245                 250                 255     


His His His His 
            260 


<210>  5
<211>  293
<212>  PRT
<213>  artificial

<220>
<223>  CPIII-Long with a Histidine Tag AMVHHHHHHSA in N-terminal

<400>  5

Ala Met Val His His His His His His Ser Ala Gly Pro Pro Gly Pro 
1               5                   10                  15      


Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val Gly Ala Ala Ala Ile 
            20                  25                  30          


Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly Phe Ala Pro Tyr Tyr Gly 
        35                  40                  45              


Asp Glu Pro Met Asp Phe Lys Ile Asn Thr Asp Glu Ile Met Thr Ser 
    50                  55                  60                  


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


Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys His 
                85                  90                  95      


Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly Cys 
            100                 105                 110         


Lys Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr 
        115                 120                 125             


Cys Ile Ser Ala Asn Pro Leu Asn Val Pro Arg Lys His Trp Trp Thr 
    130                 135                 140                 


Asp Ser Ser Ala Glu Lys Lys His Val Trp Phe Gly Glu Ser Met Asp 
145                 150                 155                 160 


Gly Gly Phe Gln Phe Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val 
                165                 170                 175     


Leu Asp Val Gln Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser 
            180                 185                 190         


Gln Gln Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln 
        195                 200                 205             


Ala Ser Gly Asn Val Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu 
    210                 215                 220                 


Gly Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu 
225                 230                 235                 240 


Glu Asp Gly Cys Thr Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe 
                245                 250                 255     


Glu Tyr Arg Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp Ile Ala 
            260                 265                 270         


Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly 
        275                 280                 285             


Pro Val Cys Phe Leu 
    290             


<210>  6
<211>  256
<212>  PRT
<213>  artificial

<220>
<223>  CPIII with a Histidine Tag AMVHHHHHHSA in N-terminal

<400>  6

Ala Met Val His His His His His His Ser Ala Asp Glu Pro Met Asp 
1               5                   10                  15      


Phe Lys Ile Asn Thr Asp Glu Ile Met Thr Ser Leu Lys Ser Val Asn 
            20                  25                  30          


Gly Gln Ile Glu Ser Leu Ile Ser Pro Asp Gly Ser Arg Lys Asn Pro 
        35                  40                  45              


Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys His Pro Glu Leu Lys Ser 
    50                  55                  60                  


Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile 
65                  70                  75                  80  


Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Ile Ser Ala Asn 
                85                  90                  95      


Pro Leu Asn Val Pro Arg Lys His Trp Trp Thr Asp Ser Ser Ala Glu 
            100                 105                 110         


Lys Lys His Val Trp Phe Gly Glu Ser Met Asp Gly Gly Phe Gln Phe 
        115                 120                 125             


Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val Leu Asp Val Gln Leu 
    130                 135                 140                 


Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser Gln Gln Ile Thr Tyr 
145                 150                 155                 160 


His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln Ala Ser Gly Asn Val 
                165                 170                 175     


Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu Gly Glu Phe Lys Ala 
            180                 185                 190         


Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu Glu Asp Gly Cys Thr 
        195                 200                 205             


Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe Glu Tyr Arg Thr Arg 
    210                 215                 220                 


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


Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly Pro Val Cys Phe Leu 
                245                 250                 255     


<210>  7
<211>  27
<212>  DNA
<213>  artificial

<220>
<223>  forward primer

<400>  7
accggtcatc atcaccacca tcactcc                                           27


<210>  8
<211>  30
<212>  DNA
<213>  artificial

<220>
<223>  reverse primer

<400>  8
tctagactcg agttataaaa agcaaacagg                                        30


<210>  9
<211>  284
<212>  PRT
<213>  artificial

<220>
<223>  protein expressed in HEK293T cells,comprising CPIII

<400>  9

Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val 
1               5                   10                  15      


Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly His 
            20                  25                  30          


His His His His His Ser Ala Asp Glu Pro Met Asp Phe Lys Ile Asn 
        35                  40                  45              


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


Ser Leu Ile Ser Pro Asp Gly Ser Arg Lys Asn Pro Ala Arg Asn Cys 
65                  70                  75                  80  


Arg Asp Leu Lys Phe Cys His Pro Glu Leu Lys Ser Gly Glu Tyr Trp 
                85                  90                  95      


Val Asp Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile Lys Val Phe Cys 
            100                 105                 110         


Asn Met Glu Thr Gly Glu Thr Cys Ile Ser Ala Asn Pro Leu Asn Val 
        115                 120                 125             


Pro Arg Lys His Trp Trp Thr Asp Ser Ser Ala Glu Lys Lys His Val 
    130                 135                 140                 


Trp Phe Gly Glu Ser Met Asp Gly Gly Phe Gln Phe Ser Tyr Gly Asn 
145                 150                 155                 160 


Pro Glu Leu Pro Glu Asp Val Leu Asp Val Gln Leu Ala Phe Leu Arg 
                165                 170                 175     


Leu Leu Ser Ser Arg Ala Ser Gln Gln Ile Thr Tyr His Cys Lys Asn 
            180                 185                 190         


Ser Ile Ala Tyr Met Asp Gln Ala Ser Gly Asn Val Lys Lys Ala Leu 
        195                 200                 205             


Lys Leu Met Gly Ser Asn Glu Gly Glu Phe Lys Ala Glu Gly Asn Ser 
    210                 215                 220                 


Lys Phe Thr Tyr Thr Val Leu Glu Asp Gly Cys Thr Lys His Thr Gly 
225                 230                 235                 240 


Glu Trp Ser Lys Thr Val Phe Glu Tyr Arg Thr Arg Lys Ala Val Arg 
                245                 250                 255     


Leu Pro Ile Val Asp Ile Ala Pro Tyr Asp Ile Gly Gly Pro Asp Gln 
            260                 265                 270         


Glu Phe Gly Val Asp Val Gly Pro Val Cys Phe Leu 
        275                 280                 


