Expression, purification and crystallization of DynE8 involved in the biosynthesis of an antitumor warhead.

• Main Author: Chen, Ming Wei.
• Other Authors: Julien Lescar
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/16344
• Institution: Nanyang Technological University

Enediynes are natural products that catalyze DNA breakage and hence are potential anticancer drugs. The polyketide backbone of the enediyne warhead ring strucure is synthesized by enediyne polyketide synthases. In this work, the enediyne polyketide synthase for dynemicin, DYNE8, was expressed, purified and crystallized. Also, co-expression with the thioesterase DYNE7 and engineered mutation at the ACP domain were designed to improve protein’s conformational homogeneity. A three-step strategy included immobilized metal-ion affinity chromatography, ion exchange, and size-exclusion chromatography. These were followed by a concentration step which could bring protein purity to 90 – 95%. However, problems such as inability to remove certain contaminants and protein aggregation were encountered. L-arginine suppressed protein aggregation without affecting protein purity. The ACP-mutant, DYNE8-ACP, yielded two crystallization-screening hits while the wildtype did not crystallize. Methods for further work are suggested towards acquiring crystals of wildtype and mutant DYNE8.