PHAGE DISPLAY SELECTION CAN DIFFERENTIATE INSECTICIDAL ACTIVITY OF SOYBEAN CYSTATINS

Plant cysteine proteinase inhibitors (phytocystatins) have been implic ated as 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 inhibi tor of papain, vicilin peptidohydrolase and insect gut proteinases. Wh en these cystatins were displayed on phage particles, papain-binding a ffinity and CPl activity of scN were substantially greater than those of set, in direct correlation with their relative CPl activity as solu ble recombinant proteins. Furthermore, scN substantially delayed cowpe a weevil (Callosobruchus maculatus (F.)) growth and development in ins ect feeding bioassays, whereas set was essentially inactive as an inse cticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200-1000-fold greater enrichment for scN relative to set . These results establish that binding affinity of cystatins can be us ed in phage display biopanning procedures to select variants with gree ter insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biologic al activity of these plant defensive proteins.