Keratinase Assay

Due to there being relatively few specific substrates for keratinases, it was decided that we would test for non-specific protease activity.

The image to the left shows a 1% gelatine plate containing our keratinase, S. aureus, and the promoter vector without the keratinase downstream. Flooding of the plate with ammonium sulphate caused precipitation and a cloudy effect, except in parts where the gelatine had not been digested.

The assay has been shown to work as a zone of clearance can be seen around the staph colony, which is known to produce keratinases. For some reason, our keratinase colony is either not producing or exporting the protein in a functional form.

Using samples taken from our keratinase colonies, we ran a protein gel to see if we could isolate a band corresponding to our protein; this is presented to the left.

There is a band clearly overexpressed in our keratinase cultures, although the size of the band is around 35kDa, which is larger than we’d expected (29.9kDa).

During export, around 100 residues (30 of which are the export tag) are meant to be cleaved from the protein to allow for refolding to create an active protein. If only the export tag were being removed, this would account almost exactly for the size difference and inactivity.