Purification and Characterization of EALN-6 Modified Ferritin Protein for Use as Drug Delivery Biomaterial

The development of novel drug delivery systems using biomaterials that are inherently compatible with human tissue is of great interest: ferritin, an iron storage protein responsible for the accumulation of excess intracellular iron, is an attractive candidate. The protein is made of 24 subunits and demonstrates high potential as a drug delivery container for its high pH and thermal stability, ability to be modified/manipulated, and its nanocage structure that includes a hollow interior perfect for the storage of medicinal drugs. Under neutral conditions, native ferritin self-aggregates into a very stable nano-cage structure and only disassembles under harshly acidic conditions (pH 2.0-3.0). Interestingly, reengineering ferritin to include a Glu-Ala-Leu-Ala (EALA) peptide repeat in place of the E-helix seems to enable a pH-induced disassembly around pH 6, rendering ferritin a more attractive drug carrier under physiologically relevant conditions. Unfortunately, experiments in the Grossoehme laboratory using the EALA-modified protein consistently yielded insoluble protein during purification attempts. Bioinformatics were used to explore alternate sequences that retain the structural properties of the EALA-repeat, but contain a larger fraction of hydrophilic amino acids. Clones were created of a series of Ftn proteins that replace the second alanine in EALA with asparagine. The newly EALN-modified protein will be used to develop experimental conditions that ensure EALN-6 ferritin remains soluble at the desired pH range. The aim of this study is to do so using two separate strategies; screening buffer additives to identify experimental conditions that maintain protein solubility over the desired pH range, and genetically adding a solubility tag to the N-terminus of EALN-6 ferritin