Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

Abstract

Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.

Figure 1

Sequence alignment of TmPlb1 orthologs. Sequences were aligned using Clustal X and shaded by BOXSHADE. Shaded residues indicate ≥75% homology (black) or ≥50% homology (gray). The G-F-S-Q-G motif is boxed, and the catalytic triad sites are indicated by arrows. Abbreviations and accession numbers are as follows: human (Homo sapiens, GenBank: NP_006321); rat (Rattus norvegicus, GenBank: NP_037138); A. flavus (Aspergillus flavus, GenBank: XP_002380983); A. fumigatus (Aspergillus fumigatus, GenBank: KMK55316); R. emersonii (Rasamsonia emersonii, GenBank: XP_013325383); T. stipitatus (Talaromyces stipitatus, GenBank: XP_002485317); S. cerevisiae (Saccharomyces cerevisiae, GenBank: AAS56265).

Figure 2

Phylogenetic analysis of PLB from various organisms. A neighbor-joining tree was constructed with MEGA version 6.0 using Poisson correction, and bootstrap values were calculated from 1000 trees. The scale bar indicates the estimated number of substitutions per 10 amino acids. Abbreviations: Aspergillus fumigatus, A. fumigatus; Aspergillus clavatus, A. clavatus; Aspergillus flavus, A. flavus; Cryptococcus neoformans, C. neoformans; Candida albicans, C. albicans; Homo sapiens, H. sapiens; Rattus norvegicus, R. norvegicus; Saccharomyces cerevisiae, S. cerevisiae; Talaromyces stipitatus, T. stipitatus; Rasamsonia emersonii, R. emersonii; phospholipase B, PLB; acyl-protein thioesterase 1, APT1; phospholipase 1, PLS1; lysophospholipase 1, Lyso1.

Figure 3

Expression of TmPLB1 in P. pastoris GS115. (A) SDS-PAGE analysis of TmPLB1 expressed in P. pastoris GS115 fermentation supernatants in shaking flask cultures. Lanes 1–8, P. pastoris induced with methanol for 6, 12, 24, 48, 72, 96, 120 and 144 h; M, standard protein marker (Fermentas, Burlington, ON, Canada). (B) Western blot analysis of TmPLB1. Lane 1, empty vector control; lane 2, TmPLB1 expression plasmids.

Figure 4

Purification of TmPLB1. Lane 1, P. pastoris GS115 fermentation supernatants induced with methanol for 96 h; lane 2, flow-through; lane 3, wash fractions 1; lane 4, wash fractions 2; lane 5, elution fractions 1; lane 6, elution fractions 2; M, protein molecular marker (kDa).