Low expression levels and instability of GPCRs and ion channels hinder protein-based screening and antibody discovery efforts. At Abilita Therapeutics, we leverage advanced protein engineering and directed-evolution EMP technology to significantly enhance the yield and thermostability of multispan membrane proteins. This results in availability of highly expressed, stable GPCRs and ion channels, enabling the effective isolation of novel therapeutic antibodies through both in vivo and in vitro approaches.

Integral membrane proteins are a class of highly druggable targets that are difficult to access in recombinant systems in amounts and quality sufficient for binding-first methods such as DNA Encoded Library (DEL) and ASMS for hit finding, and then for follow-up of hits. In this talk, I will review strategies and approaches for accessing these targets using membrane mimetics including SMALPs and other forms.

This study focuses on human focal adhesion proteins, investigating their expression and purification. We aim to elucidate the role of these proteins in cellular adhesion and signaling. Through a combination of molecular biology techniques, we successfully purified recombinant focal adhesion proteins. Our findings provide a foundation for further research into the structure, function, and potential therapeutic applications of these essential components.

Many proteins are difficult to produce in recombinant systems due to factors such as insolubility, instability, and low yield. This presentation will discuss strategies to optimize the expression of challenging protein targets.

RAS proteins are the most frequently mutated in human cancer. The role of post-translational modifications (PTMs) in RAS-dependent signaling is not fully understood. Top-down analysis of intact and modified RAS protein forms (proteoforms) provides a level of molecular detail unachievable by other proteomic methods. Previously, The NCI RAS Initiative developed-and here, we further optimized a novel top-down assay to understand cysteine reactivity and localization of PTMs on RAS proteoforms.

NendoU, a SARS-CoV-2 protein, plays a critical role in viral replication and evasion of host immune response. This presentation will explore the expression and characterization of wild-type and mutant NendoU proteins. Understanding the NendoU protein will aid in developing mitigation strategies and therapeutic interventions against the virus.

• Advancements in detergents and nanodisc technologies 
• Emerging techniques to overcome instability
• High-throughput screening and process automation approaches
• Overcoming glycosylation and post-translational challenges
• Case studies and practical challenges
  

• Choosing an appropriate QC method for your budget
• Moving on from biotin. Upcoming alternative protein labelling technologies
• Challenges in addition of site-specific PTMs (e.g. phosphorylation) and protein PTMs (e.g. ubiquitin, SUMO)
• Advances in (de)glycosylation and its analysis
  

We have developed a reproducible and scalable platform for production and purification of HIV-1 Gag VLPs to display “difficult to express” proteins in their native lipid environment and their native conformation. To enhance antigen expression, we have optimized cell lines and identified peptides that increase antigen expression in the membrane. Further, we have developed methods to site-specifically conjugate biotin molecules on VLPs without affecting antigen expression, conformation, size or purity.

The coenzyme biotin binds to streptavidin with very high affinity, and is exploited in a variety of screening and assay formats commonly used in drug discovery. Proteins can be selectively post-translationally biotinylated using the BirA biotin ligase. This is often performed post hoc, but co-expressing with BirA in vivo may lead to significant advantages, including reduced processing times and more complete modification. Here we discuss our approach for intracellular co-expression of BirA in E. coli, and intracellular and secreted co-expression in insect cells and mammalian cells.

Cell-free transcription-translation (TXTL) is a rapidly expanding technology that offers a broad range of applications. TXTL is characterized by a fast experimental turnover enabling the synthesis of proteins from DNAs encoding single genes to DNAs encoding tens of genes. I will present the last capabilities of an all-E. coli TXTL system.

Synthetic biology offers the potential to control intercellular signals such as secreted proteins in biomedicine. In this talk, I will highlight compRELEASE, a platform to control the secretion or expression of any protein of interest using endogenous 14-3-3 proteins and proteases in mammalian cells. Furthermore, the compRELEASE platform enables the compact control of multiple proteins, while minimizing the overall genetic payload-and is compatible with pre-existing synthetic protein circuits.

The Structural Biology and Protein Sciences group plays a key role within Pfizer’s oncology research organization, supporting and enabling early stage small molecule drug discovery efforts by generating recombinant proteins for hit identification, lead optimization, biochemical assay development, and structure-based drug design cycles. Addressing the challenges of resource constraints, aggressive timelines, and the competitive landscape of drug discovery, the group utilizes optimized protein production processes to support a multitude of concurrent projects in its research portfolio. This presentation will highlight some of the successful strategies utilized to generate high-quality, fit-for-purpose recombinant protein tools.

Tobacco etch virus (TEV) protease is the workhorse of protein expression. TEV-protease is remarkable in its sequence specificity, and its ability to cleave fusion tag proteins. Herein we report work on large-scale production of TEV-protease using different promotors, media, fusion tags, and expression platforms. How our pgl plus bacteria negates post-translational modification (gluconylation and phosphogluconylation), and mollifies deleterious effects on Ni2+-affinity. Our new protocols increase production yields to 400-500 mg/L TEV-protease.

This study introduces a novel self-removing affinity tag for enhanced protein production. The tag facilitates efficient purification while minimizing downstream processing steps. We demonstrate its effectiveness in various expression systems and protein targets, resulting in significantly higher yields compared to traditional affinity tags. Our findings provide a valuable tool for researchers seeking to optimize protein production and purification processes.