CLONING AND CHARACTERIZATION 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 p urified from culture filtrates by anion exchange chromatography and th e N-terminal sequence determined, Using oligodeoxyribonucleotide probe s based on this sequence and that of the highly conserved trypsin acti ve 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 seco nd proteolytic processing step at its N terminus to yield the mature e nzyme. The putative mature enzyme has extensive homology with other se rine proteases of the trypsin subclass and, in particular, with the tr ypsin characterised from Fusarium oxysporum.