Abstract
Objectives
Metal-ion independent non-specific nucleases are of high potential for applications in EDTA-containing bioprocessing workflows.
Results
A novel extracellular non-specific nuclease EcNuc from the enterobacterium Escherichia coli has been identified. The recombinant gene was expressed and the protein was purified. Maximum activity of the enzyme was detected at 41.7 °C and at an acidic pH of 5.8. EcNuc tolerates EDTA in the reaction buffer at concentrations of up to 20 mM and the activity is not impaired by high concentrations of mono- and divalent metal ions in the absence of EDTA. The viscosity of crude protein extracts after cell lysis in EDTA-containing buffers is reduced when supplemented with EcNuc.
Conclusion
Proof-of-concept has been demonstrated that a metal-ion independent non-specific nuclease can be applied for removal of nucleic acids in EDTA-containing buffers for the subsequent purification of proteins from crude extracts.
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Acknowledgements
We thank Stefan Edelburg for the help with the VICTOR™ X4 Multilabel Plate Reader.
Supporting information
Supplementary Figure 1—Codon usage optimized nucleotide sequence of EcNuc without predicted signal peptide. The deduced amino acid sequence is indicated below in 1-letter amino acid code.
Supplementary Table 1—Purification of recombinant EcNuc after expression in E. coli Veggie BL21 (DE3).
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Schmitz, S., Nölle, V. & Elleuche, S. A non-specific nucleolytic enzyme and its application potential in EDTA-containing buffer solutions. Biotechnol Lett 41, 129–136 (2019). https://doi.org/10.1007/s10529-018-2618-0
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DOI: https://doi.org/10.1007/s10529-018-2618-0