Purification and Characterization of EALN-6 Modified Ferritin Protein for Use as Drug Delivery Biomaterial
Poster Number
26
Session Title
Poster Session 2
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Faculty Mentor
Nicholas Grossoehme, Ph.D.
Abstract
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.
Course Assignment
CHEM 551 - Hurlbert & Grossoehme
Type of Presentation
Poster presentation
Grant Support?
This work was supported by grants from the National Institute of General Medical Sciences (P20GM103499-20), from the National Institutes of Health, and the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740
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.
