Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus.
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.
Nayak
Ajay P
AP
Allergy 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.
Blachere
Françoise M
FM
Hettick
Justin M
JM
Lukomski
Slawomir
S
Schmechel
Detlef
D
Beezhold
Donald H
DH
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000472:pathogenicity*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D004271:DNA, Fungal; D006460:Hemolysin Proteins; D009183:Mycotoxins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins