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Horizontal Tabs
Molecular Biology
Crystal structure 6F58
Clone Source: Michael Miley, UNC
SGC Construct ID: TBXTA-c005
Protein Region: E41-S224
Vector: pET28a
Tag: C-terminal 6HIS
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)): MGELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSHHHHHH
DNA Sequence: CCATGGGCGAACTGCGTGTGGGTCTGGAAGAAAGCGAACTGTGGCTGCGCTTTAAAGAGCTGACCAACGAAATGATCGTGACCAAAAACGGCCGCCGCATGTTTCCTGTGCTGAAAGTTAACGTGAGCGGCCTGGACCCGAACGCCATGTACAGCTTCCTGCTGGATTTCGTTGCCGCAGATAACCACCGCTGGAAGTATGTGAACGGCGAATGGGTGCCGGGTGGCAAACCGGAACCGCAGGCACCTAGCTGCGTGTATATCCACCCGGATAGCCCTAACTTCGGTGCCCATTGGATGAAAGCCCCGGTGAGCTTCAGCAAGGTGAAGCTGACCAACAAACTGAACGGCGGCGGCCAAATCATGCTGAACAGCTTACATAAATATGAACCTCGCATCCACATTGTGCGTGTGGGCGGCCCGCAACGTATGATCACCAGCCATTGCTTCCCGGAGACACAGTTCATTGCCGTTACCGCCTACCAGAACGAGGAAATTACCGCACTGAAAATTAAATACAACCCTTTTGCAAAAGCCTTCTTAGATGCCAAAGAGCGCAGCCACCACCACCATCATCACTAATGAGGATCC
Crystal structure 6F59
SGC Construct ID: TBXTA-c006
Protein Region: E41-S224
Vector: pET28a
Tag: N-6HIS;N-Thrombin;N-T7 tag;C-6HIS
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag): MGELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGDPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSHHHHHH
DNA Sequence: CCATGGGCGAACTGCGTGTGGGTCTGGAAGAAAGCGAACTGTGGCTGCGCTTTAAAGAGCTGACCAACGAAATGATCGTGACCAAAAACGGCCGCCGCATGTTTCCTGTGCTGAAAGTTAACGTGAGCGGCCTGGACCCGAACGCCATGTACAGCTTCCTGCTGGATTTCGTTGCCGCAGATAACCACCGCTGGAAGTATGTGAACGGCGAATGGGTGCCGGGTGGCAAACCGGAACCGCAGGCACCTAGCTGCGTGTATATCCACCCGGATAGCCCTAACTTCGGTGCCCATTGGATGAAAGCCCCGGTGAGCTTCAGCAAGGTGAAGCTGACCAACAAACTGAACGGCGGCGGCCAAATCATGCTGAACAGCTTACATAAATATGAACCTCGCATCCACATTGTGCGTGTGGGCGACCCGCAACGTATGATCACCAGCCATTGCTTCCCGGAGACACAGTTCATTGCCGTTACCGCCTACCAGAACGAGGAAATTACCGCACTGAAAATTAAATACAACCCTTTTGCAAAAGCCTTCTTAGATGCCAAAGAGCGCAGCCACCACCACCATCATCACTAATGAGGATCC
DNA-binding domain, Protein-only Crystal form 1 (G177D):
SGC Construct ID: TBXTA-c020
Protein Region: E41-N211
Vector: pSUMO-LIC
Tag: N-terminal 6HIS and SUMO tags followed by a SUMO protease cleavage site
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)): MCSSHHHHHHGSGSGSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGGELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYN
Sequence after tag cleavage:
GELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYN
