A Robust, Self-Digestion-Resistant LysN with Superior Activity and Cleavage Fidelity for Advanced Proteomic Workflows

Abstract

LysN is a valuable protease in proteomics because it cleaves peptide bonds N-terminal to lysine, generating peptides with physicochemical properties complementary to those produced by LysC and trypsin. However, the broader adoption of LysN in proteomic workflows has been limited by the lack of commercially available enzymes that combine high activity, low missed-cleavage rates, and sufficient stability under practical sample-processing conditions. Here, we report the recombinant production and proteomic characterization of a self-digestion-resistant and highly active LysN from Shewanella loihica (SL-LysN). Using terminomics, we mapped the mature N- and C-termini of the enzyme and established the primary structure of the active protease. We further developed a high-density fermentation, refolding, and purification workflow to obtain highly purified recombinant SL-LysN. Biochemical and proteomic benchmarking showed that SL-LysN displayed 3.3-fold higher specific activity than commercial LysN and reduced missed cleavages by approximately 80%. Notably, SL-LysN retained high activity in the presence of 8 M urea or 1% SDS and showed strong resistance to autolysis, indicating exceptional robustness for proteomic sample preparation. In complex mammalian proteome digests, SL-LysN achieved >95% cleavage specificity and a missed-cleavage rate of only 5.9%. These features address a long-standing bottleneck in N-terminal proteolysis and establish SL-LysN as a high-performance enzymatic tool for advanced proteomic workflows, including deep protein sequencing, quantitative proteomics, terminomics, de novo sequencing and analyses requiring efficient digestion under denaturing conditions.