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Monomerization of Homing Endonucleases

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.

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Monomerization of Homing Endonucleases

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  1. Monomerization of Homing Endonucleases Gene synthesis Protein Science (2003), 12: 197-206 Library size: ~1×106 J.A.C.S. (2006), 128: 2477-84.

  2. 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.

  3. 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.

  4. Thermodynamic Values of Mso24 by ITC After Correction Mso24/WTDNA WTDNA only 1. Eastburg et. al. Nucleic Acid Res. (2007), 35(21): 7209-21

  5. 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.

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