Title of Abstract

Progress Toward a Soluble pH-sensitive Ferritin Assembly System

Submitting Student(s)

Courtney Miller

Session Title

Additional Projects

Faculty Sponsor (for work done with a non-Winthrop mentor)

Nicholas Grossoehme, Ph.D.

College

College of Arts and Sciences

Department

Chemistry, Physics & Geology

Abstract

Ferritin is an iron-storage protein with properties that make it ideal for several applications including drug delivery systems (DDS). Some of these properties include: (1) it is a small and stable core structure, it has an interior cavity that can be used to transport medicinal drugs in the body, it is naturally found in humans which minimizes the potential for rejection by the body, and the possibility for genetic modifications. For the drug delivery applications, ferritin encapsulated drugs would need to be released based on an external signal or stimuli; however, native ferritin is an extremely stable protein that requires very harsh conditions (e.g., extremely acidic pH or 2.0-3.0) to trigger dissociation. Consequently, the ferritin protein requires modifications to enable the desired stimuli responsiveness. It has been reported that replacing the E-helix of ferritin with six repeating units of EALA (Glutamic acid-alanine-leucine-alanine) results in a pH switch that triggers the reversible disassembly of the nanocage structure in the 6.0-6.5 pH range. Unfortunately, this modification yields a ferritin construct with marginal solubility at neutral pH and very poor solubility below pH 6.8. The goal of the project was to test the viability of solubility tag additions onto the EALA-modified protein. Two solubility tags, Thioredoxin (Trx) tag and the E. coli Biotinylation signal sequence, were selected based on their small size and literature precedent for conferring enhanced solubility to globular proteins. Both constructs were successfully cloned. The Trx tagged protein was tested and failed to enhance the solubility to a meaningful degree.

Start Date

15-4-2022 12:00 PM

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Apr 15th, 12:00 PM

Progress Toward a Soluble pH-sensitive Ferritin Assembly System

Ferritin is an iron-storage protein with properties that make it ideal for several applications including drug delivery systems (DDS). Some of these properties include: (1) it is a small and stable core structure, it has an interior cavity that can be used to transport medicinal drugs in the body, it is naturally found in humans which minimizes the potential for rejection by the body, and the possibility for genetic modifications. For the drug delivery applications, ferritin encapsulated drugs would need to be released based on an external signal or stimuli; however, native ferritin is an extremely stable protein that requires very harsh conditions (e.g., extremely acidic pH or 2.0-3.0) to trigger dissociation. Consequently, the ferritin protein requires modifications to enable the desired stimuli responsiveness. It has been reported that replacing the E-helix of ferritin with six repeating units of EALA (Glutamic acid-alanine-leucine-alanine) results in a pH switch that triggers the reversible disassembly of the nanocage structure in the 6.0-6.5 pH range. Unfortunately, this modification yields a ferritin construct with marginal solubility at neutral pH and very poor solubility below pH 6.8. The goal of the project was to test the viability of solubility tag additions onto the EALA-modified protein. Two solubility tags, Thioredoxin (Trx) tag and the E. coli Biotinylation signal sequence, were selected based on their small size and literature precedent for conferring enhanced solubility to globular proteins. Both constructs were successfully cloned. The Trx tagged protein was tested and failed to enhance the solubility to a meaningful degree.