X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes
Final rept. 15 Sep 1997-1 Mar 2003
WEIZMANN INST OF SCIENCE REHOVOT (ISRAEL)
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The objective of the project was to establish a procedure for designing and producing species- specific protein inhibitors, i.e. chimeras, utilizing the scaffold of the three-finger toxins. Theoretical analysis of the 3D structure of the chimera of a-neurotoxin and fasciculin IT showed that the loops of fasciculin II grafted onto the core of a-neurotoxin maintain their secondary structure. This supports the feasibility of employing protein engineering, using the three-finger toxin core as a platform, for generating new proteins that mimic three-finger toxin activity but display modified specificity. We have succeeded in producing chimera II biosynthetically, by expression in E. coli, and have been able to reach adequate purity by HPLC purification. Kinetic assays showed that the chimera displays substantial inhibitory activity towards TcAChE. Due to non- specific proteolysis encountered during cleavage of the fusion protein used for expression of chimera II, it was decided to adopt a synthetic approach to its production. Chemical synthesis provided a more convenient route for obtaining chimera II, in good yield and with high purity.
- Medicine and Medical Research