		SEQUENCE LISTING

<110>  Asociacin Centro de Investigacin Cooperativa en 
       Biomateriales-CIC biomaGUNE
 
<120>  METHOD FOR THE PRODUCTION OF HUMAN THROMBIN AND USES THEREOF

<130>  PCT-05774

<160>  18    

<170>  PatentIn version 3.5

<210>  1
<211>  339
<212>  PRT
<213>  Mus musculus


<220>
<221>  MISC_FEATURE
<222>  (3)..(12)
<223>  Histidine tail

<220>
<221>  MUTAGEN
<222>  (79)..(80)
<223>  Mutated residues obtained by directed mutagenesis

<220>
<221>  MUTAGEN
<222>  (82)..(82)
<223>  Mutated residues obtained by directed mutagenesis

<400>  1

Met Gly His His His His His His His His His His Ser Ser Gly His 
1               5                   10                  15      


Ile Asp Ser Asp Ile Lys Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ser 
            20                  25                  30          


Thr Thr Asp Ala Glu Phe His Thr Phe Phe Asn Glu Lys Thr Phe Gly 
        35                  40                  45              


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


Leu Lys Asp Thr Thr Glu Lys Glu Leu Leu Asp Ser Tyr Ile Val Pro 
65                  70                  75                  80  


Arg Gly Val Glu Gly Trp Asp Ala Glu Lys Gly Ile Ala Pro Trp Gln 
                85                  90                  95      


Val Met Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser 
            100                 105                 110         


Leu Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Ile Leu Tyr 
        115                 120                 125             


Pro Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg Ile 
    130                 135                 140                 


Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Asn Val Glu Lys Ile Ser 
145                 150                 155                 160 


Met Leu Glu Lys Ile Tyr Val His Pro Arg Tyr Asn Trp Arg Glu Asn 
                165                 170                 175     


Leu Asp Arg Asp Ile Ala Leu Leu Lys Leu Lys Lys Pro Val Pro Phe 
            180                 185                 190         


Ser Asp Tyr Ile His Pro Val Cys Leu Pro Asp Lys Gln Thr Val Thr 
        195                 200                 205             


Ser Leu Leu Arg Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn 
    210                 215                 220                 


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


Leu Gln Val Val Asn Leu Pro Ile Val Glu Arg Pro Val Cys Lys Ala 
                245                 250                 255     


Ser Thr Arg Ile Arg Ile Thr Asp Asn Met Phe Cys Ala Gly Phe Lys 
            260                 265                 270         


Val Asn Asp Thr Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly 
        275                 280                 285             


Pro Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly 
    290                 295                 300                 


Ile Val Ser Trp Gly Glu Gly Cys Asp Arg Lys Gly Lys Tyr Gly Phe 
305                 310                 315                 320 


Tyr Thr His Val Phe Arg Leu Lys Arg Trp Ile Gln Lys Val Ile Asp 
                325                 330                 335     


Gln Phe Gly 
            


<210>  2
<211>  322
<212>  PRT
<213>  Homo sapiens


<220>
<221>  MISC_FEATURE
<222>  (5)..(10)
<223>  Histidine tail

<220>
<221>  MUTAGEN
<222>  (61)..(62)
<223>  Mutated residues obtained by directed mutagenesis

<220>
<221>  MUTAGEN
<222>  (64)..(64)
<223>  Mutated residues obtained by directed mutagenesis

<400>  2

Met Arg Gly Ser His His His His His His Gly Ser Val Asp Thr Ala 
1               5                   10                  15      


Thr Ser Glu Tyr Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly 
            20                  25                  30          


Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu 
        35                  40                  45              


Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr Ile Val Pro Arg Gly 
    50                  55                  60                  


Val Glu Gly Ser Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met 
65                  70                  75                  80  


Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile 
                85                  90                  95      


Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro 
            100                 105                 110         


Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys 
        115                 120                 125             


His Ser Arg Thr Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu 
    130                 135                 140                 


Glu Lys Ile Tyr Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp 
145                 150                 155                 160 


Arg Asp Ile Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp 
                165                 170                 175     


Tyr Ile His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu 
            180                 185                 190         


Leu Gln Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys 
        195                 200                 205             


Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln 
    210                 215                 220                 


Val Val Asn Leu Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr 
225                 230                 235                 240 


Arg Ile Arg Ile Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp 
                245                 250                 255     


Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe 
            260                 265                 270         


Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val 
        275                 280                 285             


Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr 
    290                 295                 300                 


His Val Phe Arg Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe 
305                 310                 315                 320 


