A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii

Abstract

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.

Keywords: Acidic endopeptidase; Disulfide mapping; Kex2; LysN; Maturation; Peptidyl-lys metalloendopeptidase; Proteomics; Trypsin; Zymogen.

Fig. 1

Anion exchange (HiTrap QFF 5 mL) chromatogram (A) of concentrated BMMY supernatant after ~96 h of induction with methanol. Pooled fractions with Tc-LysN activity are enclosed within two straight lines. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (B) of the Tc-LysN purified from culture supernatant via AEX. Lane M contains the marker proteins (broad range 10–200 kDa, NEB). Lane 1 contains culture supernatant. Lane 2 contains the AEX fractions that were collected at the trailing end of peak enclosed within two straight lines in 1A. Lane 3 contains the pooled active fractions from AEX (enclosed within two straight lines in the chromatogram). Protein load was ~2 μg protein per lane. SDS-PAGE image with enhanced contrast can be found in Online Resource 2

Fig. 2

Multiple sequence alignment (A) of LysN peptidases from Trametes coccinea, Grifola frondosa, and Armillaria mellea. Amino acid sequences of the mature LysN peptidases are underlined in gray. The Kex2 recognition sites (KR and RR) between the pro-peptides and mature proteins are boxed in green and the cleavage site is indicated by a green arrow. The TMTpro Zero labeled N-terminus amino acid, E185, of Tc-LysN is typed in green. Percent identity matrix (B) shows >50% homology across the three mature LysN peptidases

Fig. 3

pH-optimum (A) of Tc-LysN using different buffers (50 mM) with overlapping pH range. Temperature-maximum (B) of Tc-LysN determined at pH 5.0 in 50 mM sodium acetate buffer. Thermostability (C) of Tc-LysN after 18 h of incubation at specified temperatures. All data points are averages of triplicate measurements; the standard deviation was <5%