Purification of SerMNucA (Benzonase/Turbonuclease) as 10xHis, MBP or myc-tag fusions v1

During the lysis of cells, in particular of eukaryotic origin, there is a large release of nucleic acids. The presence of DNA/RNA increases the viscosity of a lysate and may pose problems during downstream processes. Removal of nucleic acids can be performed in many ways, of which one is by enzymatic digestion by nucleases. DNase/RNase are commonly added during protein purification alongside other commercial enzymes such as Benzonase (Novagen) and Turbonuclease. The latter enzymes are industrially produced or recombinant versions of the Serratia marcescens nuclease. This enzyme, hereafter referred to as SerMNucA, is able to hydrolyze both single- and double-stranded DNA and RNA with high activity.

Reported methods have shown that the nuclease can be made as inclusion bodies followed by refolding or secreted in the extracellular media. Here we report the purification of the SerMNucA nuclease in a soluble form, followed by purification from either periplasm or cell lysate. We find that purified protein from either fraction are active in a salmon sperm degradation assay with the periplasmic fraction slightly more. This protocol allows for a lab to independently produce tagged and untagged versions of the SerMNucA endonuclease in large quantities that may be required for certain experiments that may be cost prohibitive otherwise.

3 versions are reported here:

10x-His tagged MBP tagged myc tagged

The use of the enzyme is in such small quantities that repurification with the tag should not be an issue. The MBP tagged version is cleavable with TEV protease (see also: 10x-His-SuperTEV). Finally the myc-tagged version should not interfere with any common protein purification protocols.