Gly Glu 
        


<210>  3
<211>  339
<212>  PRT
<213>  Mus musculus


<220>
<221>  MISC_FEATURE
<222>  (3)..(10)
<223>  Histidine tail

<400>  3

Met Gly His His His His His His His His His His Ser Ser Gly His 
1               5                   10                  15      


Ile Asp Ser Asp Ile Lys Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ser 
            20                  25                  30          


Thr Thr Asp Ala Glu Phe His Thr Phe Phe Asn Glu Lys Thr Phe Gly 
        35                  40                  45              


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


Leu Lys Asp Thr Thr Glu Lys Glu Leu Leu Asp Ser Tyr Ile Asp Gly 
65                  70                  75                  80  


Arg Ile Val Glu Gly Trp Asp Ala Glu Lys Gly Ile Ala Pro Trp Gln 
                85                  90                  95      


Val Met Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser 
            100                 105                 110         


Leu Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Ile Leu Tyr 
        115                 120                 125             


Pro Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg Ile 
    130                 135                 140                 


Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Asn Val Glu Lys Ile Ser 
145                 150                 155                 160 


Met Leu Glu Lys Ile Tyr Val His Pro Arg Tyr Asn Trp Arg Glu Asn 
                165                 170                 175     


Leu Asp Arg Asp Ile Ala Leu Leu Lys Leu Lys Lys Pro Val Pro Phe 
            180                 185                 190         


Ser Asp Tyr Ile His Pro Val Cys Leu Pro Asp Lys Gln Thr Val Thr 
        195                 200                 205             


Ser Leu Leu Arg Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn 
    210                 215                 220                 


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


Leu Gln Val Val Asn Leu Pro Ile Val Glu Arg Pro Val Cys Lys Ala 
                245                 250                 255     


Ser Thr Arg Ile Arg Ile Thr Asp Asn Met Phe Cys Ala Gly Phe Lys 
            260                 265                 270         


Val Asn Asp Thr Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly 
        275                 280                 285             


Pro Phe Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly 
    290                 295                 300                 


Ile Val Ser Trp Gly Glu Gly Cys Asp Arg Lys Gly Lys Tyr Gly Phe 
305                 310                 315                 320 


Tyr Thr His Val Phe Arg Leu Lys Arg Trp Ile Gln Lys Val Ile Asp 
                325                 330                 335     


Gln Phe Gly 
            


<210>  4
<211>  322
<212>  PRT
<213>  Homo sapiens


<220>
<221>  MISC_FEATURE
<222>  (5)..(10)
<223>  Histidine tail

<400>  4

Met Arg Gly Ser His His His His His His Gly Ser Val Asp Thr Ala 
1               5                   10                  15      


Thr Ser Glu Tyr Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly 
            20                  25                  30          


Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu 
        35                  40                  45              


Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile 
    50                  55                  60                  


Val Glu Gly Ser Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met 
65                  70                  75                  80  


Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile 
                85                  90                  95      


Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro 
            100                 105                 110         


Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys 
        115                 120                 125             


His Ser Arg Thr Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu 
    130                 135                 140                 


Glu Lys Ile Tyr Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp 
145                 150                 155                 160 


Arg Asp Ile Ala Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp 
                165                 170                 175     


Tyr Ile His Pro Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu 
            180                 185                 190         


Leu Gln Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys 
        195                 200                 205             


Glu Thr Trp Thr Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln 
    210                 215                 220                 


Val Val Asn Leu Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr 
225                 230                 235                 240 


Arg Ile Arg Ile Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp 
                245                 250                 255     


Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe 
            260                 265                 270         


Val Met Lys Ser Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val 
        275                 280                 285             


Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr 
    290                 295                 300                 


His Val Phe Arg Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe 
305                 310                 315                 320 


Gly Glu 
        


<210>  5
<211>  924
<212>  DNA
<213>  Mus musculus


<220>
<221>  mutation
<222>  (140)..(140)
<223>  Mutated residues obtained by directed mutagenesis

<220>
<221>  mutation
<222>  (142)..(143)
<223>  Mutated residues obtained by directed mutagenesis

<220>
<221>  mutation
<222>  (148)..(149)
<223>  Mutated residues obtained by directed mutagenesis

