Prova de Enzimologia

(2 semestre de 2014)

Aluna: Sarah de Menezes Viana

(7549492)

Sequncia 1
ATGCATCGGGTTAATTGGAGAAACCTGATCGCCATTGCCGGAGCCAGCGCG CTTACCTGCC
TGTCAGCATCCGCTGCCATGGCAGCCTGCACCTACACCATCGATAGCGAATGGTCCACCGG
CTTTACCGCCAATATCACCCTCAAAAACGATACCGGTGCCGCCATCAATAACTGGAACGTG
AATTGGCAATACTCCAGCAATCGCATGACCAGCGGCTGGAATGCCAACTTCTCCGGCACCA
ACCCCTACAACGCCACCAACATGAGCTGGAACGGCAGCATCGCGCCAGGACAATCCATCTC
CTTCGGCCTCCAGGGCGAAAAAAATGGCAGCACCGCCGAGCGACCAACCGTCACCGGCGCC
GCTTGTAACAGTGCAACCACCAGCTCTGTGGCTTCCAGCTCTTCAACACCCACCACCAGTT
CATCTTCTGCATCCAGTGTGGCCTCCGCACTGCTGTTGCAAGAAGCACAAGCCGGTTTCTG
CCGTGTGGATGGCACCATCGATAATAACCACACCGGCTTTACCGGCAGTGGCTTTGCCAAC
ACCAACAATGCCCAGGGGGCAGCGGTAGTCTGGGCGATAGATGCTACCAGCAGTGGCCGTC
GCACCCTGACTATCCGCTATGCCAATGGTGGAACCGCCAATCGCAATGGCTCACTGGTGAT
TAACGGCGGCAGCAACGGTAACTATACGGTGAGTTTGCCCACGACCGGCGCCTGGACCACC
TGGCAAACCGCAACTATCGATGTGGATTTGGTACAGGGCAATAATATTGTGCAGTTGTCTG
CAACGACAGCCGAAGGCTTACCCAATATAGATTCGTTAAGTGTTGTGGGTGGTACGGTCAG
GGCAGGTAATTGCGGCAGTGTGAGCAGCAGCAGTTCCGTGCAATCGTCATCGTCATCGAAC
AGCAGTGCAGGTGCAGCAGTACCCAGTGCCGGTTGCGGTAAACCGCGCACACTGCAAAACG
GCAGGATTACATTGCAAAGCGGCGGTACACAGCGCAGCTACATCCTGGATGCACCGACTAA
CTACAACAATACTCGTCCCTATCGGGTGATCTTCGGTTATCACTGGGTCGGTGGTACCGCC
GATGATGTAGCGACCGGCCAAACGGTTCAACGCAATACCTGGGCTCACTACGGGATGAAAA
AGTTGTCTGGTGACTCCACGATTTTTGTGGCGCCACAGGGTATAGGTAACGGGTGGGGCAA
TGGTGGCGGTGCAGATGTCACGTTTACCGATAATATTCTTGCGCAATTAAAAACAGAATTG
TGTATCGACGAGAGCCGCATTTTTGCCAACGGTTTTAGCTTCGGCGGATCAATGACCTATG
CTATTGCCTGCGCCCGGGCCAATGTATTCCGCGGTGTTGCCGCCTACGGTGCAGGCTCAAT
CAGCGGTTGCTCCGGAGGCAATAGCCCTATCGCTTATTTCGGCGGCCATGGGATTCGCGAC
AATGTGTTTACCCCGGATCGCGGTCGCGCATTGCGCGATCGGTTTGTCAGAAACAATAGCT
GTACCGTCATCAACCCACCCGAACCCTCCATCGGCAGCCTGCGTCACACCTGTACCAGCTA
TCAATGCCCCAATAAAAACTATCCTGTACGCTGGTGTGCCTATGATGCCGGCCATATTGCA
GCACCCCATGATGGCTCGACTGGCGATAGTGGCAATACCTGGCTTGCCGAA GAAGCCTGGC
AATTCTTCACACAATTCTGA

ferruloyl esterase [Cellvibrio japonicus] Bactria gram negativa

NCBI Reference Sequence: WP_012488858.1
GenPept Identical Proteins Graphics
>gi|501465413|ref|WP_012488858.1| ferruloyl esterase [Cellvibrio japonicus]
MHRVNWRNLIAIAGASA
(peptdeo sinal na protena)
LTCLSASAAMAACTYTIDSEWSTGFTANITLKNDTGAAINNWNVNWQYSSNRM
TSGWNANFSGTNPYNATNMSWNGSIAPGQSISFGLQGEKNGSTAERPTVTGAACNSATTSSVASSSSTPT
TSSSSASSVASALLLQEAQAGFCRVDGTIDNNHTGFTGSGFANTNNAQGAAVVWAIDATSSGRRTLTIRY
ANGGTANRNGSLVINGGSNGNYTVSLPTTGAWTTWQTATIDVDLVQGNNIVQLSATTAEGLPNIDSLSVV
GGTVRAGNCGSVSSSSSVQSSSSSNSSAGAAVPSAGCGKPRTLQNGRITLQSGGTQRSYILDAPTNYNNT
RPYRVIFGYHWVGGTADDVATGQTVQRNTWAHYGMKKLSGDSTIFVAPQGIGNGWGNGGGADVTFTDNIL
AQLKTELCIDESRIFANGFSFGGSMTYAIACARANVFRGVAAYGAGSISGCSGGNSPIAYFGGHGIRDNV
FTPDRGRALRDRFVRNNSCTVINPPEPSIGSLRHTCTSYQCPNKNYPVRWCAYDAGHIAAPHDGSTGDSG
NTWLAEEAWQFFTQF

