Large-scale production of recombinant Saw1 in Escherichia coli

- Recombinant full length Saw1 was over-expressed and purified in Escherichia coli.
- Dynamic light scattering and differential scanning fluorimetry identified buffer conditions for optimal Saw1 stability.
- Electro-mobility shift assays indicate that purified Saw1 binds DNA with structure specificity.

Saccharomyces cerevisiae Saw1 is an essential gene in single-strand annealing - the DNA repair pathway that repairs double-strand breaks when they occur between homologous repeats. Saw1 interacts with the structure-specific nuclease Rad1-Rad10 and this results in the recruitment of this nuclease to 3′ non-homologous DNA tailed recombination intermediates. Saw1 is unstable in the absence of the Rad1-Rad10 nuclease and, hence, it has been difficult to study its specific function in vitro. In the present work, we present the combination of dynamic light scattering and differential scanning fluorimetry techniques to optimize the stability and homogeneity of recombinant Saw1. The protein expression and purification conditions identified in this study allow for higher recovery of soluble Saw1 and enable the biochemical characterization of the protein.