<400>  5
accaccgatg cggagttcca caccttcttc aatgagaaga ccttcggcct tggggaggca       60

gactgtggcc tgcggccttt gttcgagaag aagtcgttga aagacacaac cgaaaaggag      120

cttcttgact cttacatagt cccgcgcggc gtggagggct gggacgctga gaagggtatc      180

gccccctggc aggtgatgct ttttcggaag agtccccaag agctgctgtg tggggccagc      240

cttatcagtg accgatgggt cctcactgct gcccactgca ttctgtaccc accctgggac      300

aagaacttca ctgagaatga cctcctggtg cgcattggca agcattcccg aaccagatat      360

gagcggaatg ttgaaaagat ctccatgctg gaaaagatct acgtccaccc cagatataac      420

tggcgggaga acctagaccg cgatatcgct ctgctcaagc taaagaaacc tgtacccttc      480

agtgactata ttcaccccgt gtgtttgcca gacaagcaga cagtaaccag cttgctccgg      540

gctggttata aagggcgggt gacaggctgg ggcaaccttc gggagacatg gacaaccaac      600

atcaatgaga tacagcccag cgtcctgcag gtggtgaacc tgcccattgt agagcggcca      660

gtgtgcaagg cctccacccg gattcgaatt actgacaaca tgttctgtgc tggcttcaag      720

gtgaatgaca ccaagcgagg agatgcttgt gaaggtgaca gtggaggacc ttttgtcatg      780

aagagcccct ttaacaaccg ctggtatcaa atgggtattg tctcatgggg tgaaggatgt      840

gaccggaagg ggaaatacgg cttctacacg catgtgttcc gtctgaaaag gtggatacag      900

aaagtcattg atcaatttgg atag                                             924


<210>  6
<211>  969
<212>  DNA
<213>  Homo sapiens


<220>
<221>  mutation
<222>  (182)..(182)
<223>  Mutated residues obtained by directed mutagenesis

<220>
<221>  mutation
<222>  (184)..(185)
<223>  Mutated residues obtained by directed mutagenesis

<220>
<221>  mutation
<222>  (190)..(191)
<223>  Mutated residues obtained by directed mutagenesis

<400>  6
atgagaggat cgcatcacca tcaccatcac ggatccgtcg acaccgccac cagtgagtac       60

cagactttct tcaatccgag gacctttggc tcgggagagg cagactgtgg gctgcgacct      120

ctgttcgaga agaagtcgct ggaggacaaa accgaaagag agctcctgga atcctacatc      180

gtcccgcgcg gtgtggaggg ctcggatgca gagatcggca tgtcaccttg gcaggtgatg      240

cttttccgga agagtcccca ggagctgctg tgtggggcca gcctcatcag tgaccgctgg      300

gtcctcaccg ccgcccactg cctcctgtac ccgccctggg acaagaactt caccgagaat      360

gaccttctgg tgcgcattgg caagcactcc cgcaccaggt acgagcgaaa cattgaaaag      420

atatccatgt tggaaaagat ctacatccac cccaggtaca actggcggga gaacctggac      480

cgggacattg ccctgatgaa gctgaagaag cctgttgcct tcagtgacta cattcaccct      540

gtgtgtctgc ccgacaggga gacggcagcc agcttgctcc aggctggata caaggggcgg      600

gtgacaggct ggggcaacct gaaggagacg tggacagcca acgttggtaa ggggcagccc      660

agtgtcctgc aggtggtgaa cctgcccatt gtggagcggc cggtctgcaa ggactccacc      720

cggatccgca tcactgacaa catgttctgt gctggttaca agcctgatga agggaaacga      780

ggggatgcct gtgaaggtga cagtggggga ccctttgtca tgaagagccc ctttaacaac      840

cgctggtatc aaatgggcat cgtctcatgg ggtgaaggct gtgaccggga tgggaaatat      900

ggcttctaca cacatgtgtt ccgcctgaag aagtggatac agaaggtcat tgatcagttt      960

ggagagtag                                                              969


<210>  7
<211>  924
<212>  DNA
<213>  Mus musculus

<400>  7
accaccgatg cggagttcca caccttcttc aatgagaaga ccttcggcct tggggaggca       60

