Degree

Doctor of Philosophy (PhD)

Department

Biological and Agrincultural Engineering

Document Type

Dissertation

Abstract

E. coli is widely used for recombinant protein production due to its rapid growth rate, simple cultivation, and genetic tractability. However, producing large (>85 kDa) proteins with multiple disulfide bonds remains challenging. Endogenous oxidative folding enzymes in E. coli are often insufficient for eukaryotic protein synthesis and disulfide formation, which have more numerous and complex disulfide bonds than prokaryotic proteins. The CyDisCo system (cytoplasmic disulfide bond formation in E. coli), developed in 2011, enables efficient disulfide formation in E. coli using the catalysts human PDI and yeast Erv1p, producing high yields of proteins with up to 44 disulfide bonds. Despite this advancement, CyDisCo is most effective for smaller proteins, as larger disulfide-rich proteins remain difficult to produce. C-terminal agrin, a large proteoglycan containing 14 non-sequential disulfides, exemplifies this challenge. Agrin promotes CM proliferation and shows preclinical promise for heart regeneration; however, improving delivery to infarcted myocardium may enhance its therapeutic efficacy.

This work aims to develop a CFPS platform that can produce complex, disulfide-rich proteins such as agrin and engineer agrin for improved therapeutic outcomes for cardiac regeneration. Disulfide model proteins of increasing complexity (AppA, gLuc, BPTI, uPA, vtPA) were produced in CFPS supplemented with disulfide catalysts (DsbC/DsbA and PDI/Erv1p). PDI/Erv1p efficiently folded proteins with ≤5 disulfides, whereas proteins with ≥6 disulfides exhibited low solubility and activity. To address this, we developed autoinduced PDI/Erv1p-enriched extracts and combined CFPS with chaperone-rich (GroEL/GroES) extracts or an MBP fusion tag, increasing vtPA solubility and activity ~9.7-fold. Applying this optimized CFPS system to agrin yielded predominantly soluble disulfide-bonded protein. Initial results identified a region on agrin containing the EGF4 and LG3 domains as the cause of agrin insolubility; therefore, a truncated C-terminal construct lacking this region was produced in CFPS. Due to limited high-purity yields, commercial agrin (R&D Systems) was used for downstream testing. The PIGF-2123-144 [PJ1] peptide, with high affinity for ECM, was conjugated to agrin; ELISA results showed that PIGF-2123-144 conjugation significantly enhanced agrin binding to ECM.

This work establishes a simplified CFPS platform for producing large disulfide-rich proteins, better enabling the future development of therapeutic biomolecules. Additionally, PIGF-2123-144 mediated targeting of agrin to infarcted myocardium may enhance CM proliferation and improve functional regeneration.

Date

11-3-2025

Committee Chair

Yongchan, Kwon

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