DNA Sequence: ATGTGCAGCAGCCATCATCATCATCATCACGGCAGCGGCAGCGGCTCTGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATATTAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAACACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACTCAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAATGGAGGAAGAAGATGTGATTGAAGTTTACCAGGAGCAAACGGGAGGTGGAGAACTGCGTGTGGGTCTGGAAGAAAGCGAACTGTGGCTGCGCTTTAAAGAGCTGACCAACGAAATGATCGTGACCAAAAACGGCCGCCGCATGTTTCCTGTGCTGAAAGTTAACGTGAGCGGCCTGGACCCGAACGCCATGTACAGCTTCCTGCTGGATTTCGTTGCCGCAGATAACCACCGCTGGAAGTATGTGAACGGCGAATGGGTGCCGGGTGGCAAACCGGAACCGCAGGCACCTAGCTGCGTGTATATCCACCCGGATAGCCCTAACTTCGGTGCCCATTGGATGAAAGCCCCGGTGAGCTTCAGCAAGGTGAAGCTGACCAACAAACTGAACGGCGGCGGCCAAATCATGCTGAACAGCTTACATAAATATGAACCTCGCATCCACATTGTGCGTGTGGGCGGCCCGCAACGTATGATCACCAGCCATTGCTTCCCGGAGACACAGTTCATTGCCGTTACCGCCTACCAGAACGAGGAAATTACCGCACTGAAAATTAAATACAACTGAAGCGGTGGCGGTCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAACACCACCACCACCACCACTGA
DNA-binding domain, Protein-only Crystal form 2 (WT G177):
SGC Construct ID: TBXTA-c021
Protein Region: E41-N211
Vector: pSUMO-LIC
Tag: N-terminal 6HIS and SUMO tags followed by a SUMO protease cleavage site
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)): MCSSHHHHHHGSGSGSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGGELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGDPQRMITSHCFPETQFIAVTAYQNEEITALKIKYN
Sequence after tag cleavage: GELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGDPQRMITSHCFPETQFIAVTAYQNEEITALKIKYN
DNA Sequence: ATGTGCAGCAGCCATCATCATCATCATCACGGCAGCGGCAGCGGCTCTGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATATTAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAACACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACTCAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAATGGAGGAAGAAGATGTGATTGAAGTTTACCAGGAGCAAACGGGAGGTGGAGAACTGCGTGTGGGTCTGGAAGAAAGCGAACTGTGGCTGCGCTTTAAAGAGCTGACCAACGAAATGATCGTGACCAAAAACGGCCGCCGCATGTTTCCTGTGCTGAAAGTTAACGTGAGCGGCCTGGACCCGAACGCCATGTACAGCTTCCTGCTGGATTTCGTTGCCGCAGATAACCACCGCTGGAAGTATGTGAACGGCGAATGGGTGCCGGGTGGCAAACCGGAACCGCAGGCACCTAGCTGCGTGTATATCCACCCGGATAGCCCTAACTTCGGTGCCCATTGGATGAAAGCCCCGGTGAGCTTCAGCAAGGTGAAGCTGACCAACAAACTGAACGGCGGCGGCCAAATCATGCTGAACAGCTTACATAAATATGAACCTCGCATCCACATTGTGCGTGTGGGCGACCCGCAACGTATGATCACCAGCCATTGCTTCCCGGAGACACAGTTCATTGCCGTTACCGCCTACCAGAACGAGGAAATTACCGCACTGAAAATTAAATACAACTGAAGCGGTGGCGGTCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAACACCACCACCACCACCACTGA
Full-length, WT TBXT (G177)
SGC Construct ID: TBXTA-c026
Protein Region: S2- M435
Vector: pGTVL2
Tag: His6 and GST tag at the N-terminal fusion, followed by TEV protease cleavage site
Host: BL21(DE3)-R3-pRARE2
Note: Wild-type Full length TBXTA Codon Optimized DNA sequence
Sequence (with tag(s)):
MHHHHHHSSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPKSSSGVDLGTENLYFQSMSNSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM
Sequence after tag cleavage:
SMSNSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM
DNA Sequence:
CATATGCACCATCATCATCATCATTCTTCTATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGCCGACCATCCTCCAAAATCGAGCTCAGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGTCCAATAGCAGCCCTGGCACCGAAAGCGCCGGTAAGAGCCTACAGTACCGTGTGGACCATTTACTGAGCGCCGTGGAGAACGAACTACAGGCAGGCAGCGAAAAAGGTGACCCGACCGAACGCGAACTGCGTGTGGGTCTGGAAGAAAGCGAACTGTGGCTGCGCTTTAAAGAGCTGACCAACGAAATGATCGTGACCAAAAACGGCCGCCGCATGTTTCCTGTGCTGAAAGTTAACGTGAGCGGCCTGGACCCGAACGCCATGTACAGCTTCCTGCTGGATTTCGTTGCCGCAGATAACCACCGCTGGAAGTATGTGAACGGCGAATGGGTGCCGGGTGGCAAACCGGAACCGCAGGCACCTAGCTGCGTGTATATCCACCCGGATAGCCCTAACTTCGGTGCCCATTGGATGAAAGCCCCGGTGAGCTTCAGCAAGGTGAAGCTGACCAACAAACTGAACGGCGGCGGCCAAATCATGCTGAACAGCTTACATAAATATGAACCTCGCATCCACATTGTGCGTGTGGGCGGCCCGCAACGTATGATCACCAGCCATTGCTTCCCGGAGACACAGTTCATTGCCGTTACCGCCTACCAGAACGAGGAAATTACCGCACTGAAAATTAAATACAACCCTTTTGCAAAAGCCTTCTTAGATGCCAAAGAGCGCAGCGACCACAAAGAAATGATGGAGGAACCGGGCGACAGCCAGCAGCCTGGTTATAGCCAATGGGGCTGGCTGCTGCCGGGTACCAGTACACTGTGCCCGCCGGCAAACCCGCATCCTCAGTTTGGTGGCGCCCTGAGCTTACCGAGCACCCACAGTTGTGATCGCTATCCTACCCTGCGTAGCCATCGTAGCAGTCCGTACCCGAGCCCGTATGCACATCGTAACAACAGCCCGACATATAGCGACAACAGCCCGGCCTGTCTGAGCATGCTACAGAGCCATGATAACTGGAGCAGCCTGGGCATGCCGGCACATCCGAGCATGCTGCCGGTGAGTCACAATGCCAGCCCGCCGACCAGTAGCAGCCAGTATCCTAGCCTGTGGAGCGTTAGTAACGGTGCAGTGACCCCGGGTAGCCAGGCAGCAGCAGTTAGCAATGGTCTGGGCGCCCAGTTTTTTCGCGGCAGTCCGGCACATTACACCCCGCTGACCCATCCTGTGAGTGCCCCGAGCAGTAGTGGTAGCCCGCTGTATGAAGGTGCCGCAGCCGCCACCGACATTGTTGACAGCCAGTATGATGCCGCCGCACAGGGTCGCCTGATTGCAAGCTGGACCCCTGTGAGTCCGCCGAGCATGTGACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG
Full-length, G177D variant TBXT
SGC Construct ID: TBXTA-c025
Protein Region: S2- M435
Vector: pGTVL2
Tag: His6 and GST tag at the N-terminal fusion, followed by TEV protease cleavage site
Host: BL21(DE3)-R3-pRARE2
Note: G177D Full length TBXTA Codon Optimized DNA sequence
Sequence (with tag(s)):
MHHHHHHSSMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPKSSSGVDLGTENLYFQSMSNSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGDPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM
Sequence after tag cleavage:
SMSNSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGDPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM
DNA Sequence:
CATATGCACCATCATCATCATCATTCTTCTATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGCCGACCATCCTCCAAAATCGAGCTCAGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGTCCAATAGCAGCCCTGGCACCGAAAGCGCCGGTAAGAGCCTACAGTACCGTGTGGACCATTTACTGAGCGCCGTGGAGAACGAACTACAGGCAGGCAGCGAAAAAGGTGACCCGACCGAACGCGAACTGCGTGTGGGTCTGGAAGAAAGCGAACTGTGGCTGCGCTTTAAAGAGCTGACCAACGAAATGATCGTGACCAAAAACGGCCGCCGCATGTTTCCTGTGCTGAAAGTTAACGTGAGCGGCCTGGACCCGAACGCCATGTACAGCTTCCTGCTGGATTTCGTTGCCGCAGATAACCACCGCTGGAAGTATGTGAACGGCGAATGGGTGCCGGGTGGCAAACCGGAACCGCAGGCACCTAGCTGCGTGTATATCCACCCGGATAGCCCTAACTTCGGTGCCCATTGGATGAAAGCCCCGGTGAGCTTCAGCAAGGTGAAGCTGACCAACAAACTGAACGGCGGCGGCCAAATCATGCTGAACAGCTTACATAAATATGAACCTCGCATCCACATTGTGCGTGTGGGCGACCCGCAACGTATGATCACCAGCCATTGCTTCCCGGAGACACAGTTCATTGCCGTTACCGCCTACCAGAACGAGGAAATTACCGCACTGAAAATTAAATACAACCCTTTTGCAAAAGCCTTCTTAGATGCCAAAGAGCGCAGCGACCACAAAGAAATGATGGAGGAACCGGGCGACAGCCAGCAGCCTGGTTATAGCCAATGGGGCTGGCTGCTGCCGGGTACCAGTACACTGTGCCCGCCGGCAAACCCGCATCCTCAGTTTGGTGGCGCCCTGAGCTTACCGAGCACCCACAGTTGTGATCGCTATCCTACCCTGCGTAGCCATCGTAGCAGTCCGTACCCGAGCCCGTATGCACATCGTAACAACAGCCCGACATATAGCGACAACAGCCCGGCCTGTCTGAGCATGCTACAGAGCCATGATAACTGGAGCAGCCTGGGCATGCCGGCACATCCGAGCATGCTGCCGGTGAGTCACAATGCCAGCCCGCCGACCAGTAGCAGCCAGTATCCTAGCCTGTGGAGCGTTAGTAACGGTGCAGTGACCCCGGGTAGCCAGGCAGCAGCAGTTAGCAATGGTCTGGGCGCCCAGTTTTTTCGCGGCAGTCCGGCACATTACACCCCGCTGACCCATCCTGTGAGTGCCCCGAGCAGTAGTGGTAGCCCGCTGTATGAAGGTGCCGCAGCCGCCACCGACATTGTTGACAGCCAGTATGATGCCGCCGCACAGGGTCGCCTGATTGCAAGCTGGACCCCTGTGAGTCCGCCGAGCATGTGACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG
Plasmid depositions in Addgene
construct ID |
Domain |
variant |
Vector |
tags |
Crystallized |
Boundaries |
Addgene |
TBXTA-c005 |
DBD |
WT |
pET28a |
C-His6 |
Yes/6F58 |
E41-S224 |
139754 |
TBXTA-c006 |
DBD |
G177D |
pET28a |
C-His6 |
Yes/6F59 |
E41-S224 |
139755 |
TBXTA-c019 |
DBD |
WT |
pSUMO-LIC |
N-His-SUMO |
E41-A214 |
139756 |
|
TBXTA-c020 |
DBD |
WT |
pSUMO-LIC |
N-His-SUMO |
Yes/ Fragment screening |
E41-N211 |
139757 |
TBXTA-c021 |
DBD |
G177D |
pSUMO-LIC |
N-His-SUMO |
Yes/ Fragment screening |
E41-N211 |
139758 |
TBXTA-c022 |
DBD |
G177D |
pSUMO-LIC |
N-His-SUMO |
E41-A214 |
139759 |
|
TBXTA-c023 |
DBD |
WT |
pGTVL2 |
N-His-GST-TEV |
E41-D225 |
139760 |
|
TBXTA-c024 |
DBD |
G177D |
pGTVL2 |
N-His-GST-TEV |
E41-D225 |
139761 |
|
TBXTA-c025 |
FL |
G177D |
pGTVL2 |
N-His-GST-TEV |
S2- M435 |
139762 |
|
TBXTA-c026 |
FL |
WT |
pGTVL2 |
N-His-GST-TEV |
S2- M435 |
139763 |
|
TBXTA-c027 |
FL |
G177D |
pHGT-Bio |
N-His-GST-TEV, C-Bio |
S2- M435 |
139764 |
|
TBXTA-c028 |
FL |
WT |
pHGT-Bio |
N-His-GST-TEV, C-Bio |
S2- M435 |
139765 |
|
TBXTA-c043 |
DBD |
WT |
pNIC-Bio3 |
N-His-TEV, C-Bio |
E41- D225 |
139766 |
|
TBXTA-c044 |
DBD |
G177D |
pNIC-Bio3 |
N-His-TEV, C-Bio |
E41- D225 |
139767 |
Protein Expression and Purification
Medium: Terrific Broth (TB) Merck with 4 ml of glycerol
Antibiotics: Kanamycin, 50 µg/ml
From the glycerol stock, bacteria were inoculated in 15 ml of 1 x TB in a 50 ml tube with Kanamycin 0.05 mg/ml and 0.034 mg/ml of chloramphenicol and grown overnight in a shaker at 37°C, 250rpm. The following day, 4 ml of the overnight culture were inoculated in 1L of TB. The bacteria grew in an incubator at 37°C, shaking 180 rpm. Once the OD reached 2-3, IPTG (300uM) was added to the media and left overnight at 18°C, shaking 180 rpm. The pellets were harvested the next following day.