# Measure
max. C
max. Y
max. S
mean S
D

Position
Value
Cutoff signal peptide?
18
0.202
18
0.430
13
0.972
1-17
0.932
1-17
0.701 0.450 YES

Name=Sequence SP='YES' Cleavage site between pos. 17 and 18: ASA-LT D=0.701
D-cutoff=0.450 Networks=SignalP-noTM # data

Clivar entre 17 e 18 17x3 = 51

Vermelho = peptdeo sinal
Azul = primers
ATGCATCGGGTTAATTGGAGAAACCTGATCGCCATTGCCGGAGCCAGCGCG CTTACCTGCC
TGTCAGCATCCGCTGCCATG GCAGCCTGCACCTACACCATCGATAGCGAATGGTCCACCGG
CTTTACCGCCAATATCACCCTCAAAAACGATACCGGTGCCGCCATCAATAACTGGAACGTG
AATTGGCAATACTCCAGCAATCGCATGACCAGCGGCTGGAATGCCAACTTCTCCGGCACCA
ACCCCTACAACGCCACCAACATGAGCTGGAACGGCAGCATCGCGCCAGGACAATCCATCTC
CTTCGGCCTCCAGGGCGAAAAAAATGGCAGCACCGCCGAGCGACCAACCGTCACCGGCGCC
GCTTGTAACAGTGCAACCACCAGCTCTGTGGCTTCCAGCTCTTCAACACCCACCACCAGTT
CATCTTCTGCATCCAGTGTGGCCTCCGCACTGCTGTTGCAAGAAGCACAAGCCGGTTTCTG
CCGTGTGGATGGCACCATCGATAATAACCACACCGGCTTTACCGGCAGTGGCTTTGCCAAC
ACCAACAATGCCCAGGGGGCAGCGGTAGTCTGGGCGATAGATGCTACCAGCAGTGGCCGTC
GCACCCTGACTATCCGCTATGCCAATGGTGGAACCGCCAATCGCAATGGCTCACTGGTGAT
TAACGGCGGCAGCAACGGTAACTATACGGTGAGTTTGCCCACGACCGGCGCCTGGACCACC
TGGCAAACCGCAACTATCGATGTGGATTTGGTACAGGGCAATAATATTGTGCAGTTGTCTG
CAACGACAGCCGAAGGCTTACCCAATATAGATTCGTTAAGTGTTGTGGGTGGTACGGTCAG
GGCAGGTAATTGCGGCAGTGTGAGCAGCAGCAGTTCCGTGCAATCGTCATCGTCATCGAAC
AGCAGTGCAGGTGCAGCAGTACCCAGTGCCGGTTGCGGTAAACCGCGCACACTGCAAAACG
GCAGGATTACATTGCAAAGCGGCGGTACACAGCGCAGCTACATCCTGGATGCACCGACTAA
CTACAACAATACTCGTCCCTATCGGGTGATCTTCGGTTATCACTGGGTCGGTGGTACCGCC
GATGATGTAGCGACCGGCCAAACGGTTCAACGCAATACCTGGGCTCACTACGGGATGAAAA
AGTTGTCTGGTGACTCCACGATTTTTGTGGCGCCACAGGGTATAGGTAACGGGTGGGGCAA
TGGTGGCGGTGCAGATGTCACGTTTACCGATAATATTCTTGCGCAATTAAAAACAGAATTG
TGTATCGACGAGAGCCGCATTTTTGCCAACGGTTTTAGCTTCGGCGGATCAATGACCTATG
CTATTGCCTGCGCCCGGGCCAATGTATTCCGCGGTGTTGCCGCCTACGGTGCAGGCTCAAT
CAGCGGTTGCTCCGGAGGCAATAGCCCTATCGCTTATTTCGGCGGCCATGGGATTCGCGAC
AATGTGTTTACCCCGGATCGCGGTCGCGCATTGCGCGATCGGTTTGTCAGAAACAATAGCT
GTACCGTCATCAACCCACCCGAACCCTCCATCGGCAGCCTGCGTCACACCTGTACCAGCTA
TCAATGCCCCAATAAAAACTATCCTGTACGCTGGTGTGCCTATGATGCCGGCCATATTGCA
GCACCCCATGATGGCTCGACTGGCGATAGTGGCAATACCTGGCTTGCCGAA GAAGCCTGGC
AATTCTTCACACAATTCTGA

The Sequence Manipulation Suite: Reverse Complement

Results for 30 residue sequence starting
"GAAGCCTGGC".
5-3 da fita de baixo revertida:
TCAGAATTGTGTGAAGAATTGCCAGGCTTC
5-3 da fita de cima (forward):
CTTACCTGCCTGTCAGCATCCGCTGCCATG

Primer forward
5'- CTT ACC TGC CTG TCA GCA TCC G -3
5'- CGG ATG CTG ACA GGC AGG TAA G -3
Melt temperature: 60C

Primer reverse
Sequence: 5'- CTT ACC TGC CTG TCA GCA TCC G -3'
Complement: 5'- CGG ATG CTG ACA GGC AGG TAA G -3'
Melt temperature: 60C

