                         SEQUENCE LISTING

<110>  Universitt Leipzig
 
<120>  human trypsinogen with reduced autoactivation and its use in an 
       immunoassay

<130>  ule106wo

<150>  EP13187194.9
<151>  2013-10-02

<160>  18    

<170>  PatentIn version 3.5

<210>  1
<211>  232
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  PRSS1-sc, a supercharged variant of human trypsinogen-1

<400>  1

Met Gly Phe Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Asn Cys Ala 
1               5                   10                  15      


Ala His Ser Val Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe 
            20                  25                  30          


Cys Gly Gly Ser Leu Ile Asn Glu Gln Trp Val Val Ser Ala Gly His 
        35                  40                  45              


Cys Tyr Lys Ser Arg Ile Gln Val Arg Leu Gly Glu His Asn Ile Lys 
    50                  55                  60                  


Val Leu Glu Gly Asn Glu Gln Phe Ile Asn Ala Ala Lys Ile Ile Arg 
65                  70                  75                  80  


His Pro Gln Tyr Asp Arg Lys Thr Leu Asn Asn Asp Ile Met Leu Ile 
                85                  90                  95      


Lys Leu Ser Ser Arg Ala Val Ile Asn Ala Lys Val Ser Thr Ile Ser 
            100                 105                 110         


Leu Pro Thr Ala Pro Pro Ala Thr Gly Thr Leu Cys Leu Ile Ser Gly 
        115                 120                 125             


Trp Gly Asn Thr Ala Ser Ser Gly Ala Lys Asn Pro Asp Leu Leu Gln 
    130                 135                 140                 


Cys Leu Asn Ala Pro Val Leu Ser Gln Ala Lys Cys Glu Ala Ser Tyr 
145                 150                 155                 160 


Pro Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe Leu Glu Gly 
                165                 170                 175     


Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn 
            180                 185                 190         


Gly Gln Leu Gln Gly Val Val Ser Trp Gly Asp Gly Cys Ala Gln Lys 
        195                 200                 205             


Asn Lys Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val Lys Trp Ile 
    210                 215                 220                 


Lys Asn Thr Ile Ala Ala Asn Ser 
225                 230         


<210>  2
<211>  699
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  PRSS1-sc, DNA encoding a supercharged variant of human 
       trypsinogen-1

<400>  2
atgggctttg atgatgatga caagatcgtt gggggctaca actgtgcggc gcattctgtc       60

ccctaccagg tgtccctgaa ttctggctac cacttctgtg gtggctccct catcaacgaa      120

cagtgggtgg tatcagcagg ccactgctac aagtcccgca tccaggtgag actgggagag      180

cacaacatca aagtcctgga ggggaatgag cagttcatca atgcagccaa gatcatccgc      240

cacccccaat acgacaggaa gactctgaac aatgacatca tgttaatcaa gctctcctca      300

cgtgcagtaa tcaacgccaa agtgtccacc atctctctgc ccaccgcccc tccagccact      360

ggcacgctgt gcctcatctc tggctggggc aacactgcga gctctggcgc caaaaaccca      420

gacctgctgc agtgcctgaa cgctcctgtg ctgagccagg ctaagtgtga agcctcctac      480

cctggaaaga ttaccagcaa catgttctgt gtgggcttcc ttgagggagg caaggattca      540

tgtcagggtg attctggtgg ccctgtggtc tgcaatggac agctccaagg agttgtctcc      600

tggggtgatg gctgtgccca gaagaacaag cctggagtct acaccaaggt ctacaactat      660

gtgaaatgga ttaagaacac catagctgcc aatagctaa                             699


<210>  3
<211>  232
<212>  PRT
<213>  Homo sapiens

<400>  3

Met Gly Phe Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Asn Cys Glu 
1               5                   10                  15      


Glu Asn Ser Val Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe 
            20                  25                  30          


Cys Gly Gly Ser Leu Ile Asn Glu Gln Trp Val Val Ser Ala Gly His 
        35                  40                  45              


Cys Tyr Lys Ser Arg Ile Gln Val Arg Leu Gly Glu His Asn Ile Glu 
    50                  55                  60                  


Val Leu Glu Gly Asn Glu Gln Phe Ile Asn Ala Ala Lys Ile Ile Arg 
65                  70                  75                  80  


