title: 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.
authors.affiliation: Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Linghua
authors.initials: L
authors.lastname: Li
authors.affiliation: Research Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Fengyu
authors.initials: F
authors.lastname: Hu
authors.affiliation: Department of Clinical Laboratory, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Wanshan
authors.initials: W
authors.lastname: Chen
authors.affiliation: Research Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Huali
authors.initials: H
authors.lastname: Lei
authors.affiliation: Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Xiejie
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Weiping
authors.initials: W
authors.lastname: Cai
authors.affiliation: Research Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D008245:Lysophospholipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D008245:Lysophospholipase