Sequncia com os primers

CTT ACC TGC CTG TCA GCA TCC G GCAGCCTGCACCTACACCATCGATAGCGAATGGTCCACCGG
CTTTACCGCCAATATCACCCTCAAAAACGATACCGGTGCCGCCATCAATAACTGGAACGTGAATTGGCAATACTCCAGCAATCGCA
TGACCAGCGGCTGGAATGCCAACTTCTCCGGCACCAACCCCTACAACGCCACCAACATGAGCTGGAACGGCAGCATCGCGCCAG
GACAATCCATCTCCTTCGGCCTCCAGGGCGAAAAAAATGGCAGCACCGCCGAGCGACCAACCGTCACCGGCGCC
GCTTGTAACAGTGCAACCACCAGCTCTGTGGCTTCCAGCTCTTCAACACCCACCACCAGTTCATCTTCTGCATCCAGTGTGGCCT
CCGCACTGCTGTTGCAAGAAGCACAAGCCGGTTTCTGCCGTGTGGATGGCACCATCGATAATAACCACACCGGCTTTACCGGCA
GTGGCTTTGCCAACACCAACAATGCCCAGGGGGCAGCGGTAGTCTGGGCGATAGATGCTACCAGCAGTGGCCGTC
GCACCCTGACTATCCGCTATGCCAATGGTGGAACCGCCAATCGCAATGGCTCACTGGTGATTAACGGCGGCAGCAACGGTAACTA
TACGGTGAGTTTGCCCACGACCGGCGCCTGGACCACCTGGCAAACCGCAACTATCGATGTGGATTTGGTACAGGGCAATAATATT
GTGCAGTTGTCTGCAACGACAGCCGAAGGCTTACCCAATATAGATTCGTTAAGTGTTGTGGGTGGTACGGTCAG
GGCAGGTAATTGCGGCAGTGTGAGCAGCAGCAGTTCCGTGCAATCGTCATCGTCATCGAACAGCAGTGCAGGTGCAGCAGTAC
CCAGTGCCGGTTGCGGTAAACCGCGCACACTGCAAAACGGCAGGATTACATTGCAAAGCGGCGGTACACAGCGCAGCTACATCC
TGGATGCACCGACTAACTACAACAATACTCGTCCCTATCGGGTGATCTTCGGTTATCACTGGGTCGGTGGTACCGCC
GATGATGTAGCGACCGGCCAAACGGTTCAACGCAATACCTGGGCTCACTACGGGATGAAAAAGTTGTCTGGTGACTCCACGATT
TTTGTGGCGCCACAGGGTATAGGTAACGGGTGGGGCAATGGTGGCGGTGCAGATGTCACGTTTACCGATAATATTCTTGCGCAA
TTAAAAACAGAATTGTGTATCGACGAGAGCCGCATTTTTGCCAACGGTTTTAGCTTCGGCGGATCAATGACCTATG
CTATTGCCTGCGCCCGGGCCAATGTATTCCGCGGTGTTGCCGCCTACGGTGCAGGCTCAATCAGCGGTTGCTCCGGAGGCAATA
GCCCTATCGCTTATTTCGGCGGCCATGGGATTCGCGACAATGTGTTTACCCCGGATCGCGGTCGCGCATTGCGCGATCGGTTTG
TCAGAAACAATAGCTGTACCGTCATCAACCCACCCGAACCCTCCATCGGCAGCCTGCGTCACACCTGTACCAGCTA
TCAATGCCCCAATAAAAACTATCCTGTACGCTGGTGTGCCTATGATGCCGGCCATATTGCAGCACCCCATGATGGCTCGACTGGCG
ATAGTGGCAATACCTGGCTTGCCGAA CTT ACC TGC CTG TCA GCA TCC G

Escolha do vetor
Vetor escolhido: bacteriano

Stios de Restrio
#

Xho I:

Primer inicial com stio restritivo no comeo:

C TCGA G

Nde I:

Primer final com stio restritivo no comeo:

CA TA TG

Enzyme
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

AatII
AccI
AclI
AcuI
AfeI
AflII
AflIII
AleI
ApaI
ApaLI
ApoI
AscI
AseI
AsiSI
AvrII
BaeGI
BamHI
BbsI
BbvCI
BciVI
BclI
BcoDI
BfaI
BglII
BlpI
BmgBI
BmtI
Bpu10I
BpuEI
BsaAI
BsaBI
BsaI

33 BsaXI
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107

BseRI
BseYI
BsiHKAI
BsiWI
BsmAI
BsmBI
BspCNI
BspHI
BsrBI
BsrGI
BssHII
BssSI
BstAPI
BstBI
BstEII
BstYI
BstZ17I
Bsu36I
DdeI
DraI
DraIII
DrdI
EagI
Eco53kI
EcoNI
EcoO109I
EcoRI
EcoRV
FauI
FseI
HincII
HindIII
HpaI
MfeI
MluI
MmeI
MscI
NdeI
NheI
NotI
NsiI
NspI
PacI
PaeR7I
PciI
PflFI
PmeI
PmlI
PpuMI
PshAI
PsiI
PspOMI
PspXI
PstI
PvuII
RsrII
SacI
SalI
SbfI
ScaI
SexAI
SfcI
SfiI
SmlI
SnaBI
SpeI
SphI
StuI
SwaI
Tth111I
XbaI
XhoI
XmnI
ZraI

