Graduation Year

2026

Document Type

Thesis

Degree

M.S.C.H.

Degree Name

MS in Chemical Engineering (M.S.C.H.)

Degree Granting Department

Chemical, Biological and Materials Engineering

Major Professor

Lawrence A. Stern, Ph.D.

Committee Member

Rituparna Samanta, Ph.D.

Committee Member

Piyush Koria, Ph.D.

Keywords

Cell Signaling, Endoplasmic Reticulum Sequestration, Enzymatic Modification, Protein Engineering, ZAP70

Abstract

Developing and optimizing platforms to study complex protein-protein interactions is important to maintain fidelity of the modeled activity. Yeast surface display has been used as a high-throughput platform to model substrate phosphorylation by tyrosine kinases, a key enzymatic process that is essential for most cell signaling pathways. However, existing characteristics of the yeast surface display platform limit the ease-of-use, reproducibility, and feasibility of screening enzymatic modification processes like tyrosine phosphorylation. The cytoplasmic domain of T cell costimulatory receptor CD28 and lymphocyte kinase (LCK) were used as a model enzyme-substrate pair to showcase molecular cloning issues that can hinder reproducibility and phenotype assessment, and the resulting challenges in developing proper negative controls for promiscuous protein-protein interactions. Linker for activation of T cells (LAT), zeta-chain-associated protein kinase 70 (ZAP70), and LCK were used to understand the platform’s capabilities in modeling larger dual-enzyme phosphorylation cascades and optimize construct architecture for improved functional readout. Several controls were developed to assess protein function in the display platform compared to documented kinase function from literature, with a peptide library being generated to differentiate between candidate substrate peptides for ZAP70 and LCK. These studies together provide several important insights on the limitations of the endoplasmic reticulum sequestration yeast display platform and showcase the steps required to optimize a platform to a targeted protein-protein interaction for further engineering.

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