Purification and biochemical characterisation of Psp1, an extracellular protease produced by the oilseed rape pathogen Pyrenopeziza brassicae

Abstract

An extracellular serine protease, designated Pyrenopeziza brassicae serine protease 1 (Psp1), was purified by a combination of ammonium sulphate precipitation and fast protein liquid chromatography (FPLC) to yield a single 34 kDa band on SDS–PAGE which showed proteolytic activity following renaturation. The N-terminal sequence of the purified protein was similar to that of several fungal serine proteases, with the highest identity (84% over 13aa) with PrtA from Aspergillus nidulans. The effect of protease inhibitors on Psp1 confirmed that it is a member of the subtilisin (S8) family of serine proteases. Characterisation of Psp1 activity on a range of synthetic di- tri- and oligo-peptide substrates indicated mixed trypsin/chymotrypsin specificity. A renaturable 34 kDa protease, corresponding to Psp1, was observed in cotyledons 7 days and 13 days post infection with P. brassicae.