Specificity
GACGTC
GTMKAC
AACGTT
CTGAAG(N)14NN
AGCGCT
CTTAAG
ACRYGT
CACNNNNGTG
GGGCCC
GTGCAC
RAATTY
GGCGCGCC
ATTAAT
GCGATCGC
CCTAGG
GKGCMC
GGATCC
GAAGACNNNNNN
CCTCAGC
GTATCC(N)5N
TGATCA
GTCTCNNNNN
CTAG
AGATCT
GCTNAGC
CACGTC
GCTAGC
CCTNAGC
CTTGAG(N)14NN
YACGTR
GATNNNNATC
GGTCTCNNNNN
NNN(N)9AC(N)5CTCC(N)7NNN
GAGGAG(N)8NN
CCCAGC
GWGCWC
CGTACG
GTCTCNNNNN
CGTCTCNNNNN
CTCAG(N)7NN
TCATGA
CCGCTC
TGTACA
GCGCGC
CACGAG
GCANNNNNTGC
TTCGAA
GGTNACC
RGATCY
GTATAC
CCTNAGG
CTNAG
TTTAAA
CACNNNGTG
GACNNNNNNGTC
CGGCCG
GAGCTC
CCTNNNNNAGG
RGGNCCY
GAATTC
GATATC
CCCGCNNNNNN
GGCCGGCC
GTYRAC
AAGCTT
GTTAAC
CAATTG
ACGCGT
TCCRAC(N)18NN
TGGCCA
CATATG
GCTAGC
GCGGCCGC
ATGCAT
RCATGY
TTAATTAA
CTCGAG
ACATGT
GACNNNGTC
GTTTAAAC
CACGTG
RGGWCCY
GACNNNNGTC
TTATAA
GGGCCC
VCTCGAGB
CTGCAG
CAGCTG
CGGWCCG
GAGCTC
GTCGAC
CCTGCAGG
AGTACT
ACCWGGT
CTRYAG
GGCCNNNNNGGCC
CTYRAG
TACGTA
ACTAGT
GCATGC
AGGCCT
ATTTAAAT
GACNNNGTC
TCTAGA
CTCGAG
GAANNNNTTC
GACGTC

Sequncia com os stios de

restrio
C TCGA G CTT ACC TGC CTG TCA GCA TCC G GCAGCCTGCACCTACACCATCGATAGCGAATGGTCCACCGG
CTTTACCGCCAATATCACCCTCAAAAACGATACCGGTGCCGCCATCAATAACTGGAACGTGAATTGGCAATACTCCAGCAATCGCA
TGACCAGCGGCTGGAATGCCAACTTCTCCGGCACCAACCCCTACAACGCCACCAACATGAGCTGGAACGGCAGCATCGCGCCAG
GACAATCCATCTCCTTCGGCCTCCAGGGCGAAAAAAATGGCAGCACCGCCGAGCGACCAACCGTCACCGGCGCC
GCTTGTAACAGTGCAACCACCAGCTCTGTGGCTTCCAGCTCTTCAACACCCACCACCAGTTCATCTTCTGCATCCAGTGTGGCCT
CCGCACTGCTGTTGCAAGAAGCACAAGCCGGTTTCTGCCGTGTGGATGGCACCATCGATAATAACCACACCGGCTTTACCGGCA
GTGGCTTTGCCAACACCAACAATGCCCAGGGGGCAGCGGTAGTCTGGGCGATAGATGCTACCAGCAGTGGCCGTC
GCACCCTGACTATCCGCTATGCCAATGGTGGAACCGCCAATCGCAATGGCTCACTGGTGATTAACGGCGGCAGCAACGGTAACTA
TACGGTGAGTTTGCCCACGACCGGCGCCTGGACCACCTGGCAAACCGCAACTATCGATGTGGATTTGGTACAGGGCAATAATATT
GTGCAGTTGTCTGCAACGACAGCCGAAGGCTTACCCAATATAGATTCGTTAAGTGTTGTGGGTGGTACGGTCAG
GGCAGGTAATTGCGGCAGTGTGAGCAGCAGCAGTTCCGTGCAATCGTCATCGTCATCGAACAGCAGTGCAGGTGCAGCAGTAC
CCAGTGCCGGTTGCGGTAAACCGCGCACACTGCAAAACGGCAGGATTACATTGCAAAGCGGCGGTACACAGCGCAGCTACATCC
TGGATGCACCGACTAACTACAACAATACTCGTCCCTATCGGGTGATCTTCGGTTATCACTGGGTCGGTGGTACCGCC
GATGATGTAGCGACCGGCCAAACGGTTCAACGCAATACCTGGGCTCACTACGGGATGAAAAAGTTGTCTGGTGACTCCACGATT
TTTGTGGCGCCACAGGGTATAGGTAACGGGTGGGGCAATGGTGGCGGTGCAGATGTCACGTTTACCGATAATATTCTTGCGCAA
TTAAAAACAGAATTGTGTATCGACGAGAGCCGCATTTTTGCCAACGGTTTTAGCTTCGGCGGATCAATGACCTATG
CTATTGCCTGCGCCCGGGCCAATGTATTCCGCGGTGTTGCCGCCTACGGTGCAGGCTCAATCAGCGGTTGCTCCGGAGGCAATA
GCCCTATCGCTTATTTCGGCGGCCATGGGATTCGCGACAATGTGTTTACCCCGGATCGCGGTCGCGCATTGCGCGATCGGTTTG
TCAGAAACAATAGCTGTACCGTCATCAACCCACCCGAACCCTCCATCGGCAGCCTGCGTCACACCTGTACCAGCTA
TCAATGCCCCAATAAAAACTATCCTGTACGCTGGTGTGCCTATGATGCCGGCCATATTGCAGCACCCCATGATGGCTCGACTGGCG
ATAGTGGCAATACCTGGCTTGCCGAA CTT ACC TGC CTG TCA GCA TCC G CA TA TG

Peso Molecular e PI

Number of amino acids: 558

Molecular weight: 57998.3
Theoretical pI: 7.61

N-glicosilaes
# name
Cmax
Sequence 0.092

pos ?
17 N

Ymax
0.209

pos ?
17 N

Smax
0.944

pos ?
1
Y

Smean ?
0.578 Y

D
?
0.393 N

Asn-Xaa-Ser/Thr sequons in the sequence output below are highlighted in

blue. Asparagines predicted to be N-glycosylated are highlighted in red.

