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.

<p>Molecular typing revealed the emergence of <em>pvl</em>-positive sequence type 22 methicillin-susceptible <em>Staphylococcus aureus</em> in Urumqi, Northwestern China</p>

“…A previous investigation of MRSA from inpatients in China found that 28.6% of isolates were pvl –positive 7. Several other recent studies also showed that about 10% of MRSA isolates were pvl -positive 2,48. Our results showed that only 3.0% of MRSA isolates were pvl -positive, which is very different from these other studies 2,48.…”

“…Several other recent studies also showed that about 10% of MRSA isolates were pvl -positive 2,48. Our results showed that only 3.0% of MRSA isolates were pvl -positive, which is very different from these other studies 2,48. However, 34.4% of our MSSA isolates harbored the pvl gene, similar to the prevalences reported by recent studies in Chongqing 38…”

“…Livestock-associated clone ST398 and ST188 are the most prevalent in Urumqi. It is probably because a great number of people who live in this area are engaged in livestock husbandry or related activities.ST121 is mainly distributed in Africa, Asia, and Europe and has been characterized as a hypervirulent clone causing SSTIs 2,45. In the present study, all ST121 clones were MSSA isolates.…”

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

“…Previous studies suggested that DNA priming immunization induces higher levels of antigen-specific B-cell responses. 25,26 Consistently, the Cells/DNA method resulted in the highest number of antigenspecific scFv clones in this study (Figure 4a), although functional experiments will be necessary to determine if these scFv clones translate to better mAbs.…”

A Thermolabile Phospholipase B from Talaromyces marneffei GD-0079: Biochemical Characterization and Structure Dynamics Study

“…He et al 2016 performed expression and characterization of Talaromyces marneffei active phospholipase B using Pichia pastoris GS115 expression system. Using Ni-affinity chromatography, the protein concentration of active TmPLB1 was 240.4 mg L −1 of the culture medium [43]. It usually takes 5-7 days for expression of recombinant protein in Pichia pastoris expression system.…”