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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.
Procedures
• Gene Synthesis
Two copies of ORFs (~65% identity)
optimized based on E.coli codon usage
with an internal MCS was synthesized
• Insertion of a linker library with 60
to 80 amino acid residues between
two copies of ORFs.
• High-throughput screening or/and
in vivo selection to identify active
HEs in monomeric form.
• Characterization of individual
selectants.
ACCGGT ACTAGTG GGTACC
AgeI
SpeI
KpnI
The Linker Library
V170
G175
Random sequences
encoding 60-120
amino acids
• After three-round selection against
SH2 ligand using phage display, a group
of peptides with 60-80 amino acid
residues were identified to have
minimal effects on SH2 domain stability.
SH2 domain
Scalley-Kim M. et. al. Protein Science (2003), 12: 197-206
In vivo Selection
Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84.
In Vivo Selection
• Control experiments
HEs
Positive 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
a Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84
Note: Two copies of target site were introduced into pCcdB vector.
Linkers Length of Monomeric HEs
Linker Length
18
16
Cre library
Mso library
# of linkers
14
12
10
8
6
4
2
0
13
33
53
Linker Length (Aa)
73
• 19 various linkers were identified from Cre library with length from 13 Aa to 73 Aa.
• 13 various linkers were identified from Mso library with length at 13 and 33 Aa.
The Composition of Randomized Portion of the Linkers
Amino Acids distribution (random portion)
25
Crelinker
Msolinker
20
%
15
10
5
0
Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr
Aa names
• Ala, Lys, Asn, Pro, and Thr dominant.
The Composition of Randomized Portion of the Linkers
Cre Library Aa Distribution
12
Percentage (%)
10
Selected
Naïve
8
6
4
2
0
Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr
Amino Acid
• Arg is selected for, and Gly is against.
The Composition of Randomized Portion of the Linkers
Mso Library Aa Distribution
20
18
Percentage (%)
16
14
Selected
Naïve
12
10
8
6
4
2
0
Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln Arg Ser Thr Val Trp Tyr
Amino Acid
• Thr is selected for, and Gly, Ser are against.
In vitro Cleavage
• 14 monomeric Cres, and 8 monomeric Msos were cloned into expression
vectors, respectively.
• All but one (Msomono#96) show in vitro cleavage activity
Verification of Protein Molecular Organization
Inactive
Active
In vitro Cleavage of Single Active Site Knock-out
Monomeric HEs
• Single active site knock-out mutation eliminate the cleavage activity in
Cremono#15, Msomono#24 and #27 completely, while partially in Cremono#6.
• All single active site knock-out mutants show nicking activity against
supercoiled substrate.
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
73 Aa linker doesn’t.
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.
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).
Monomeric CreI linkers
Linker
#2
#3
#6
#7
#14
Sequence
Frequency
TGSGSGSTNMKPPVRAFEPTGVRSRGSGSGSGT (33Aa)
6/84
TGSGSGSKSQAVAHPTDGQRDFGAKGSGSGSGT (33Aa)
5/84
TGSGSGSKPAGGDAPRLMQGVNRIDGSGSGSGT (33Aa)
19/84
TGSGSGSGSGSGT (13Aa)
29/84
TGSGSGSNPRNSPNSKTSMPIDVNNGSAYSMQSNRGYVKEEYLHRGSGS
GSGT (53Aa)
1/84
#15
#19
#45
#48
#53
#56
TGSGSGSKTKNMSPKANIERTPENKGSGSGSGT (33Aa)
7/84
TGSGSGSSTKERTNLKDNMTIDKPRGSGSGSGT (33Aa)
1/84
TGSGSGSKDVTQANRTYIPRENASRGSGSGSGT (33Aa)
1/84
TGSGSGSTDQAGHDPGAKTAKPMLGGSGSGSGT (33Aa)
1/84
TGSGSGSNYAAKPIPSAGQLETSHNGSGSGSGT (33Aa)
3/84
TGSGSGSIPQTQFHLVLGAAATRDNGSGISETNPRDPTQVSDKNIGSTVT
GQVVRTDSLEENKANGSGSGSGT (73Aa)
2/84
#65
TGSGSGSKTKNMSPSANIERTPDNKGSGSGSGT (33Aa)
1/84
Continued
Monomeric CreI linkers
Linker
Sequence
#81 TGSGSGSKYEGKAILSAGQLDTSYKGSGSGSGT (33Aa)
#90 TGSGSGSNNKSSHPQGDVEQKHQHSGSGSGSGT (33Aa)
Frequency
1/84
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 MsoI linkers
Linker
#3
#5
#14
#15
#24
#25
#27
#28
#29
#43
#55
#70
#96
Sequence
Frequency
TGSGSGSTAAKPPVRTTDGMESTFMGSGSGSGT(33Aa)
1/57
TGSGSGSGSGSGT(13Aa)
15/57
TGSGSGSAYTTTTDEAPTLVKPRHNGSGSGSGT (33Aa)
1/57
TGSGSGSKPTALNPWNIDRTTIPAKGSGSGSGT (13Aa)
6/57
TGSGSGSKHPTLTLPTTTSQENLPNGSGSGSGT (33Aa)
3/57
TGSGSGSRFAGESHVNNTTKTTKLEGSGSGSGT (33Aa)
9/57
TGSGSGSKTKNPHPENPGQSMTQAKGSGSGSGT (33Aa)
1/57
TGSGSGSRFAGESHVNNTTKTTKLEGSGSGSGT (33Aa)
3/57
TGSGSGSTHTTRHNRTPTAPNYRPIGSGSGSGT (33Aa)
1/57
TGSGSGSGFANKYNVDHNPLSNMNSGSGSGSGT (33Aa)
1/57
TGSGSGSKTKNPHPWNPDRSTTPAKGSGSGSGT (33Aa)
1/57
TGSGSGSTTQAPPTMTYTRGVATTDGSGSGSGT (33Aa)
1/57
TGSGSGSNLGAENAQSASQKDDALRGSGSGSGT (33Aa)
1/57