Functional characterisation of neprosin via site-directed mutagenesis and in planta analysis

T. Y. TING, H. H. GOH

Plant Functional Genomics Research Group, Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Malaysia

Neprosin is a novel protease found in the carnivorous Nepenthes plant pitcher fluid. Due to its preference to cleave after the C-terminal of proline residue in a polypeptide chain, it has the potential as a treatment for coeliac disease by detoxifying gliadin which can trigger an immune response in the intestine. Recently, we reported neprosin to be a glutamic peptidase with a catalytic dyad comprising glutamic acids via an extensive in silico analysis. Neprosin has two uncharacterized domains, namely the neprosin activation domain (PF14365) followed by the catalytic neprosin domain (PF03080). There are over 4,200 proteins with this domain architecture that are found almost exclusively in plant species, including the model plant Arabidopsis thaliana (50 proteins), which suggests biological functions of neprosins beyond prey digestion. Since neprosins from N. rafflesiana (Nr) have not been characterised, we aim to express and purify recombinant Nr (rNr1) in Escherichia coli for in vitro enzyme characterisation. In planta analysis using A. thaliana will allow identification of the biological function of neprosin via site-directed mutagenesis. Here, we present our results on the in silico analysis of neprosin amino acid sequences and structures, overexpression of rNr1, and on-column refolding of rNr1 using immobilised metal affinity chromatography.