O-glicosilaes

Sequncia 2
ATGGCGAAGTTCCAATCGTTGCTCTCCTGCGTCACCCTTCTTTTCGCCGCCTCAGCCCATG
CGGGCATTGGCCCCAAGGCCGACCTTACCATTTCCAACGCGAACATCGCCCCTGATGGCTA
CACCCGTGCCGCCGTTGTGGTGAATGGTGTCTTCCCTGGGCCGCTCATCACAGGGAACAAG
GGAGACCGTTTCCAGCTGAATGTCATCGACCAACTGACGAACCACACAATGCTGAAGACCA
CCAGCATTCATTGGCATGGCTTTTTCCAGAAGGGCACGAACTGGGCGGATGGTCCCGCGTT
CATCAACCAGTGTCCGATTGCTAGCGGGCACTCGTTCCTCTACGATTTCCAGGTTCCGGAT
CAGGCCGGCACTTTTTGGTACCACAGCCATCTCTCCACGCAGTACTGTGACGGTCTCAGGG
GTCCATTCGTGGTATATGACCCTAAGGACCCCCTCAAGGGACTGTACGACGTCGACAACGA
CTCGACTGTGATCACCCTCTCCGACTGGTATCACGTGGCTGCCAGGCTTGGACCGAGCTTC
CCGCTCGGCTCGGACTCGACTCTCATCAATGGCCTTGGCCGTAGCACTACCAACGCTACCG
CCGGCCTCGCTGTTATCAACGTCACACAGGGCAAACGTTATCGCTTCCGCCTTGTGTCCTT
GTCATGCGACCCCAACTACACCTTCAGCATCGACGGCCATGACATGTCCGTTATTGAGGCG
GATGGTATTGCAACGCAACCCGTGACCGCGAACGCTATTCAAATCTTCTCTGCTCAACGAT
ATTCTTTCGTGCTGACTGCAAATCAGACAATTGGCAACTATTGGATTCGCGCCAACCCGAG
CTTTGGAAATATTGGTTTCACGAATGGAATCAACTCTGCCATCCTGCGCTACTCGGGAGCG
GATCCCATCGAACCTACGACGGCCCAACAAACCACACAGAACCTCCTCAATGAGGTCGACC
TCCACCCCTTTGTCGCTAAACAGACGCCTGGCCGCGCTACACAGGGTGGTACCGATGTGGC
CATCAACATGGTCTTCAACTTTAACGGCTCGAACTTCTTCATCAACAACGCGTCCTTCACG
CCTCCCACTGTCCCTGTCCTCCTTCAGATTTTGAGCGGCGCACAGGCCGCCCAGGACCTCC
TGCCTTCCGGAAGTGTCTACACGCTGCCGATCAACAAGTCCATCGAGCTCACCTTCCCCGC
CACGGTCAACGCCCCCGGGGCTCCCCACCCCTTCCACCTGCACGGTCATTCGTTCGCTGTG
GTCCGCAGCGCCGGCTCCACAGAATACAACTATAACAATCCCGTATGGCGCGACGTCGTTT
CGACCGGCACCCCTGCAGCGGGCGACAACGTCACGATCCGCTTCCAGACCGACAACCCCGG
ACCGTGGTTCCTCCATTGCCACATCGACTTCCACCTCGAGGCGGGCTTCGCTGTCGTGTTC
GCCGAGGACACCGCTGATACTTCTCTGGCGAACCATGTCCCACAAGCATGGTCGGATCTTT
GCCCGACGTACGATGCGCTCTCGGCTGATGATCACTGA

laccase [Ganoderma lucidum] Fungo filamentoso

GenBank: ACR24357.1
GenPept Identical Proteins Graphics
>gi|237861575|gb|ACR24357.1| laccase [Ganoderma lucidum]
MAKFQSLLSCVTLLFAASAHA (peptdeo sinal)
GIGPKADLTISNANIAPDGYTRAAVVVNGVFPGPLITGNKGDRFQLNVI
DQLTNHTMLKTTSIHWHGFFQKGTNWADGPAFINQCPIASGHSFLYDFQVPDQAGTFWYHSHLSTQYCD
G LRGPFVVYDPKDPLKGLYDVDNDSTVITLSDWYHVAARLGPSFPLGSDSTLINGLGRSTTNATAGLAVIN
VTQGKRYRFRLVSLSCDPNYTFSIDGHDMSVIEADGIATQPVTANAIQIFSAQRYSFVLTANQTIGNYWI
RANPSFGNIGFTNGINSAILRYSGADPIEPTTAQQTTQNLLNEVDLHPFVAKQTPGRATQGGTDVAINMV
FNFNGSNFFINNASFTPPTVPVLLQILSGAQAAQDLLPSGSVYTLPINKSIELTFPATVNAPGAPHPFHL
HGHSFAVVRSAGSTEYNYNNPVWRDVVSTGTPAAGDNVTIRFQTDNPGPWFLHCHIDFHLEAGFAVVFAE
DTADTSLANHVPQAWSDLCPTYDALSADDH

# Measure
max. C
max. Y
max. S
mean S
D

Position
22
22
16
1-21
1-21

Value
0.644
0.747
0.933
0.868
0.812

Cutoff signal peptide?

