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
we have explored building 2-kinase phosphorylation cascades in the yeast ER. Recent progress
in extending these techniques toward cell-surface substrate modification and re-
compartmentalizing intracellular interactions will be discussed. Second, I will describe 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 and a PD-L1-Atezolizumab 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.

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.