His Pro Gln Tyr Asp Arg Lys Thr Leu Asn Asn Asp Ile Met Leu Ile 
                85                  90                  95      


Lys Leu Ser Ser Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser 
            100                 105                 110         


Leu Pro Thr Ala Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly 
        115                 120                 125             


Trp Gly Asn Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp Glu Leu Gln 
    130                 135                 140                 


Cys Leu Asp Ala Pro Val Leu Ser Gln Ala Lys Cys Glu Ala Ser Tyr 
145                 150                 155                 160 


Pro Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe Leu Glu Gly 
                165                 170                 175     


Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Asn 
            180                 185                 190         


Gly Gln Leu Gln Gly Val Val Ser Trp Gly Asp Gly Cys Ala Gln Lys 
        195                 200                 205             


Asn Lys Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val Lys Trp Ile 
    210                 215                 220                 


Lys Asn Thr Ile Ala Ala Asn Ser 
225                 230         


<210>  4
<211>  699
<212>  DNA
<213>  Homo sapiens

<400>  4
atgggctttg atgatgatga caagatcgtt gggggctaca actgtgagga gaattctgtc       60

ccctaccagg tgtccctgaa ttctggctac cacttctgtg gtggctccct catcaacgaa      120

cagtgggtgg tatcagcagg ccactgctac aagtcccgca tccaggtgag actgggagag      180

cacaacatcg aagtcctgga ggggaatgag cagttcatca atgcagccaa gatcatccgc      240

cacccccaat acgacaggaa gactctgaac aatgacatca tgttaatcaa gctctcctca      300

cgtgcagtaa tcaacgcccg cgtgtccacc atctctctgc ccaccgcccc tccagccact      360

ggcacgaagt gcctcatctc tggctggggc aacactgcga gctctggcgc cgactaccca      420

gacgagctgc agtgcctgga cgctcctgtg ctgagccagg ctaagtgtga agcctcctac      480

cctggaaaga ttaccagcaa catgttctgt gtgggcttcc ttgagggagg caaggattca      540

tgtcagggtg attctggtgg ccctgtggtc tgcaatggac agctccaagg agttgtctcc      600

tggggtgatg gctgtgccca gaagaacaag cctggagtct acaccaaggt ctacaactat      660

gtgaaatgga ttaagaacac catagctgcc aatagctaa                             699


<210>  5
<211>  235
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  hEPl-sc-C112S, a supercharged variant of human enteropeptidase

<400>  5

Ile Val Gly Gly Ser Asp Ala Lys Glu Gly Ala Trp Pro Trp Val Val 
1               5                   10                  15      


Gly Leu Tyr Tyr Asp Asp Arg Leu Leu Cys Gly Ala Ser Leu Val Ser 
            20                  25                  30          


Ser Asp Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Leu 
        35                  40                  45              


Glu Pro Ser Lys Trp Thr Ala Ile Leu Gly Leu His Met Lys Ser Asn 
    50                  55                  60                  


Leu Thr Ser Pro Gln Thr Val Pro Arg Leu Ile Asp Glu Ile Val Ile 
65                  70                  75                  80  


Asn Pro His Tyr Asn Arg Arg Arg Lys Asp Asn Asp Ile Ala Met Met 
                85                  90                  95      


His Leu Glu Phe Lys Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Ser 
            100                 105                 110         


Leu Pro Glu Glu Asn Gln Val Phe Pro Pro Gly Arg Asn Cys Ser Ile 
        115                 120                 125             


Ala Gly Trp Gly Thr Val Val Tyr Gln Gly Thr Thr Ala Asp Ile Leu 
    130                 135                 140                 


Gln Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Arg Cys Gln Gln Gln 
145                 150                 155                 160 


Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr Glu 
                165                 170                 175     


Glu Gly Gly Ile Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met 
            180                 185                 190         


Cys Gln Glu Asn Asn Arg Trp Phe Leu Ala Gly Val Thr Ser Phe Gly 
        195                 200                 205             


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


Arg Phe Thr Glu Trp Ile Gln Ser Phe Leu His 
225                 230                 235 


<210>  6
<211>  5
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 3, a substrate of human trypsin-1

