Modification of luciferase to be a substrate for plant aspartic proteinase

The possibility of using firefly luciferase as a substrate for an aspartic proteinase was explored. Several amino acid modifications to the C-terminus of the luciferase were created on the basis of the known substrate of the Arabidopsis thaliana aspartic proteinase, pro-(barley lectin), One lucifera se with the sequence Arg-Asp-Gly-Val-Phe-Ala-Ala instead of the native Arg- Glu-Ile-Leu-Ile-Lys-Ala at position -15 to -9 relative to the C-terminus of native luciferase was found to possess 17% of the original luciferase acti vity. When this modified luciferase was incubated with the aspartic protein ase, a specific loss in activity occurred that was not observed with the or iginal luciferase. However, both-enzymes seemed very sensitive to the acidi c conditions required for aspartic proteinase activity. The other versions of luciferase with different numbers of pro-(barley lectin) amino acids wer e not active luciferases, This provided information on the structural requi rements of the C-terminal portion of the protein for luciferase activity, T he luciferase proteins were also monitored during the digestion by using We stern blots and some were shown to be substrates for the aspartic proteinas e. Contrary to what had been expected, the modified luciferase that incorpo rated the pro-(barley lectin) sequences was not simply cleaved at the engin eered site but at additional positions in the protein. The Arabidopsis aspa rtic proteinase cleaved two other standard protein substrates at many sites , suggesting that this proteinase could have a role in the degradation of p roteins in addition to processing propeptides in plants.