Structural investigation of Folliculin and Ornithine decarboxylase

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

Initial degradation issues during purification of ODC and AZ has prevented crystallization of the complex. We have significantly improved purification techniques to reduce the degradation of each protein by co-expression of ODC and AZ in Ecoli. Initial crystallization trials have yielded multiple crystal forms. Currently optimizing and finding new crystallization conditions are in progress. We utilized H/D exchange technique coupled with MALDI/TOF experiments to analyze their surface residues. We have purified ODC monomer, ODC dimer, AZ, and ODC/AZ complex to see the changes in surface residues that are buried in the interface. We have identified the ODC/AZ hetero dimer interface. A manuscript is in preparation to explain the identified heterodimer interface and explain their in-vivo importance of these residues. We created a dual tag clone for easy purification of FLCN protein and we have improved the protein stability by expressing the protein as a complex. This resulted in extreme reduction in degraded products. Currently we are able to purify the proteins as a stable complex and have initial crystals of the complex and the conditions are being optimized.