monomerization of homing endonucleases targets: crei, msoi, and ceui. goal: generation of...
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Monomerization of Homing Endonucleases
• Targets: CreI, MsoI, and CeuI.
• Goal: Generation of catalytically active monomeric HEs by connecting two
copies of protein with a linker.
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Procedures
• Gene Synthesis Two copies of ORFs (~65% identity)
optimized based on E.coli codon usage
with an internal MCS was synthesizedACCGGT ACTAGTG GGTACC AgeI SpeI KpnI
• Insertion of a linker library with 60 t
o 80 amino acid residues between two
copies of ORFs.
• High-throughput screening or/and
in vivo selection to identify active HE
s in monomeric form.
• Characterization of individual select
ants.
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The Linker Library
Scalley-Kim M. et. al. Protein Science (2003), 12: 197-206
SH2 domain
V170
G175
Random sequences encoding 60-120 amino acids
• After three-round selection against SH
2 ligand using phage display, a group of
peptides with 60-80 amino acid residues
were identified to have minimal effects
on SH2 domain stability.
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In vivo Selection
Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84.
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In Vivo Selection
HEsPositive Selection
(Active HEs)
Background
(Inactive HEs)
SceIa 20-40% 2.0×10-5
CreI 30-40% 1.5×10-4
MsoI ~30% 1.0×10-4
• Control experiments
a Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84
Note: Two copies of target site were introduced into pCcdB vector.
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Li nker Length
0
2
4
6
8
10
12
14
16
18
13 33 53 73
Li nker Length (Aa)
# of linkers
Cre l i braryMso l i brary
Linkers Length of Monomeric HEs
• 19 various linkers were identified from Cre library with length from 13 Aa to 73 A
a.
• 13 various linkers were identified from Mso library with length at 13 and 33 Aa.
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The Composition of Randomized Portion of the Linkers
Ami no Aci ds di st r i but i on ( r andom por t i on)
0
5
10
15
20
25
Al a Cys Asp Gl u Phe Gl y Hi s I l e Lys Leu Met Asn Pr o Gl n Ar g Ser Thr Val Tr p Tyr
Aa names
%
Cr el i nkerMsol i nker
• Ala, Lys, Asn, Pro, and Thr dominant.
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The Composition of Randomized Portion of the Linkers
• Arg is selected for, and Gly is against.
Cr e Li br ar y Aa Di st r i but i on
0
2
4
6
8
10
12
Al a Cys Asp Gl u Phe Gl y Hi s I l e Lys Leu Met Asn Pr o Gl n Ar g Ser Thr Val Tr p Tyr
Ami no Aci d
Percentage (%)
Sel ect ed
Naïve
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The Composition of Randomized Portion of the Linkers
• Thr is selected for, and Gly, Ser are against.
Mso Li br ar y Aa Di st r i but i on
0
2
4
6
8
10
12
14
16
18
20
Al a Cys Asp Gl u Phe Gl y Hi s I l e Lys Leu Met Asn Pr o Gl n Ar g Ser Thr Val Tr p Tyr
Ami no Aci d
Percentage (%)
Sel ect ed
Naïve
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In vitro Cleavage
• 14 monomeric Cres, and 8 monomeric Msos were cloned into expression ve
ctors, respectively.
• All but one (Msomono#96) show in vitro cleavage activity
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Verification of Protein Molecular Organization
Inactive
Active
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In vitro Cleavage of Single Active Site Knock-out Monomeric HEs
• Single active site knock-out mutation eliminate the cleavage activity in Cremon
o#15, Msomono#24 and #27 completely, while partially in Cremono#6.
• All single active site knock-out mutants show nicking activity against supercoil
ed substrate.
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In vitro Cleavage of Single Active Site Knock-out Monomeric HEs
• Monomeric HEs with 13 Aa linker show non-specific cleavage.
• Monomeric Cre with 53 Aa linker shows cleavage activity, while the one with 7
3 Aa linker doesn’t.
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Conclusion
• Majority of monomeric HEs show in vitro cleavage activity, validating the in vivo
selection system.
• Monomeric Hes with different linkers behave differently in term of the oligomeric
organization.
Future Works:
• Complete the cleavage profile of single active site knock-out mutants.
• In vivo activity in human cells using DR-GFP reporter system.
