title: Novel Application of Peptidyl-Lys Metallopeptidase as a C-Terminal Processing Protease.
abstract: Adding fusion partners to proteins or peptides can aid or be a necessity to facilitate recombinant expression, folding, or purification. Independent of the reason it is desirable to remove the fusion partner to restore native functionality. Processing proteases catalyze the removal of fusion partners, however, most of these proteases have substrate specificity for the N-terminal of the scissile bond, leaving non-native termini if fusions are added to the C-terminal. The peptidyl-lys metallopeptidease of Armillaria mellea (Am-LysN) is unusual by having substrate specificity for the C-terminal side of the scissile peptide bond, allowing it to generate native C-termini. Am-LysN has strict specificity for lysine in P1', making all lysines of a protein or peptide a potential degradation site, however there are a number of amino acid side chains which lower hydrolysis significantly when located adjacent to the lysine. In this study we show that Am-LysN can be used as a processing protease to remove C-terminal extensions of peptides with no internal lysine to generate native Ctermini. Furthermore we show that removal of C-terminal extensions on peptides containing internal lysines can be achieved with little degradation of the product depending on the adjacent amino acids. These results demonstrate the utility of LysN allowing for novel ways to use fusion technology in the production of recombinant proteins.
authors: Anders S R
authors: AS
authors: Ødum
authors: Søren
authors: S
authors: Østergaard
authors: Inga
authors: I
authors: Nørby
authors: Morten
authors: M
authors: Meldal
authors: The Global Research, Novo Nordisk AS, Novo Nordisk Park, 2760 Måløv, Denmark. kjdo@novonordisk.com.
authors: Kjeld
authors: K
authors: Olesen
mesh_terms: D055392:Armillaria / Q000737:chemistry / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D010455:Peptides; C505389:AMMP protein, Armillaria mellea; D045726:Metalloproteases; D008239:Lysine