Post-secretory processing and metabolism of neuropeptide or peptide hormones occurs predominantly through the action of peptidases that are attached to the plasma membrane. General features of membrane peptidases, or ectoenzymes, are that their active sites face toward the extracellular surface. Many of these are zinc metallopeptidases, which play key roles in the processing and metabolism of a diverse range of peptides. Neprilysin (NEP: EC 3.4.24.11) is a unique membrane endopeptidase anchored in the lipid bilayer by a short hydrophobic anchor at the N-terminus, and extracellular active site. They have the potential to participate in a range of cardiovascular functions, and re implicated in the control of hypertension, pain perception and cartilage and bone metabolism. We are using C. elegans as a model organism to explore the function of NEP-like genes in the organism, so that we may elucidate the function of NEPs in higher organisms. Spatio/temporal GFP and Lac Z reporter data indicates that T25B6.2 (a homologue to human NEP) is highly predominantly expressed in the intestine in all postembryonic stages. Our primary aim is to biochemically analyse the recombinant protein to establish its potential substrates. Previous attempts to generate heterologous recombinant protein in bacterial, yeast (Pichia pastoris) and mammalian (CHO) systems have proved unsuccessful. Currently, we are attempting to ectopically express native T25B6.2 by transforming C. elegans with genomic DNA tagged with two epitopes (Flag/His or C-Myc/His) to allow detection and purification. A trial run using GFP (green fluorescent protein) containing these tags has been very successful. In the experiments, GFP protein expression was controlled using either the heat shock promoter (hsp 16-41) or a strong constitutive promoter (
unc-54). The entire coding region was placed downstream of the promoter and appropriate tags (Flag/His or C-Myc/His) were introduced at the C-terminus by PCR. High expression levels were seen with both promoters and western blot analysis confirmed detection was successful using both Flag and His tags. Purification via a Ni-NTA column was also successful as the GFP protein could be purified from whole worm extracts to apparent homogeneity. Having established this expression and purification technique with GFP, we are currently analysing T25B6.2 and other C. elegans zinc metallopeptidases.