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.
