A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
- 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-terminalA 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.…
Document Type: | Article (reviewed) |
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Zitierlink: | https://opus.hs-offenburg.de/8266 | Bibliografische Angaben |
Title (English): | A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii |
Author: | Uzair AhmedStaff Member, Tobias Stadelmann, Daniel Heid, Berit Würtz, Jens Pfannstiel, Katrin OchsenreitherORCiD, Thomas EiseleStaff MemberORCiDGND |
Year of Publication: | 2024 |
Date of first Publication: | 2024/01/13 |
Publisher: | SpringerNature |
First Page: | 1 |
Last Page: | 12 |
Parent Title (English): | Applied Microbiology and Biotechnology |
Volume: | 108 |
ISSN: | 0175-7598 (Print) |
ISSN: | 1432-0614 (Elektronisch) |
DOI: | https://doi.org/10.1007/s00253-023-12986-3 |
URN: | https://urn:nbn:de:bsz:ofb1-opus4-82665 |
Language: | English | Inhaltliche Informationen |
Institutes: | Fakultät Maschinenbau und Verfahrenstechnik (M+V) |
Collections of the Offenburg University: | Bibliografie |
Tag: | Acidic endopeptidase; Disulfide mapping; Kex2; Lys-N; Maturation; Peptidyl-lys metalloendopeptidase; Proteomics; Trypsin; Zymogen | Formale Angaben |
Relevance for "Jahresbericht über Forschungsleistungen": | Wiss. Zeitschriftenartikel reviewed: Listung in Master Journal List |
Open Access: | Open Access |
Gold | |
Licence (German): | Creative Commons - CC BY - Namensnennung 4.0 International |