Rapid Cloning For Protein Crystallography Using Type IIS Restriction Enzymes

Abstract

Recombinant proteins are used routinely in macromolecular crystallographic experiments. Efficiency in cloning thus becomes a valuable resource to a macromolecular crystallographer. The use of type IIS restriction enzymes (outside cutters) concurrent with ligation allows for rapid cloning. A vector can be modified easily to incorporate sites for outside cutters, such as BspQI or BsaI. Critical and unique to our cloning method is the upshift of reaction incubations to 50 °C where the ligase is inactivated while the restriction enzyme is still active. The result is that very low background of undesired cloning intermediates is observed. Multiple DNA molecules can be simultaneously joined with a simplicity and effectiveness that rivals overlap PCR when BsaI is used. Using type IIS enzymes thus provides great control, flexibility, and simplicity in cloning strategies.