0.450 YES

Name=Sequence SP='YES' Cleavage site between pos. 21 and 22: AHA-GI D=0.812 D-cutoff=0.450 Networks=SignalPnoTM # data

Clivar entre 21 e 22 21x3 = 63

Vermelho = peptdeo sinal
Azul = primers
ATGGCGAAGTTCCAATCGTTGCTCTCCTGCGTCACCCTTCTTTTCGCCGCCTCAGCCCATG
CG GGCATTGGCCCCAAGGCCGACCTTACCATT TCCAACGCGAACATCGCCCCTGATGGCTA
CACCCGTGCCGCCGTTGTGGTGAATGGTGTCTTCCCTGGGCCGCTCATCACAGGGAACAAG
GGAGACCGTTTCCAGCTGAATGTCATCGACCAACTGACGAACCACACAATGCTGAAGACCA
CCAGCATTCATTGGCATGGCTTTTTCCAGAAGGGCACGAACTGGGCGGATGGTCCCGCGTT
CATCAACCAGTGTCCGATTGCTAGCGGGCACTCGTTCCTCTACGATTTCCAGGTTCCGGAT
CAGGCCGGCACTTTTTGGTACCACAGCCATCTCTCCACGCAGTACTGTGACGGTCTCAGGG
GTCCATTCGTGGTATATGACCCTAAGGACCCCCTCAAGGGACTGTACGACGTCGACAACGA
CTCGACTGTGATCACCCTCTCCGACTGGTATCACGTGGCTGCCAGGCTTGGACCGAGCTTC
CCGCTCGGCTCGGACTCGACTCTCATCAATGGCCTTGGCCGTAGCACTACCAACGCTACCG
CCGGCCTCGCTGTTATCAACGTCACACAGGGCAAACGTTATCGCTTCCGCCTTGTGTCCTT
GTCATGCGACCCCAACTACACCTTCAGCATCGACGGCCATGACATGTCCGTTATTGAGGCG
GATGGTATTGCAACGCAACCCGTGACCGCGAACGCTATTCAAATCTTCTCTGCTCAACGAT
ATTCTTTCGTGCTGACTGCAAATCAGACAATTGGCAACTATTGGATTCGCGCCAACCCGAG
CTTTGGAAATATTGGTTTCACGAATGGAATCAACTCTGCCATCCTGCGCTACTCGGGAGCG
GATCCCATCGAACCTACGACGGCCCAACAAACCACACAGAACCTCCTCAATGAGGTCGACC
TCCACCCCTTTGTCGCTAAACAGACGCCTGGCCGCGCTACACAGGGTGGTACCGATGTGGC
CATCAACATGGTCTTCAACTTTAACGGCTCGAACTTCTTCATCAACAACGCGTCCTTCACG
CCTCCCACTGTCCCTGTCCTCCTTCAGATTTTGAGCGGCGCACAGGCCGCCCAGGACCTCC
TGCCTTCCGGAAGTGTCTACACGCTGCCGATCAACAAGTCCATCGAGCTCACCTTCCCCGC
CACGGTCAACGCCCCCGGGGCTCCCCACCCCTTCCACCTGCACGGTCATTCGTTCGCTGTG
GTCCGCAGCGCCGGCTCCACAGAATACAACTATAACAATCCCGTATGGCGCGACGTCGTTT
CGACCGGCACCCCTGCAGCGGGCGACAACGTCACGATCCGCTTCCAGACCGACAACCCCGG
ACCGTGGTTCCTCCATTGCCACATCGACTTCCACCTCGAGGCGGGCTTCGCTGTCGTGTTC
GCCGAGGACACCGCTGATACTTCTCTGGCGAACCATGTCCCACAAGCATGGTCGGATCTTT
GCCCGACGTACGATGCGCTCTCGGCTGATGATCACTGA

The Sequence Manipulation Suite: Reverse Complement

Results for 30 residue sequence starting "TACGATGCGC".
5-3 da fita de baixo revertida:
TCAGTGATCATCAGCCGAGAGCGCATCGTA
5-3 da fita de cima (forward):
GGCATTGGCCCCAAGGCCGACCTTACCATT

Primer forward
Sequence: 5'- GGC ATT GGC CCC AAG GCC G -3'
Complement: 5'- CGG CCT TGG GGC CAA TGC C -3'
Melt temperature: 65.3C

Primer reverse
Sequence: 5'- TCA GTG ATC ATC AGC CGA GAG CGC ATC GTA 3
Complement: 5'- TAC GAT GCG CTC TCG GCT GAT GAT CAC
TGA -3'
Melt temperature: 65.3C

