Expressing, Purifying, and Characterizing a Flavodoxin from the Solvent-Producing Clostridium Acetobutylicum

Flavodoxins (Fld) are relatively small redox proteins responsible for facilitating the transport of electrons in certain types of bacteria and algae. In order to understand how these proteins control the flow of electrons between different redox partners in organisms with multiple electron transfer proteins, we must understand how their sequence and structure controls their partner specificity. While structures have been reported for Flds, we do not understand their structural properties sufficiently enough to predict electron flow in cells.
This project aims to overexpress a flavodoxin from Clostridium acetobutylicum (CacFld1) in E. coli cells, purify it using chromatography techniques, screen for conditions that yield CacFld1 crystals, and acquire structural data using the crystals. To express CacFld1, the gene encoding this protein was synthesized and placed under a T7 phage promoter which is inducible by IPTG. Optimal expression conditions of CacFld1 were found and purification of the protein by anion exchange chromatography was attempted. Future studies entail finding the conditions in which CacFld1 crystallizes and analysis of its structure through x-ray crystallography techniques.