We describe production of human A2A adenosine receptor (A2AAR), a G protein-coupled receptor (GPCR), samples in lipid nanodiscs for both NMR spectroscopy and single molecule fluorescence (SMF) spectroscopy. We explain steps shared between the two sample preparation strategies, including expression and isolation of A2AAR and assembly of A2AAR in lipid nanodiscs and procedures for incorporation of either 19F-NMR or fluorescence probes.

We are developing approaches to produce full-length functional forms of recombinant membrane-bound proteins using nanodisc technologies coupled with cell-free co-translation. This approach focuses on utilizing high-density apolipoproteins or styrene-maleic anhydride polymers (SMALPs) or branched polyethylene glycol-based telodendrimers, which can all form nanodisc scaffolds in the presence of phospholipids. Discs range in size from 10-40 nm and overcome problems associated with poor solubility. These methods are also adaptable to large functional protein complexes. Data will be presented for a chlamydial major outer membrane protein, a Yersinia pestis type III secretion system, and a synthetic voltage-gated calcium channel. 

The aßT cell receptor recognition of peptide presented by major histocompatibility complex molecules leads to intracellular signaling events that activate the T lymphocyte to generate an immune system response against virally-infected or cancerous cells. It is critical to understand these TCR processes to uncover T cell-specific therapeutics. Thus, innovative methodologies have been developed to produce the TCR transmembrane proteins to gain insight into their role in TCR signal regulation. Previously, these subunits were very difficult to produce and isolate and were intractable for NMR spectroscopic analysis, but with newly established methods these insights into TCR function are possible.

Astex has a state-of-the-art cryo-EM facility that has opened the door to challenging human membrane protein targets. The reliable production of high-quality functionally relevant protein is essential for the determination of reproducible and meaningful high-resolution liganded structures by single particle cryoEM. I will share our experience in expressing, purifying, and characterizing these challenging membrane protein targets and how this is enabling structure-based drug discovery.

Membrane proteins are important drug targets (GPCRs, ion channels, transporters), yet are notoriously difficult to work with. We developed a nano-membrane platform technology (Salipro) that stabilizes these important membrane proteins. We will present novel data on Salipro-CXCRs complexes, as well as case studies on other membrane protein types, to illustrate how Salipro nanoparticles enable the development of next-generation therapeutics, for example via SPR, phage display, B cell sorting and cryoEM.

Moderna is developing over 40 mRNA vaccines and therapeutics, all of which require recombinant proteins. mRNA drug-products can deliver proteins to patients that are otherwise not easily produced on a large-scale (multi-subunit complexes, multi-pass membrane proteins, novel fusions). This mRNA advantage presents unique challenges for laboratory-scale recombinant protein production due to the diversity and complexity of targets. We overcome these challenges by continually building and utilizing a robust expression/purification toolbox.

Recombinant protein production is an integral part of drug discovery. As therapeutic targets become more challenging, we are constantly looking for ways to triage protein variants more efficiently, while reducing cost and shortening timelines. To reduce the burden on large-scale protein production and to allow for faster triaging of multiple variants, we have developed a mid-scale platform that enables delivery of small quantities of proteins for biochemical and structural screening.

Matrix metalloproteinases (MMPs) have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to their overexpression. To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed to produce active, soluble MMPs. A summary of recent methods used to overcome these challenges and improve the yields of soluble active MMPs will be discussed.

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here we present high throughput and avidity-based methods that allow the capture of membrane protein interactions with the goal of discovering and interrogating mechanisms that govern extracellular cross-talk. These specialized screens revealed previously unknown ligands for immune checkpoints receptors like PVR and B7-H3, thereby presenting novel targets for immunotherapy.

Expanding the chemistries available in antibodies using genetic code expansion offers opportunities to discover function-disrupting covalent antibodies, enzyme inhibitors, and other “hybrids” that combine complementary features of antibodies and small molecules. Biosynthesis and efficient characterization of these proteins is an ongoing challenge hindering hybrid discovery and application. This talk will describe advances in the biosynthesis and evaluation of hybrids using yeast-based, high-throughput approaches.

Large molecule R&D efforts are increasingly shifting to molecules of greater complexity in design, structure, and engineering. To keep pace with the rapid iterative design of these complex molecules, increased throughput and automation-based technologies to deliver high-quality protein reagents are critical to identifying the best therapeutic candidates in early discovery pipelines. This presentation will focus on efforts to build semi-automated, design-of-experiment based platforms to meet the demands of complex purifications.