Browsing by Person "Eisele, Thomas"

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A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
(2024) Ahmed, Uzair; Stadelmann, Tobias; Heid, Daniel; Würtz, Berit; Pfannstiel, Jens; Ochsenreither, Katrin; Eisele, Thomas
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