Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.
Metarhizium anisophilae (Ma) secretes a range of proteases when grown in vitro on insect cuticle. A trypsin-like serine protease, PR2, was purified from culture filtrates by anion exchange chromatography and the N-terminal sequence determined. Using oligodeoxyribonucleotide probes based on this sequence and that of the highly conserved trypsin active site, a gene was isolated from a lambda EMBL3 genomic library of Ma isolate ME1. Sequencing of the gene and RT-PCR revealed that the gene contains two introns which are 94 and 40 bp long. The deduced protein consists of 254 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme. The putative mature enzyme has extensive homology with other serine proteases of the trypsin subclass and, in particular, with the trypsin characterised from Fusarium oxysporum.
Microbial Pathogenicity Group, School of Biology and Biochemistry, University of Bath, UK.
S L
SL
Smithson
I C
IC
Paterson
A M
AM
Bailey
S E
SE
Screen
B A
BA
Hunt
B D
BD
Cobb
R M
RM
Cooper
A K
AK
Charnley
J M
JM
Clarkson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D007313:Insecta / Q000382:microbiology; D003904:Mitosporic Fungi / Q000235:genetics*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D021382:Protein Sorting Signals; D014357:Trypsin