Monomerization of Homing Endonucleases

Gene synthesis

Protein Science (2003), 12: 197-206

J.A.C.S. (2006), 128: 2477-84.

Library size: ~1×106

The Length and Aa Distribution of Linkers

• 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.

Monomeric Mso24

• mMso24 has a 33 Aa linker

between two Mso domains.

• mMso24 shows comparable site-

specific cleavage activity as WT

Mso.

• mMso24 exists as a monomer in

solution, verified by size-exclusion

chromatography.

Thermodynamic Values of Mso24 by ITC

Mso24/WTDNA WTDNA only After Correction

ProteinKD

nM

∆H

kcal/mol

∆S

cal/mol/deg

-T∆S

kcal/mol

∆G

kcal/mol

MsoWT1 21±5.0 12.7±0.27 77±1.1 -23.3 -10.6

mMso24 40.2±9.6 14.7±0.4 82.4 -25.0 -10.3

1. Eastburg et. al. Nucleic Acid Res. (2007), 35(21): 7209-21

Current Projects• Resolve mMso24 structure.

• Expression and purification of mCre3.

• Crystallography study.

• Biochemical study:

Binding affinity by isothermal titration calorimetry (ITC).

Thermal stability by circular dichroism (CD).

• Binding and Cleavage specificity matrix.

• Establish stable cell lines containing GFP-Cre and GFP-Mso cassettes.

Future Projects

• Engineering monomeric Cre and Mso towards novel DNA targets

using combinatorail and computational methos.