Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis - A novel type of class II hydrophobin

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusif ormis. The predicted amino-acid sequence of the protein, dubbed CFTH1, show s a putative signal sequence for secretion, followed by three class II hydr ophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cu ltures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of bot h untreated mature CFTH1 and of a fragment obtained by trypsin digestion re vealed that CFTH1 is not processed between the hydrophobin domains. Mass sp ectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-t ranslational modification but not glycosylation. Purified CFTH1 self-assemb led at hydrophilic/hydrophobic interfaces and, after assembly at a water/ai r interface, it was found to be highly surface active. Antibodies raised ag ainst CFTH1 localized the protein in a mucilageous coat surrounding submerg ed vegetative hyphae in liquid shaken culture and, as a discrete layer of a bout 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.