Production of meganucleases by cell-free protein synthesis for functional and structural studies

Meganucleases are highly specific endonucleases that recognize and cleave long DNA sequences, making them powerful tools for gene targeting. We describe the production of active recombinant meganucleases suitable for functional and structural studies using a batch-based cell-free protein synthesis method. Isotopic labeling of the I-CreI meganuclease is demonstrated opening the way for structural and ligand binding studies in solution by nuclear magnetic resonance (NMR)2 which was previously hampered by the problems associated with the toxicity of the enzyme for Escherichia coli limiting its growth. The method can be adapted for the synthesis of soluble engineered variants that are produced as inclusion bodies in bacterial cells, thus facilitating their purification as soluble proteins instead of using denaturing-refolding protocols.

► Active meganucleases can be produced in extracts of Esherichia coli cells.
► Solubility of engineered meganucleases is improved in extracts with chaperones.
► Isotopic labeling of meganucleases for NMR studies is achieved in modified extracts.