Peptidyl‐Lys metalloendopeptidase (Lys‐N) purified from dry fruit of Grifola frondosa demonstrates “mirror”digestion property with lysyl endopeptidase (Lys‐C)

Abstract

RationaleLys‐N, also known as lysine‐specific metalloendopeptidase, functions as the “sister” enzyme of lysyl endopeptidase (Lys‐C) in proteomic research. Its digestion specificity at the N‐terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)‐based de novo sequencing of proteins.MethodsHere we present a complete production process of highly purified Lys‐N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification.ResultsThe overall yield of the process was approximately 6.7 mg Lys‐N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys‐N exhibited high enzymatic activity and specificity.ConclusionsThe novel production process provides for the first time the extraction of Lys‐N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS‐based proteomics and other biological studies.

Discrimination between Alpha-Synuclein Protein Variants with a Single Nanometer-Scale Pore

“…, native) αS (αS_wt) is shown in Scheme . To produce the desired enzyme-induced cleavage fragments for αS_wt and the single-point mutant version (αS_E46K), we used the recombinant zinc metalloprotease LysN from Grifola frondosa , which cleaves proteins at the amino terminus side of lysine residues. − Thus, LysN cleavage of wild-type αS would theoretically create 16 total polypeptides with varying numbers of amino acid residues and composition, of which 13 are unique (Table ). Note that one of these fragments, which contains 13 amino acids highlighted in yellow (residues 45 to 57, inclusive), is where the E46K point mutation considered here would be located.…”