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Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris
Yan He1, Linghua Li1, Fengyu Hu2
1Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong Province, China.
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.
Insights
This study reports the successful expression and purification of TmPlb1, a novel phospholipase from Talaromyces marneffei. The findings provide a foundation for understanding fungal virulence factors and developing new antifungal strategies.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- Phospholipase B is a known virulence factor in pathogenic fungi like Candida albicans.
- The role of phospholipase B in Talaromyces marneffei, a thermally dimorphic fungus, was previously unclear.
Purpose of the Study:
- To characterize the novel phospholipase gene TmPlb1 from Talaromyces marneffei.
- To investigate the expression and activity of TmPlb1 in a heterologous system.
Main Methods:
- Gene cloning and expression in Pichia pastoris GS115.
- Real-time quantitative reverse-transcription PCR (qRT-PCR) for expression analysis.
- Protein purification using Ni-affinity chromatography and activity assays.
Main Results:
- TmPlb1 expression was 1.85-fold higher in the yeast phase compared to the mycelial phase.
- TmPLB1 protein was successfully expressed, purified to 240.4 mg/L, and demonstrated phospholipase A1 and A2 activities.
- The protein possesses a conserved catalytic triad and G-X-S-X-G domain.
Conclusions:
- The study provides the first report on the expression and characterization of TmPlb1 from Talaromyces marneffei.
- High-purity, active TmPLB1 was obtained, enabling further research into its role in fungal virulence.
- This work lays the groundwork for understanding T. marneffei pathogenesis and potential therapeutic targets.