gactgtggcc tgcggccttt gttcgagaag aagtcgttga aagacacaac cgaaaaggag      120

cttcttgact cttacataga cgggcgcatc gtggagggct gggacgctga gaagggtatc      180

gccccctggc aggtgatgct ttttcggaag agtccccaag agctgctgtg tggggccagc      240

cttatcagtg accgatgggt cctcactgct gcccactgca ttctgtaccc accctgggac      300

aagaacttca ctgagaatga cctcctggtg cgcattggca agcattcccg aaccagatat      360

gagcggaatg ttgaaaagat ctccatgctg gaaaagatct acgtccaccc cagatataac      420

tggcgggaga acctagaccg cgatatcgct ctgctcaagc taaagaaacc tgtacccttc      480

agtgactata ttcaccccgt gtgtttgcca gacaagcaga cagtaaccag cttgctccgg      540

gctggttata aagggcgggt gacaggctgg ggcaaccttc gggagacatg gacaaccaac      600

atcaatgaga tacagcccag cgtcctgcag gtggtgaacc tgcccattgt agagcggcca      660

gtgtgcaagg cctccacccg gattcgaatt actgacaaca tgttctgtgc tggcttcaag      720

gtgaatgaca ccaagcgagg agatgcttgt gaaggtgaca gtggaggacc ttttgtcatg      780

aagagcccct ttaacaaccg ctggtatcaa atgggtattg tctcatgggg tgaaggatgt      840

gaccggaagg ggaaatacgg cttctacacg catgtgttcc gtctgaaaag gtggatacag      900

aaagtcattg atcaatttgg atag                                             924


<210>  8
<211>  969
<212>  DNA
<213>  Homo sapiens

<400>  8
atgagaggat cgcatcacca tcaccatcac ggatccgtcg acaccgccac cagtgagtac       60

cagactttct tcaatccgag gacctttggc tcgggagagg cagactgtgg gctgcgacct      120

ctgttcgaga agaagtcgct ggaggacaaa accgaaagag agctcctgga atcctacatc      180

gacgggcgca ttgtggaggg ctcggatgca gagatcggca tgtcaccttg gcaggtgatg      240

cttttccgga agagtcccca ggagctgctg tgtggggcca gcctcatcag tgaccgctgg      300

gtcctcaccg ccgcccactg cctcctgtac ccgccctggg acaagaactt caccgagaat      360

gaccttctgg tgcgcattgg caagcactcc cgcaccaggt acgagcgaaa cattgaaaag      420

atatccatgt tggaaaagat ctacatccac cccaggtaca actggcggga gaacctggac      480

cgggacattg ccctgatgaa gctgaagaag cctgttgcct tcagtgacta cattcaccct      540

gtgtgtctgc ccgacaggga gacggcagcc agcttgctcc aggctggata caaggggcgg      600

gtgacaggct ggggcaacct gaaggagacg tggacagcca acgttggtaa ggggcagccc      660

agtgtcctgc aggtggtgaa cctgcccatt gtggagcggc cggtctgcaa ggactccacc      720

cggatccgca tcactgacaa catgttctgt gctggttaca agcctgatga agggaaacga      780

ggggatgcct gtgaaggtga cagtggggga ccctttgtca tgaagagccc ctttaacaac      840

cgctggtatc aaatgggcat cgtctcatgg ggtgaaggct gtgaccggga tgggaaatat      900

ggcttctaca cacatgtgtt ccgcctgaag aagtggatac agaaggtcat tgatcagttt      960

ggagagtag                                                              969


<210>  9
<211>  28
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Mouse Prethrombin-2 Forward Primer

<400>  9
cgcggatcca ccaccgatgc ggagttcc                                          28


<210>  10
<211>  30
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Mouse Prethrombin-2 Reverse Primer

<400>  10
ccgctcgagc tatccaaatt gatcaatgac                                        30


<210>  11
<211>  45
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer for site-specific mutations of mouse prethrombin-2
       gene

<400>  11
cttcttgact cttacatagt cccgcgcggc gtggagggct gggac                       45


<210>  12
<211>  45
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer for site-specific mutations of mouse prethrombin-2
       gene

<400>  12
gtcccagccc tccacgccgc gcgggactat gtaagagtca agaag                       45


<210>  13
<211>  25
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Human Prethrombin-2 Forward Primer

<400>  13
acgcgtcgac accgccacca gtgag                                             25


<210>  14
<211>  27
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Human Prethrombin-2 Reverse Primer

<400>  14
cccaagcttc tactctccaa actgatc                                           27


<210>  15
<211>  44
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Forward primer for site-specific mutations of human prethrombin-2
       gene

<400>  15
cctggaatcc tacatcgtcc cgcgcggtgt ggagggctcg gatg                        44


<210>  16
<211>  44
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Reverse primer for site-specific mutations of human prethrombin-2
       gene

<400>  16
catccgagcc ctccacaccg cgcgggacga tgtaggattc cagg                        44


<210>  17
<211>  6
<212>  PRT
<213>  Homo sapiens

<400>  17

Ile Asp Gly Arg Ile Val 
1               5       


<210>  18
<211>  6
<212>  PRT
<213>  Homo sapiens

<400>  18

Ile Val Pro Arg Gly Val 
1               5       







