monomerization of homing endonucleases
DESCRIPTION
Monomerization of Homing Endonucleases. Gene synthesis. Protein Science (2003), 12: 197-206. Library size: ~1 ×10 6. J.A.C.S. (2006), 128: 2477-84. The Length and Aa Distribution of Linkers. 19 various linkers were identified from Cre library with length from 13 Aa to 73 Aa. - PowerPoint PPT PresentationTRANSCRIPT
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.