Sequncia com os primers

GGC ATT GGC CCC AAG GCC G

TCCAACGCGAACATCGCCCCTGATGGCTACACCCGTGCCGCCGTTGTGGTGAATGGTGTCTTCCC
TGGGCCGCTCATCACAGGGAACAAGGGAGACCGTTTCCAGCTGAATGTCATCGACCAACTGACG
AACCACACAATGCTGAAGACCACCAGCATTCATTGGCATGGCTTTTTCCAGAAGGGCACGAACTG
GGCGGATGGTCCCGCGTTCATCAACCAGTGTCCGATTGCTAGCGGGCACTCGTTCCTCTACGATT
TCCAGGTTCCGGATCAGGCCGGCACTTTTTGGTACCACAGCCATCTCTCCACGCAGTACTGTGAC
GGTCTCAGGGGTCCATTCGTGGTATATGACCCTAAGGACCCCCTCAAGGGACTGTACGACGTCGA
CAACGACTCGACTGTGATCACCCTCTCCGACTGGTATCACGTGGCTGCCAGGCTTGGACCGAGC
TTCCCGCTCGGCTCGGACTCGACTCTCATCAATGGCCTTGGCCGTAGCACTACCAACGCTACCG
CCGGCCTCGCTGTTATCAACGTCACACAGGGCAAACGTTATCGCTTCCGCCTTGTGTCCTT
GTCATGCGACCCCAACTACACCTTCAGCATCGACGGCCATGACATGTCCGTTATTGAGGCG
GATGGTATTGCAACGCAACCCGTGACCGCGAACGCTATTCAAATCTTCTCTGCTCAACGAT
ATTCTTTCGTGCTGACTGCAAATCAGACAATTGGCAACTATTGGATTCGCGCCAACCCGAG
CTTTGGAAATATTGGTTTCACGAATGGAATCAACTCTGCCATCCTGCGCTACTCGGGAGCG
GATCCCATCGAACCTACGACGGCCCAACAAACCACACAGAACCTCCTCAATGAGGTCGACC
TCCACCCCTTTGTCGCTAAACAGACGCCTGGCCGCGCTACACAGGGTGGTACCGATGTGGC
CATCAACATGGTCTTCAACTTTAACGGCTCGAACTTCTTCATCAACAACGCGTCCTTCACG
CCTCCCACTGTCCCTGTCCTCCTTCAGATTTTGAGCGGCGCACAGGCCGCCCAGGACCTCC
TGCCTTCCGGAAGTGTCTACACGCTGCCGATCAACAAGTCCATCGAGCTCACCTTCCCCGC
CACGGTCAACGCCCCCGGGGCTCCCCACCCCTTCCACCTGCACGGTCATTCGTTCGCTGTG
GTCCGCAGCGCCGGCTCCACAGAATACAACTATAACAATCCCGTATGGCGCGACGTCGTTT
CGACCGGCACCCCTGCAGCGGGCGACAACGTCACGATCCGCTTCCAGACCGACAACCCCGG
ACCGTGGTTCCTCCATTGCCACATCGACTTCCACCTCGAGGCGGGCTTCGCTGTCGTGTTC
GCCGAGGACACCGCTGATACTTCTCTGGCGAACCATGTCCCACAAGCATGGTCGGATCTTT
GCCCGACG TCA GTG ATC ATC AGC CGA GAG CGC ATC GTA

Escolha do vetor
Vetor escolhido: fungos
filamentosos
(Emprega-se bactria antes para
replicar o vetor)

Stios de Restrio
#

Xbal

Primer inicial com stio

restritivo no comeo:

Hind III:

Primer final com stio restritivo

no comeo:

Enzyme

Specificity

1 AfeI

AGCGCT

2 AflII

CTTAAG

3 AgeI

ACCGGT

4 AhdI

GACNNNNNGTC

5 AleI

CACNNNNGTG

6 AlwNI

CAGNNNCTG

7 ApaI

GGGCCC

8 ApaLI

GTGCAC

9 ApoI

RAATTY

10 AscI

GGCGCGCC

11 AseI

ATTAAT

12 AsiSI

GCGATCGC

13 AvrII

CCTAGG

14 BbvCI

CCTCAGC

15 BcgI

NN(N)10CGA(N)6TGC(N)10NN

16 BciVI

GTATCC(N)5N

17 BglII

AGATCT

18 BlpI

GCTNAGC

19 BmgBI

CACGTC

20 BpmI

CTGGAG(N)14NN

21 Bpu10I

CCTNAGC

22 BsaBI

GATNNNNATC

23 BseYI

CCCAGC

24 BsiWI

CGTACG

25 BsmBI

CGTCTCNNNNN

26 BspDI

ATCGAT

27 BspHI

TCATGA

28 BspQI

GCTCTTCNNNN

29 BsrGI

TGTACA

30 BssHII

GCGCGC

31 BssSI

CACGAG

32 BstAPI

GCANNNNNTGC

33 BstBI

TTCGAA

34 BstEII

GGTNACC

35 BstZ17I

GTATAC

36 BtsI

GCAGTGNN

37 ClaI

ATCGAT

38 CspCI

NN(N)11CAA(N)5GTGG(N)10NN

39 DraI

TTTAAA

40 DrdI

GACNNNNNNGTC

41 EagI

CGGCCG

42 EarI

CTCTTCNNNN

43 EcoP15I

CAGCAG(N)25NN

44 EcoRI

GAATTC

45 EcoRV

GATATC

46 FseI

GGCCGGCC

47 FspI

TGCGCA

48 HindIII

AAGCTT

49 HpaI

GTTAAC

50 KasI

GGCGCC

51 NarI

GGCGCC

52 NcoI

CCATGG

53 NdeI

CATATG

54 NotI

GCGGCCGC

55 NruI

TCGCGA

56 NsiI

ATGCAT

57 PacI

TTAATTAA

58 PflMI

CCANNNNNTGG

59 PluTI

GGCGCC

60 PmeI

GTTTAAAC

61 PshAI

GACNNNNGTC

62 PsiI

TTATAA

63 PspOMI

GGGCCC

64 PvuI

CGATCG

65 SacII

CCGCGG

66 SapI

GCTCTTCNNNN

67 SbfI

CCTGCAGG

68 SexAI

ACCWGGT

69 SfiI

GGCCNNNNNGGCC

70 SfoI

GGCGCC

71 SgrAI

CRCCGGYG

72 SnaBI

TACGTA

73 SpeI

ACTAGT

74 SphI

GCATGC

75 StuI

AGGCCT

76 SwaI

ATTTAAAT

77 XbaI

TCTAGA

78 XcmI

CCANNNNNNNNNTGG

Peso Molecular e PI

Number of amino acids: 499

Molecular weight: 53981.1
Theoretical pI: 5.43

N-glicosilaes

# name Cmax pos ?

Sequence 0.611 24 Y

Ymax
0.050

pos ?
38 N

Smax
0.097

pos ?
4 N

Smean ?
0.049 N

D
?
0.050 N

O-glicosilaes

Respostas

1)

