Biochemical assays are a cornerstone of our analysis. We generally use recombinant proteins derived from cDNA expression in E. coli or, if necessary, yeast or insect cells. Generally we use poly-His-tagged constructs (or other fusions) to provide straightforward purification of recombinant proteins. Our characterization of His-tagged ACH2 and the collaborative investigation of 4CL and 4CL-like proteins illustrate this approach. We currently have full-length clones for 45 of the 59 genes in our set and are ready to begin a high-throughput screen for substrates of the CoA ligases.
We recognized that the physiological substrate for every ligase is present in Arabidopsis plants. Of course, the compound may be restricted to a particular organ or stage of plant development. The basis of our screen is that a recombinant ligase is allowed to select its substrate from plant extracts and the substrate is then identified after purification of the CoA thioester.