Protein Purification: DNA-binding domain constructs
The pellet was re-suspended in 250 ml of Lysis Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM Imidazole, 5% Glycerol and 1 mM TCEP). The cells, on ice, were sonicated for 20 minutes with 5 seconds pulse ON and 10 seconds pulse OFF with 35% of amplitude and centrifuged for 25 minutes at 66700 x g. The supernatant was incubated for an hour at 4°C, with Nickel beads pre-washed with Lysis buffer. After one hour of batch-binding, the tubes containing the lysate centrifuged at 700 x g at 4°C for 5 minutes and the supernatant discarded. This step was repeated twice with, respectively, 100 ml and 50 ml of Lysis Buffer.
Beads were loaded on a gravity column with 20 ml of Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% Glycerol and 1 mM TCEP) and, followed by two elution of 10 ml each with Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM Imidazole, 5% Glycerol and 1 mM TCEP). After an SDS-PAGE gel, the elution containing the protein was concentrated with an Amicon 10kDa concentrator and loaded on a Hi Load 16/600 Superdex 75 pg column at 1 ml/min, collecting 2-ml fractions. The fractions containing the protein were pooled together and concentrated with an Amicon 10kDa concentrator to 10 mg/ml was reached. Protein aliquots were flash frozen in Liquid Nitrogen and stored at -80°C. The protein was confirmed by ESI-TOF intact mass spectrometry:
6F58: Predicted: 21987; observed: 21856 – corresponding to expected mass with loss of N-terminal methionine
6F59: Predicted: 22045; observed: 21914 – corresponding to expected mass with loss of N-terminal methionine.
Protein Purification for DNA-free Crystal forms 1 and 2
The pellet was re-suspended in 250 ml of Lysis Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM Imidazole, 5% Glycerol and 1 mM TCEP). The cells, on ice, were sonicated for 20 minutes with 5 seconds pulse ON and 10 seconds pulse OFF with 35% of amplitude and centrifuged for 25 minutes at 66700 x g. The supernatant was incubated for an hour at 4°C, with Nickel beads pre-washed with Lysis buffer. After one hour of batch-binding, the tubes containing the lysate centrifuged at 700 x g at 4°C for 5 minutes and the supernatant discarded. This step was repeated twice with, respectively, 100 ml and 50 ml of Lysis Buffer.
Beads were loaded on a gravity column with 20 ml of Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% Glycerol and 1 mM TCEP) and, followed by two elution of 10 ml each with Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM Imidazole, 5% Glycerol and 1 mM TCEP). Fractions containing TBXT were pooled and SUMO protease was added to a final mass ratio 1:150. Cleavage was performed overnight during dialysis into dialysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % Glycerol, 1 mM TCEP) using 3500 MWCO snakeskin dialysis tubing.
After dialysis the protein was concentrated with an Amicon 10kDa concentrator and loaded on a Hi Load 16/600 Superdex 75 pg column. The flow-rate of the gel filtration was 1 ml/min and the volume of the fractions collected was 2 ml. The fractions containing the protein were pulled together and concentrated with an Amicon 10kDa concentrator, until the concentration 12 mg/ml was reached. Protein aliquots were stored at -80°C after being flash frozen in Liquid Nitrogen. The protein was confirmed by ESI-TOF intact mass spectrometry
Crystal form 1, TBXTA-c020: Predicted: 19571.7; observed: 19571.8
Crystal form 2, TBXTA-c021: Predicted: 19629.7; observed: 19629.8
Protein Purification: Full-length protein constructs TBXTA-c025, TBXTA-c026
The cell pellet was re-suspended in 250 ml of Lysis Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM Imidazole, 5% Glycerol and 1 mM TCEP). The cells were sonicated on ice for 20 minutes with 5 seconds pulse ON and 10 seconds pulse OFF with 35% of amplitude and centrifuged for 25 minutes at 66700 x g. The supernatant was incubated for an hour at 4°C with Nickel-sepharose beads pre-washed with Lysis buffer. After one hour of batch-binding, the beads were collected by centrifugation (700 x g at 4°C for 5 minutes) and the supernatant was discarded. The beads were washed with 100 ml and 50 ml of Lysis Buffer.