Stios de restrio

Stios de reconhecimento para enzimas de restrio so curtas sequncias

reconhecidas e clivadas por vrias endonucleases. As enzimas de
restrio catalisam a quebra da ligao fosfoster entre o oxignio na
posio 3 de uma base e o fosfato ligado ao oxignio dan posio 5 da
base seguinte. O DNA a ser inserido deve ser cortado com as mesmas
duas enzimas empregadas para linearizar (abrir) o vetor, de forma que as
extremidades no se tornem incompatveis.

A escolha dos stios de restrio deve ser cuidadosa, para que no se

escolha um stio que clive a sequncia de DNA que quero inserir no vetor.
Alm do que, quanto mais nas extremidades do stio de insero do vetor
melhor. Analisando os vetores escolhidos para as sequncias I e II
(bacteriano e fungo filamentoso), vemos que nos seus stios,
respectivamente, as extremidades so Xho I e Nco I, e Xbal e HindIII. No
entanto, na minha sequncia de DNA no existe a enzima Nco I, o que me
leva a optar pela seguinte (mais prxima da extremidade), Nce I.

2) Clula Hospedeira

A escolha da clula hospedeira depende da natureza do DNA de interesse. A

replicao da sequncia deve ser feita previamente em uma bactria, como a
Escherichia coli DH5-Alpha, que aumenta a estabilidade da insero e melhora
a qualidade do plasmdeo de DNA. As mutaes que essa linhagem da E. coli
apresenta (dlacZ, Delta M15, Delta lacZYA-argF, U169, recA1, endA1,
hsdR17(rK-mK+), supE44, thi-1, gryA96, relA1) correspondem s distintas
caractersticas que tornam a linhagem DH5-Alpha excelente para replicao
das sequncias.
Em seguida escolhe-se o vetor, de acordo com a natureza do DNA de interesse,
e outras caractersticas desejveis, como:

Baixo peso molecular (para facilitar o isolamento do vetor intacto);

Marcadores selecionveis;

Um ou mais stios de restrio em que o fragmento de DNA possa ser

inserido

As clulas hospedeiras escolhidas para carregar o vetor e expressar o DNA de

interesse foi, respectivamente, a Escherichia coli TOP10 (a sequencia de
bactria) e Aspergillus nidulans (sequncia de fungo filamentoso).

5) Tcnicas de purificao

As etapas necessrias para purificao de protenas consistem em extrao, separao e concentrao. A extrao consiste
em promover a liberao da protena do material biolgico; a separao das protenas pode ocorrer por caractersticas como
solubilidade, tamanho, carga eltrica ou afinidade.

Centrifugao diferencial (em velocidades progressivamente mais altas) fracionam o homogneo de clulas em seus
componentes (ncleos, citoesqueletos, mitocndrias, lisossomos, peroxissomos, microssomos, vesculas celulares, ribossomos,
viros e macromolculas grandes).

O Salting out um mtodo preliminar que pode levar precipitao pela perda da camada de solvatao devido presena
de um sulfato de amnia, por exemplo. Em seguida pode ser usada uma coluna cromatogrfica de filtrao (excluso com
base no peso molecular/tamanho da protena) ou troca inica (com base no ponto isoeltrico).

Para a sequncia 1 (peso molecular de 57kD) ou 2 (peso molecular de 53kD) com um gel Sephadex 200 (com separao de 5kD
a 600kD) ou Bio-Gel P100 (de 5kD a 200kD) ou Sepharose 6B (10kD a 4000kD).

J se fosse empregada uma resina para cromatografia de troca inica, que adsorve as protenas com carga contraria resina
enquanto as protenas com mesma carga saem da coluna, e posteriormente elui as protenas adsorvidas, poderamos
empregar uma resina DEAE celulose (pH<10), as protenas de pI 7,61 e 5,43 adquirem carga negativa e so adsorvidas,
enquanto as com cargas positivas so rapidamente eluidas.

Atravs de um espectrofotmetro possvel detectar os fragmentos de aminocidos presentes na amostra para se verificar se
h apenas a protena de interesse na amostra, ou seja, se a separao foi efetiva.

6) Grfico

A protena 1 (57kD) eluir em, aproximadamente, 23mL,

enquanto que a protena 2 (53kD) elui aos 27mL.

8) Atividade enzimtica

Sequncia 1 (ferruloyl esterase)

No consta no banco de dados

Sequncia 2 (laccase)

9) Como saber se a protena de interesse?

Se ela participa as reaes que a protena de interesse

deveria catalisar, pode-se deduzir que ela a protena
de interesse.

10) Estrutura 3D

Sequncia 1 (ferruloyl esterase de Aspergillus niger)

Sequncia 2 (laccase de Lentinus tigrinus)