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 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.

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.

In vivo Selection

Doyon JB et. al. J.A.C.S. (2006), 128: 2477-84.

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.

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.

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.

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

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

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

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 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.

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.

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 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

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

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