“Protein Engineering Approaches to Study Cell Signaling”

A CBE seminar by Lawrence Stern, Ph.D.

Abstract

Engineered proteins have had profound impacts in the clinical setting. This class of molecules is vast, including a wide variety of functionalities, such as 1) synthetic receptors that turn engineered inputs into augmented natural outputs, 2) inhibitory proteins that block cell-cell and cell-ligand interactions from affecting cell behavior, and 3) molecular imaging agents capable of detecting disease with drastically improved sensitivity and specificity compared to conventional means. Although these advances have been great, many challenges remain in the pipeline of engineering new proteins to meet clinical demands. In this seminar, I will discuss my lab’s efforts to address these challenges through the development and application of new high-throughput screening platforms using yeast surface display. First, I will describe our recent work in the characterization of kinase-substrate interaction resulting in substrate phosphorylation by adapting a previously described endoplasmic reticulum sequestration screening strategy. These phosphorylation events are the currency of cell signaling, the understanding of which is imperative both for mapping natural cell signaling networks and for successful synthetic receptor engineering. We have demonstrated the ability to 1) display full-length receptor intracellular domains on the yeast surface, 2) robustly enrich phosphorylated intracellular domains from dilute pools using magnetic selection, 3) profile substrate specificities for a tyrosine kinase, and 4) demonstrate the ability to build 2-kinase phosphorylation cascades in the yeast ER. Second, I will discuss our efforts in developing a yeast surface display platform that uses inhibition rather than target binding as a selective pressure. To date, most efforts to engineer new inhibitory proteins start with high-throughput screening for binding function, followed by labor-intensive low-throughput screening to identify inhibitory activity. We have demonstrated the ability to simultaneously display a large receptor extracellular domain and a candidate inhibitory protein on the yeast surface. Application of an EGFR-Erbitux model system leads to robust detection of the exclusion of a titrated soluble competitor. Recent progress in further optimizing this system for affinity ranges and construct architecture will be discussed.

Career Bio

Dr. Lawrence A. Stern earned Bachelor of Science degrees in Chemical Engineering and Chemistry at Virginia Tech and a Ph.D. in Chemical Engineering at the University of Minnesota under the mentorship of Dr. Ben Hackel. He completed postdoctoral study in the T Cell Therapeutics Research Laboratory at City of Hope under the mentorship of Dr. Christine Brown and Dr. Stephen Forman. After completing his studies, Dr. Stern began his independent research career in January 2020 at the University of South Florida as an Assistant Professor in the Department of Chemical, Biological and Materials Engineering. His lab applies protein engineering and high-throughput screening techniques to answer questions in immune cell signaling, building toward the augmentation of cell-based immunotherapy. He has received several awards including a USF Faculty Outstanding Research Achievement Award, an NIGMS MIRA R35, an NSF CAREER Award and an ORAU Ralph E. Powe Junior Faculty Enhancement Award.