Purification and Characterization of EALN-6 Modified Ferritin Protein for Use as Drug Delivery Biomaterial
Poster Number
26
Faculty Mentor
Nicholas Grossoehme, Ph.D.; grossoehmen@winthrop.edu
College
College of Arts and Sciences
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.
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Course Assignment
CHEM 551 - Hurlbert
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
Start Date
16-4-2021 12:30 PM
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.
