BioProject

Nucleases, i.e. enzymes that catalyze the hydrolysis of phosphodiester bonds in nucleic acids, are essential tools in molecular biology and biotechnology. More...

Nucleases, i.e. enzymes that catalyze the hydrolysis of phosphodiester bonds in nucleic acids, are essential tools in molecular biology and biotechnology. Staphylococcus aureus nuclease (MNase) is particularly interesting due to its thermostability and Ca2+-dependence, making it the prime choice for applications where nuclease modulation is critical, such as ribosome profiling in bacteria and halophilic archaea. The latter poses a technical and economical challenge, as high salt reaction conditions negatively impact MNase activity, necessitating large amounts of nuclease to be used for achieving efficient cleavage. Here, we set out to generate an optimized production protocol for two forms of MNase — fully processed MNaseA and the 19 aa propeptide containing MNaseB — and to biochemically benchmark them against a commercial nuclease. Our results show that both MNases are highly active in normal reaction conditions, but MNaseA maintains higher enzymatic activity in high salt concentrations than MNaseB. MNaseA also retains >90% of its activity after multiple freeze-thaw cycles when stored at -80°C in a buffer containing 5% glycerol. Importantly, ribosome profiling experiments in Haloferax volcanii demonstrated that MNaseA produces ribosome footprints highly comparable to those obtained with the commercial nuclease, making it a suitable alternative for high-salt ribosome profiling applications. In conclusion, our method can be easily implemented for efficient MNaseA production, thereby providing access to an effective, robust, and cost-efficient alternative to commercial nucleases, as well as facilitating future translation studies into halophilic organisms. Overall design: We have generated a construct design and optimized expression conditions and biochemical assaying to produce and characterize MNaseA (abbreviated MNA) for robust and reproducible utilization in molecular biology methods that require the presence of high salt concentrations. Furthermore, we compared purified MNA to a commercially available nuclease (Roche MNase, abbreviated MNR) and, as a proof-of-concept, applied it for Ribo-seq of halophilic model archaeon H. volcanii (Hv). In brief, we harvested Hv culture, cryogenically lysed the cells, and isolated ribosomes from lysate. Ribosomes were then footprinted with either MNA or MNR, then separated along a sucrose gradient. Ribosome footprints were isolated from gradient fractions corresponding to single ribosomes and used for library preparation. Our subsequent analysis of ribosome footprints produced by either MNA or MNR digestion shows that MNA matches and exceeds the performance of its commercial counterpart. Consequently, this work provides a simple protocol for the efficient production of highly active MNaseA, making future Ribo-seq studies of halophilic organisms more accessible. Less...