Degree

Doctor of Philosophy (PhD)

Department

Biological Engineering

Document Type

Dissertation

Abstract

The epidermal skin barrier is fundamentally maintained by the precise, sequential processing of profilaggrin during keratinocyte differentiation. Disruptions in these structural pathways can be associated with numerous pathogenic skin barrier disorders. However, studying these highly repetitive, tandem-repeat proteins presents challenges in production via traditional prokaryotic hosts. This work addresses these limitations through an integrated biological engineering approach by combining enzymatic kinetic validation with cell-free protein synthesis.

First, an E. coli-based cell-free platform was optimized for the scalable expression of an engineered dimeric filaggrin variant, avoiding traditional cellular toxicity constraints to successfully cleave the tandem repeats into a monomeric form. Concurrently, the peptidyl arginine deiminases (PADs) responsible for citrullination were produced in cells as well as the cell-free system. The catalytic activity of these enzymes was validated through kinetic assays using synthetic substrate mimics of the target residue. Finally, these enzymes were paired with monomeric filaggrin, first in a purified, highly regulated in-vitro environment, then subsequently in the cell-free system, to achieve a citrullinated filaggrin product.

Ultimately, this work establishes a highly scalable framework for the production and processing of structural epidermal proteins, offering a foundational pathway for developing therapeutics that target diseased epidermis. Moreover, it demonstrates the versatility of crude extract-based cell-free systems in synthesizing traditionally difficult bio-products including human proteases, large enzymes, and highly post-translationally modified proteins.

Date

7-7-2026

Committee Chair

Kwon, Yongchan

LSU Acknowledgement

1

LSU Accessibility Acknowledgment

1

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Available for download on Wednesday, July 07, 2027

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