• Structural study.
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In vitro Cleavage
• WT CreI and MsoI were expressed from pET16b vector in BL21(DE3).
• Monomeric CreI and MsoI were expressed from pBAD vector in TOP10.
• Proteins were purified Ni-NTA kit (Qiagen).
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Monomeric CreI linkers
Linker Sequence Frequency
#2 TGSGSGSTNMKPPVRAFEPTGVRSRGSGSGSGT (33Aa) 6/84
#3 TGSGSGSKSQAVAHPTDGQRDFGAKGSGSGSGT (33Aa) 5/84
#6 TGSGSGSKPAGGDAPRLMQGVNRIDGSGSGSGT (33Aa) 19/84
#7 TGSGSGSGSGSGT (13Aa) 29/84
#14 TGSGSGSNPRNSPNSKTSMPIDVNNGSAYSMQSNRGYVKEEYLHRGSGSGSGT (53Aa)
1/84
#15 TGSGSGSKTKNMSPKANIERTPENKGSGSGSGT (33Aa) 7/84
#19 TGSGSGSSTKERTNLKDNMTIDKPRGSGSGSGT (33Aa) 1/84
#45 TGSGSGSKDVTQANRTYIPRENASRGSGSGSGT (33Aa) 1/84
#48 TGSGSGSTDQAGHDPGAKTAKPMLGGSGSGSGT (33Aa) 1/84
#53 TGSGSGSNYAAKPIPSAGQLETSHNGSGSGSGT (33Aa) 3/84
#56 TGSGSGSIPQTQFHLVLGAAATRDNGSGISETNPRDPTQVSDKNIGSTVTGQVVRTDSLEENKANGSGSGSGT (73Aa)
2/84
#65 TGSGSGSKTKNMSPSANIERTPDNKGSGSGSGT (33Aa) 1/84
Continued
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Linker Sequence Frequency
#81 TGSGSGSKYEGKAILSAGQLDTSYKGSGSGSGT (33Aa) 1/84
#90 TGSGSGSNNKSSHPQGDVEQKHQHSGSGSGSGT (33Aa) 1/84
#102 TGSGSGSTSARLYPQTTATMNDSTMGSGSGSGT (33Aa) 1/84
#119 TGSGSGSNPAMLADPKNTGLATGAIGSGSGSGT (33Aa) 1/84
#121 TGSGSGSNDTEMSSWTAERRTPRPTGSGSGSGT (33Aa) 1/84
#124 TGSGSGSNPGVRSPRNNDLPDHRLIGSGSGSGT (33Aa) 1/84
#125 TGSGSGSNAGNLPSRENNTSKHSAEGSGSGSGT (33Aa) 2/84
Monomeric CreI linkers
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Monomeric MsoI linkers
Linker Sequence Frequency
#3 TGSGSGSTAAKPPVRTTDGMESTFMGSGSGSGT(33Aa) 1/57
#5 TGSGSGSGSGSGT(13Aa) 15/57
#14 TGSGSGSAYTTTTDEAPTLVKPRHNGSGSGSGT (33Aa) 1/57
#15 TGSGSGSKPTALNPWNIDRTTIPAKGSGSGSGT (13Aa) 6/57
#24 TGSGSGSKHPTLTLPTTTSQENLPNGSGSGSGT (33Aa) 3/57
#25 TGSGSGSRFAGESHVNNTTKTTKLEGSGSGSGT (33Aa) 9/57
#27 TGSGSGSKTKNPHPENPGQSMTQAKGSGSGSGT (33Aa) 1/57
#28 TGSGSGSRFAGESHVNNTTKTTKLEGSGSGSGT (33Aa) 3/57
#29 TGSGSGSTHTTRHNRTPTAPNYRPIGSGSGSGT (33Aa) 1/57
#43 TGSGSGSGFANKYNVDHNPLSNMNSGSGSGSGT (33Aa) 1/57
#55 TGSGSGSKTKNPHPWNPDRSTTPAKGSGSGSGT (33Aa) 1/57
#70 TGSGSGSTTQAPPTMTYTRGVATTDGSGSGSGT (33Aa) 1/57
#96 TGSGSGSNLGAENAQSASQKDDALRGSGSGSGT (33Aa) 1/57