Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus
Ajay P Nayak1, Françoise M Blachere, Justin M Hettick
1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 1095 Willowdale Rd, Morgantown, WV 26505, USA.
Abstract:
Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly β-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the β-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.
Insights
Researchers identified and characterized terrelysin, a novel fungal hemolysin from Aspergillus terreus. This study details its production, purification, and structural properties, confirming its role as a potential virulence factor.
Area of Science:
- Mycology
- Molecular Biology
- Protein Chemistry
Background:
- Fungal hemolysins are recognized as significant virulence factors.
- Aegerolysins, a family of fungal proteins, are known for their cytolytic activity against erythrocytes and other cells.
Purpose of the Study:
- To identify and characterize a novel hemolysin from Aspergillus terreus, named terrelysin.
- To investigate the production, purification, and structural properties of recombinant terrelysin (rTerrelysin).
Main Methods:
- Genomic sequence analysis to identify the terrelysin sequence via homology.
- RNA isolation, cDNA synthesis, and PCR amplification of the terrelysin open reading frame.
- Recombinant protein production in Escherichia coli using the pASK-IBA6 vector, followed by purification.
- Mass determination using MALDI-TOF MS, secondary structure analysis via circular dichroism, and thermal denaturation studies.
- Generation of polyclonal antibodies for detection in fungal extracts.
Main Results:
- The terrelysin sequence was identified through homology searches in the Aspergillus terreus genome.
- Recombinant terrelysin (rTerrelysin) was successfully produced and purified, with a measured mass of 16,428 Da.
- Circular dichroism indicated a predominantly β-sheet secondary structure, stable up to 65°C.
- Polyclonal antibodies confirmed the presence of a ~16.5 kDa protein in Aspergillus terreus mycelial extracts.
Conclusions:
- Terrelysin is a novel hemolysin from Aspergillus terreus belonging to the aegerolysin family.
- The recombinant protein exhibits structural characteristics consistent with its family, including thermal stability.
- The findings support terrelysin's role as a potential fungal virulence factor, with implications for understanding fungal pathogenesis.
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