A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii.
Acidic endopeptidase
Disulfide mapping
Kex2
LysN
Maturation
Peptidyl-lys metalloendopeptidase
Proteomics
Trypsin
Zymogen
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
Dec 2024
Dec 2024
Historique:
received:
23
08
2023
accepted:
24
12
2023
revised:
11
12
2023
medline:
17
1
2024
pubmed:
17
1
2024
entrez:
17
1
2024
Statut:
ppublish
Résumé
A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5-7.5 and ≥30% activity between pH values 8.5-10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN's activity up to ~100% and ~50%, respectively. Tc-LysN's thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)'s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides. KEY POINTS: •A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity •Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants •Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting.
Identifiants
pubmed: 38229299
doi: 10.1007/s00253-023-12986-3
pii: 10.1007/s00253-023-12986-3
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
103Informations de copyright
© 2024. The Author(s).
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