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

Figures & data

Table 1 Sequences of primers used in this study

Primer	Sequence	Length (bp)
qF1	5′-ATCGTCCTACCTGTTACTT-3′	148
qR1	5′-ATGCTTCTGCGTCATATC-3′	148
Act1F	5′-TGATGAGGCACAGTCTAAGC-3′	155
Act1R	5′-CTTCTCTCTGTTGGACTTGG-3′	155
5′AOX1	5′-GACTGGTTCCAATTGACAAGC-3′	492
3′ AOX1	5′-GCAAATGGCATTCTGACATCC-3′	492

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).