The beads were then transferred to a gravity column with 20 ml of Wash Buffer 1 (50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% Glycerol and 1 mM TCEP). The beads were washed with 20 ml of a Wash Buffer 2 (50 mM HEPES pH 7.5, 500 mM NaCl, 60 mM Imidazole, 5% Glycerol and 1 mM TCEP). Washes were followed by 4 elution of 10 ml each with Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol and 1 mM TCEP) with increasing concentrations of Imidazole: 100 mM, 200 mM Imidazole, 300 mM and 500 mM Imidazole.
The 4 elutions were dialysed overnight in Dialysis Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol and 1 mM TCEP) with 0.5 mg of TEV for 20 mg of protein in a Cold Room.
The beads were washed with 20 ml of Lysis Buffer. The dialysed elution was loaded on the beads and washed with 20 ml of Lysis Buffer. The imidazole gradient was repeated 10 ml of Wash 1, 10 ml of Wash 2, followed by the same 10 ml elutions prior to the dialysis (100 mM Imidazole, 200 mM Imidazole, 300 mM Imidazole and 500 mM Imidazole). The fractions were analysed by SDS-PAGE; the cleaved protein was found in wash1 and wash 2.
The combined fractions were concentrated with an Amicon 30kDa concentrator and loaded on a Hi Load 16/600 Superdex 200 pg column with Gel Filtration Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol and 1 mM TCEP) at 1.2 ml/min, collecting 2 ml fractions. The fractions containing the protein were pooled together and concentrated with an Amicon 30kDa concentrator, to 10 mg/ml. Protein aliquots were stored at -80°C after being flash frozen in Liquid Nitrogen. The protein was confirmed by ESI-TOF intact mass spectrometry
TBXTA-c025: Predicted: 47789; observed: 47790.
TBXTA-c026: Predicted: 47731; observed: 47732
Structure Determination
6F58:
Crystallisation: A self-complementary DNA oligonucleotide 5’- AATTTCACACCTAGGTGTGAAATT was dissolved to 1mM, heated to 95°C on a heat block and allowed to cool slowly over 2hrs. The protein and DNA were mixed in a 1:1.1 molar ratio (assuming a duplex DNA molecule) and sitting drop vapour diffusion crystallisation trials were set up with a Mosquito (TTP Labtech) crystallisation robot at a final concentration of 6.6 mg/ml. TBXT crystallised at 4°C in conditions containing 40% PEG300 -- 0.1M citrate pH 4.2. Crystals were loop mounted and cryo-cooled by plunging directly into liquid nitrogen.
Data Collection: Data were collected to 2.2Å resolution at Diamond light source beamline
I04-1 and processed using XDS
Data Processing: The structure was solved by molecular replacement using the program PHASER and the structure of Xenopus laevis brachyury (1XBR) as a search model. Refinement was performed using PHENIX REFINE to a final Rfactor = 24.2%, Rfree = 28.8%.
6F59 (G177D variant):
Crystallisation: A self-complementary DNA oligonucleotide 5’- GAATTTCACACCTAGGTGTGAAATTC was dissolved to 1mM, heated to 95°C on a heat block and allowed to cool slowly over 2hrs. The protein and DNA were mixed in a 1:1.1 molar ratio (assuming a duplex DNA molecule) and sitting drop vapor diffusion crystallsation trials were set up with a Mosquito (TTP Labtech) crystallization robot at a final concentration of 8 mg/ml. TBXT crystallised at 4°C in conditions containing 56% MPD, 0.1 M SPG pH 6.0. Crystals were loop mounted and cryo-cooled by plunging directly into liquid nitrogen.
Data Collection: Data were collected to 2.1Å resolution at Diamond light source beamline
I04-1 and processed using DIALS.
Data Processing: The structure was solved by molecular replacement using the program PHASER and the structure of Xenopus laevis brachyury (1XBR) as a search model. Refinement was performed using PHENIX REFINE to a final Rfactor = 22.1%, Rfree = 25.2%.