<400>  6

Gly Arg Ala Ser Cys 
1               5   


<210>  7
<211>  6
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 4, a substrate of human trypsin-1

<400>  7

Gly Arg Ala Ser Thr Cys 
1               5       


<210>  8
<211>  7
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 5, a substrate of human trypsin-1

<400>  8

Gly Arg Ala Ser Thr Ser Cys 
1               5           


<210>  9
<211>  3
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 6, a substrate of human trypsin-1

<400>  9

Gly Arg Cys 
1           


<210>  10
<211>  4
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 7, a substrate of human trypsin-1

<400>  10

Gly Ser Arg Cys 
1               


<210>  11
<211>  5
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 8, a substrate of human trypsin-1

<400>  11

Gly Ser Arg Ala Cys 
1               5   


<210>  12
<211>  6
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 9, a substrate of human trypsin-1

<400>  12

Gly Ser Arg Ala Ser Cys 
1               5       


<210>  13
<211>  7
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 10, a substrate of human trypsin-1

<400>  13

Gly Ser Arg Ala Ser Thr Cys 
1               5           


<210>  14
<211>  11
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 11, a substrate of human trypsin-1

<400>  14

Gly Ser Ala Thr Arg Ala Ser Thr Glu Lys Cys 
1               5                   10      


<210>  15
<211>  10
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 12, a substrate of human trypsin-1

<400>  15

Gly Pro Ala Arg Leu Ala Ile Ser Glu Cys 
1               5                   10  


<210>  16
<211>  3
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  peptide 13, a substrate of human trypsin-1

<400>  16

Gly Pro Arg 
1           


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

<220>
<223>  peptide 14, a substrate of human trypsin-1

<400>  17

Ser Arg Ala Lys 
1               


<210>  18
<211>  5932
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  pET28 vector encoding the supercharged variant of human 
       trypsinogen-1

<400>  18
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg       60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc      120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg      180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc      240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt      300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc      360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta      420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt      480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta      540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat      600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa      660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc      720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga      780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc      840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac      900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac      960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat     1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag     1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca     1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac     1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg     1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca     1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac     1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa     1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga     1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg     1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc     1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag     1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc     1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg     1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac     1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga     1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt     1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag     2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg     2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta     2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc     2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg     2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta     2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg     2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct     2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag     2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc     2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag     2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt     2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa     2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg     2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg     2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc     2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta     3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca     3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc     3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc     3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa     3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc     3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac     3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca     3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta     3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa     3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat     3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca     3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa     3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt     3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg     3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca     3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta     3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg     4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat     4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct     4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg     4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat     4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc     4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca     4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg     4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt     4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg     4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct     4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga     4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg     4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc     4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg     4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg     4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga     4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa     5040

ttttgtttaa ctttaagaag gagatatacc atgggctttg atgatgatga caagatcgtt     5100

gggggctaca actgtgcggc gcattctgtc ccctaccagg tgtccctgaa ttctggctac     5160

cacttctgtg gtggctccct catcaacgaa cagtgggtgg tatcagcagg ccactgctac     5220

aagtcccgca tccaggtgag actgggagag cacaacatca aagtcctgga ggggaatgag     5280

cagttcatca atgcagccaa gatcatccgc cacccccaat acgacaggaa gactctgaac     5340

aatgacatca tgttaatcaa gctctcctca cgtgcagtaa tcaacgccaa agtgtccacc     5400

atctctctgc ccaccgcccc tccagccact ggcacgctgt gcctcatctc tggctggggc     5460

aacactgcga gctctggcgc caaaaaccca gacctgctgc agtgcctgaa cgctcctgtg     5520

ctgagccagg ctaagtgtga agcctcctac cctggaaaga ttaccagcaa catgttctgt     5580

gtgggcttcc ttgagggagg caaggattca tgtcagggtg attctggtgg ccctgtggtc     5640

tgcaatggac agctccaagg agttgtctcc tggggtgatg gctgtgccca gaagaacaag     5700

cctggagtct acaccaaggt ctacaactat gtgaaatgga ttaagaacac catagctgcc     5760

aatagctaac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga     5820

aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc     5880

tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at             5932


