Zoetis is working with a canine cytokine, which is important in the disease of interest, but its expression is challenging. The protein was expressed in different hosts, but none of them produced functionally active cytokine. For this reason, three cell-free expression systems were tested. Two of the proteins showed binding, but only one was functionally active. Choosing the best expression system is critical and is key for antibody screening.

We will discuss cell-free methods using various forms of nanodisc such as apolipoprotein, telodendrimers, and SMALPs to support and refold challenging membrane proteins, including large mammalian proteins over 200 kDa. This includes proteins like MOMP, CAR-T receptors, voltage-gated ion channels, and SARS-CoV-2 RBD that were all expressed in E. coli lysates and solubilized in synthetic or natural lipids. These approaches significantly enhance protein stability, solubility, and biological functionality, outperforming traditional refolding methods. Our strategy enables efficient production of therapeutic membrane proteins and supports new solutions for producing complex, high-molecular-weight mammalian proteins, addressing key challenges in protein biochemistry and biotechnology.

G protein-coupled receptors (GPCRs) regulate important physiological processes and are attractive targets for drug development. Generation of selective antibodies against GPCRs is challenging due to their structural complexities and low immunogenicity. CHS-114 is a human afucosylated IgG1 monoclonal antibody targeting CCR8, that preferentially depletes intratumor CCR8+Tregs, relieving immunosuppression . CHS-114, is a differentiated antibody with no off-target binding and in clinical studies has demonstrated acceptable safety profile to date, anti-tumor activity and immune activation.

Complex multipass membrane proteins are targets for many potential therapeutic antibodies. However, producing these proteins in a native-like state for antibody discovery and development presents a significant challenge. Presently, there is no single expression system or immunogen that can consistently support antibody discovery strategies and thus requires a diversified repertoire to efficiently target these complex membrane protein targets. This presentation will present key challenges and solutions for these complex targets.

As most of the low hanging fruit has been picked, people are now finding it harder and harder to express and purify protein targets. These include multi-protein complexes and transmembrane protein complexes. Many of these target proteins require co-expression of chaperone proteins as well. I will focus my talk on two targets: the Alpha 7 nicotinic receptor (requires co-expression of a chaperone), and the Ghrelin GPCR complex (4 proteins, a chaperone, and a nanobody to help stabilize the complex).

Glycoproteins take part in nearly every biological process and, when not properly maintained, can lead to disease states. Glycosylation of these important proteins has been shown to influence protein structure, dynamics, protein-protein interactions, and recognition by host immunity. In order to study these affects, full-length recombinantly expressed membrane proteins that contain N-glycosylation consensus sequences have been glycosylated in vitro by N-glycosyltransferase in the presence of membrane mimetic environments.

The RAS proteins are important molecular switches in the cell that control cell growth and several basic cell functions. The RAS family consists of four isoforms (KRAS4b, HRAS, KRAS4a, NRAS), and mutations are involved several types of human cancer, such as colorectal, lung, and skin. A key element to HRAS, KRAS4a, and NRAS activation is loading of a molecule of GTP and localization to the plasma membrane. This last element is facilitated by the post-translational modification of the c-terminus, where three c-terminal residues are cleaved, and a cysteine is both farnesylated and methylated to generate a hydrophobic lipid tail that can insert into the membrane. Using our insect cell expression platform, we describe a protocol that leads to milligram quantities of protein. Production of these three proteins is important to novel drug-screening campaigns.

Efficient biologics development depends on optimal protein expression in mammalian cells. We evaluated five codon usage strategies for 21 human and murine glycoproteins expressed from our open-source pTipi2.1 vector in HEK293 cells. Small-scale screens revealed no benefit of codon optimization over native sequences, while RNA stability-focused schemes reduced yields. In large-scale production, biased codon usage occasionally improved yields. Thus, codon optimization is non-essential, but exploring multiple strategies can enhance consistency.

• Alternative proteases for fusion tag removal
• Cost-effective reagent-based QC methods
• Protein-labeling technologies
• Up-and-coming fusion tags and promising expression systems
• Is there one hypothetical reagent that would be transformational to your protein production efforts??

• What is the use of codon optimization? Revisiting gene synthesis
• HTP cloning methods; Golden gate assembly, Gibson cloning, etc
• Expression systems revisited (Old and up and coming)Expression chaperones, fusion proteins
• Open source cell lines, vectors and media​​

Efficient recombinant-protein production requires strategies that enhance yield while reducing experimental trials. This work demonstrates how metabolomics and dynamic flux balance analysis accelerate process optimization. By mapping substrate utilization and identifying metabolic hubs, targeted supplementation boosted protein productivity by 12-fold. This approach enables rapid, cost-effective bioprocess development. We also present a novel framework to harmonize metabolomics datasets from repositories such as Metabolomics Workbench, enabling broader comparative analyses across studies.

AI-based protein design enables generation of novel antibodies and antigens with improved properties, but successful translation depends on scalable recombinant production. We present our integrated workflow for expressing and characterizing AI-designed proteins, including antibodies and solubilized multi-pass membrane protein mimics (solMPMPs). By combining structure-guided design with high-throughput expression and screening, we evaluate design success and identify key failure modes related to expression, folding, and manufacturability. Our experience underscores the value of coupling computational tools with robust experimental pipelines to improve hit rates, especially for complex or traditionally intractable targets.

Membrane protein targets are recombinantly expressed in a soluble, native-like conformation using a machine learning-guided scaffolding approach. These membrane protein surrogates are experimentally validated to retain native ligand binding and are expressed in human cells to support post-translational modifications. This strategy enables soluble production of previously intractable targets and has led to the successful discovery of highly specific antibodies against challenging membrane proteins.

Histatins comprise a family of ~12 histidine-rich peptides naturally present in human saliva. Their antimicrobial properties have attracted significant interest as potential therapeutics for combating oral infections. Recombinant expression of histatin peptides with E. coli has traditionally used cyanogen bromide to cleave the desired peptide sequence from a fusion protein. This talk will present an immobilized enzyme approach for obtaining histatin peptides that obviates the need for cyanogen bromide. The applicability of this approach to the production of other peptides, and its practicality in terms of yield, cost, and environmental impact, will also be discussed.

Conjugate vaccines, composed of pathogen glycans attached to immunogenic carrier proteins, are effective tools to prevent bacterial infections. However, many conjugate vaccines produce poor immune responses, and new methods are required to optimize vaccine design for stronger immunogenicity. We developed an in vitro workflow coupling cell-free gene expression and AlphaLISA to rapidly characterize and engineer post-translational modifications, including glycosylation. We used our workflow to engineer oligosaccharyltransferases involved in protein glycan coupling technology, leading to the identification of mutant enzymes and sites within a vaccine carrier protein that enable high efficiency production of glycosylated proteins. We then scaled up the cell-free production of vaccines for in vivo immune evaluation. Our method accelerates the characterization of post-translational modifications and the engineering of enzymes for more efficient production of therapeutic proteins.