Crystal form 1 for fragment screens (G177D, no DNA)
Crystallisation: The protein was adjusted to 7.5 mg/ml and sitting drop vapor diffusion crystallisation trials were set up with a Mosquito (TTP Labtech) crystallisation. TBXT crystallised at 4°C in conditions containing 32% PEG400, 0.1M acetate pH 4.5, 0.1 M cadmium chloride. Crystals were loop mounted and cryo-cooled by plunging directly into liquid nitrogen.
Fragment soaking: Fragments from the DSI-Poised library were added to the crystallisation drops by acoustic dispensing using an ECHO acoustic liquid handler from a 500 mM stock concentration dissolved in DMSO to a final concentration of 10%. Soaking times varied from 1.5 to 4 hours.
Data Collection: Data were collected at Diamond light source beamline I04-1 and processed using the XChem Explorer pipeline.
Data Processing: Structures were solved by difference Fourier synthesis using the XChem Explorer pipeline. Fragment hits were identified using the PanDDA program. Refinement was performed using REFMAC.
Crystal form 2 for fragment screens (WT, no DNA)
Crystallisation: The protein was adjusted to 16 mg/ml and sitting drop vapor diffusion crystallisation trials were set up with a Mosquito (TTP Labtech) crystallisation robot. TBXT crystallised at 4°C in conditions containing 30% PEG1000, 0.1M SPG pH 7.0. Crystals were loop mounted and cryo-cooled by plunging directly into liquid nitrogen.
Fragment soaking: Fragments from the DSI-Poised library were added to the crystallisation drops by acoustic dispensing using an ECHO acoustic liquid handler from a 500 mM stock concentration dissolved in DMSO to a final concentration of 10%. Soaking times varied from 1.5 to 4 hours.
Data Collection: Data were collected at Diamond light source beamline I04-1 and processed using the XChem Explorer pipeline.
Data Processing: Structures were solved by difference Fourier synthesis using the XChem Explorer pipeline. Fragment hits were identified using the PanDDA program. Refinement was performed using REFMAC.
Assays
Electrophoretic mobility shift assays (EMSA)
The following oligonucleotide pair was used:
TA50-F CATGCATGCAGGGAATTTCACACCTAGGTGTGAAATTCCCATTCGTGCGA
TA50-R TCGCACGAATGGGAATTTCACACCTAGGTGTGAAATTCCCTGCATGCATG
To reduce the formation of hairpin structures, the oligos were annealed at a high concentration (200 µM each) in 10 mM tris-HCl, pH 7.5, 50 mM NaCl by heating to 95°C in a dry block and leaving to cool to room temperature. The dsDNA was subsequently labelled with T4 polynucleotide kinase (NEB) and γ-32P-ATP. The labelled DNA was separated from the remaining ATP/ADP using a BioRad MicroBiospin P-6 column equilibrated in annealing buffer. EMSA buffer was: 25 mM HEPES, pH 7.4, 10% glycerol, 75 mM NaCl, 0.1% tween 20, 1 mM TCEP.
Protein was diluted serially in this buffer and mixed with 1-5 nM of DNA diluted in the same buffer. After 10-minute incubation on ice, the samples (5 µl) were loaded on a pre-run 8% polyacrylamide gel (40:1 acrylamide/bis) in chilled TAE buffer (40 mM TRIS base, 20 mM acetic acid, 1 mM EDTA). The gel tanks were placed in an ice bucket and run for 75 minutes at 150V. The dried gels were exposed overnight using a BioRad phosphorimager screen.
Ligand binding by Surface Plasmon Resonance (SPR)
Machine
Biacore S200
Sensor surface
Series S SA sensor, Immobilized with ~1500 RU of biotinylated G177D TBXTA full length protein (immobilization at approximately 20 nM for 100 seconds)
Running buffer
10 mM Hepes pH 7.5, 150 mM NaCl, 1 mM DTT, 1% DMSO.
Protein:
Protein sequence
SMSNSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGDPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSMSSKGGYGLNDIFEAQKIEWHE*
*AviTag biotin conjugation sequence
The protein was expressed from construct TBXTA-c027, encoding a C-terminal Avitag which is biotinylated in E. coli when co-expressed with BirA enzyme in the presence of biotin. (Keates et al. PMID: 22027370). The protein was purified as described for TBXTA-c026.
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