Figure 2. Expression of active rOaAEP1_b in E. coli.

(a) Sequence of OaAEP1_b predicted from O. affinis genomic DNA. Predicted ER signal sequence shown in red; N-terminal propeptide shown in blue; the putative signal peptidase cleavage site is indicated by ∇ and autocatalytic processing sites by ▾. The mature OaAEP1 cyclase domain is underlined and the C-terminal auto-processing region is indicated with broken underline. The putative catalytic dyad is shown in bold and labelled with *. (b) An rOaAEP1-containing anion exchange fraction pre-and post-activation at pH 4.5 (5 h, 37 °C) was tested for activity against the wt IQF peptide (14 μM). Baseline fluorescence from a no substrate control has been subtracted and the relative fluorescence intensity (RFU) at t=90 min is reported. The average of two technical replicates is shown and error bars report the range (c) Activated rOaAEP1_b purified by cation exchange was analysed by SDS–PAGE and (i) Instant blue staining or (ii) Western blotting with anti-OaAEP1_b (residues D₄₇–P₄₇₄) polyclonal rabbit serum.