Summary: Plant cysteine proteinase inhibitors (phytocystatins) have been implicated to be defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases (Zhao et al., 1996). When these cystatins were displayed on phage particles, papain binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble unconjugated recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage displayed soyacystatins resulted in a 200- to 1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.

Figure legend: Phage display and biopanning selection of insecticidal phytocystatins. Phytocystatins are plant defensive molecules with demonstrated insecticidal activity against hemipteran and coleopteran insects. Soybean cystatins (soyacystatins) can be expressed as functionally active pIlI fusions on the surface of filamentous phage particles and that binding affinity of the soyacystatin-pIII fusions to an immobilized cysteine proteinase, papain, is directly correlated with relative insecticidal efficacy. These results are evidence that phage display selection can be used for directed molecular evolution of plant defensive proteins that interact specifically with biochemical targets of the pest. Illustrated in the left panel is a diagrammatic sketch of biopanning selection based on binding affinity of variant phage-displayed fusion proteins to an immobilized target. Elution conditions will fractionate protein variants with highest binding affinity for the substrate. The right panel (with left and right columns) illustrates results from a representative experiment of biopanning selection to papain of a mixture containing pBluescript (control), and two soyacystatins that have high and low binding affinity to the proteinase; original mixture (top) and after one (center) or two (bottom) rounds of biopanning. Left column, enrichment for the soyacystatin with highest binding affinity based on probe hybridization and right column, selective elimination of control bacterial colonies (blue) during biopanning.