(629f) Exploring Human Transmembrane Protein Domains for Synthetic Protein Engineering through Massive Parallel Reporter Assay

Motivation: The field of synthetic biology and cellular engineering rely on the use of synthetic receptors and proteins to impart novel cellular functions, such as CAR-T receptors or synthetic protein circuits. One such synthetic protein system is the RELEASE (Retained Endoplasmic Cleavable Secretion) platform (Fig. 1A), which controls the surface expression of any protein of interest in response to protease activity. The RELEASE platform contains 4 core-components: an ER-facing linker protein, a transmembrane domain, a cytosolic linker protein containing a protease cleavage site and a cytoplasmic ER retention motif. While each of these domains are often treated as modular building blocks, transmembrane domains remain an unexplored and critical component of many synthetic proteins including RELEASE. Despite being primarily composed of hydrophobic amino acids, transmembrane domains play a crucial role in protein function, surface expression, and mediating the transport and sorting of integral membrane proteins. To fully understand the potential effect of the transmembrane domain on protein retention and surface expression of synthetic proteins, we have performed a Massive Parallel Reporter Assay (MPRA) to probe 6000 different human-derived transmembrane domains.

Methods: By coupling a synthetic surface marker (human IgG1 FC region) to the RELEASE platform, we bioinformatically mined the online human UNIPROT database to generate a protein library of different RELEASE variants each with unique transmembrane domains (Fig. 1B). These variants were then delivered into an inducible reporter cell line via lentiviral transduction and used to measure changes in the retention and surface expression of IgG1 following magnetic separation and next generation sequencing (Fig. 1C). To measure retention scores, we took ratios of the calculated enrichment scores for each library element between uninduced and induced cells. In addition to probing the effect of transmembrane domains alone, we also included variants with their respective intracellular regions (juxtamembrane) to measure their effects on protein retention and surface expression.

Results: We observed that the transmembrane domain had a significant effect on both protein retention and surface expression (Group 1 – Fig. 1D). Furthermore, the presence of the juxtamembrane region had a prominent effect on improving the retention scores of the respective transmembrane domains (Group 2 – Fig. 1E). We are now in the processes of adapting machine learning algorithms to determine key parameters such as specific residues and positions important for these observed differences in protein retention and surface expression.

Conclusions: Using this MPRA, we will determine the key design parameters of transmembrane domains to help guide future engineering of synthetic signalling platforms, such as CAR-T receptors and other synthetic receptors.

Figure Caption: A) Schematic of RELEASE platform for controlling the surface expression of any protein of interest. B) RELEASE variants with different transmembrane domains conjugated to the FC domain of IgG1. C) Massive Parallel Reporter Assay workflow using RELEASE. Scatter plots showing correlations between surface expression (Surface Enrichment Score) and protein retention (Retention Score). RELEASE variants with D) transmembrane domains alone or E) transmembrane domains and their juxtamembrane regions are highlighted compared to the complete data set.