Biotech scientists often get mixed results when using fusion proteins to attempt high yield production of difficult-to-express proteins. After our own experience struggling to improve yield with traditional fusion tags, we developed a new kind of fusion tag – synthetic, custom, and optimized for the protein of interest. Using this novel tag, we observed an increase in expression yields of notoriously difficult-to-express targets like amyloid beta and a designed membrane pore peptide, 2 and 10 fold, respectively when compared to SUMO.

Antibody fragments such as scFvs have great potential as crystallization chaperones for structural biology due to their ability to stabilise targets, trap certain conformations and/or promote crystal packing. Here we present a few examples of using scFvs to determine 3D structures through X-ray crystallography and discuss properties of the molecule that could be improved for higher success rates. Furthermore, production of biotinylated antigens and scFvs have also been optimized.

-Although antibodies still predominate in the clinic, there is a shift in drug development pipelines towards more complex, next generation biologics (NGBs)

-Such NGBs often do not express well in traditional expression platforms

-In this presentation, we will discuss two solutions which are included in our GS toolbox and are designed to especially help overcome challenges with NGBs: GS® pXC Multigene Vectors and GS piggyBac^®. 

Cryo-EM has demonstrated great potential to elucidate high-resolution structures of challenging membrane protein targets with fast turnaround time. In the current study, we utilized cryo-EM as a major tool to identify the optimal reconstitution systems for two difficult membrane proteins. Our results indicated that even though these membrane proteins showed decent biophysical properties in many conditions, only through high-resolution cryo-EM structural studies were we able to identify conditions that confer the greatest conformational stability.

Native ion mobility mass spectrometry (IM-MS) is an emerging biophysical technique to probe membrane protein complexes and their interactions with lipids and other small molecules. I will demonstrate how IM-MS can be used as an invaluable method to optimize purification of target proteins. I will then highlight our work using native IM-MS to not only determine binding thermodynamics but also cooperative and allosteric mechanisms for membrane protein-ligands interactions.

COVID-19 is a global health crisis caused by SARS-CoV-2, and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S). We characterize the production of the SARS-CoV-2 S protein in ExpiCHO-S Cells™, and highlight downstream biological and clinical uses of purified recombinant S protein. These include examining interactions with proposed binding partners within the human secretome, analysis of S protein glycosylation patterns, the development of clinical assays to detect SARS-CoV-2 antibodies, and analysis of the innate and humoral immune responses to SARS-CoV-2.

Manufacturing of protein therapeutics, such as biologics, antibodies and subunit vaccines, is often hindered by difficulties in recombinant protein production, especially for membrane-bound proteins. Messenger RNA therapeutics harness the body’s own cells to produce and deliver therapeutic protein molecules. We have successfully developed a platform using HDL mimetics (termed nanolipoprotein particles, NLPs) to deliver self-amplifying mRNA constructs in vivo and achieve robust expression efficiency.

It remains challenging to transfer intact physiological macromolecules from their native sources into suitably stabilizing in vitro environments. To address this, we developed an interactome capture platform that is akin to a crystallographic screen. The approach will be summarized in this talk, leveraging research vignettes. Our long-term objective is to enable robust, tunable transfer of target endogenous macromolecules from their in vivo milieus into test tubes, for biochemical, structural, and mechanistic studies.

Sino Biological is a global leader in development and manufacturing of ISO9001 and ISO13485-certified reagents, including recombinant proteins and antibodies. An overview of the company’s state-of-the-art technologies for optimizing protein expression will be given addressing production challenges as well as key influencing factors and will include several example case studies.

Covalently circularized nanodiscs and DNA-corralled nanodiscs have opened up the possibility of engineering large nanodiscs of sizes up to 90 nm. These large nanodiscs are extending the applicability of nanodisc technology from studying small membrane proteins to acting as a surrogate membrane to investigate structural and functional aspects of viral entry. We will present the recent technical developments leading to construction of large nanodiscs and show some of the viral entry applications.

Employing nanolipoprotein particles (NLPs), aka nanodiscs, to yield membrane proteins in stable, native-like states has become common practice to facilitate biochemical and biophysical characterization, and NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques. We show how cell-free reactions can be modified to render control over nanoparticle size, monodispersity and complex organization in support of producing functional membrane proteins.

G-protein coupled receptors (GPCRs) are the largest family of drug targets and the targets of >35% of all drugs. Biochemical and structural studies of GPCRs have been hampered by the difficulty of recombinantly producing and purifying GPCRs. I provide an overview of using cell-free expression systems to produce GPCRs. I also describe our lab’s success with producing the GPCR, G-protein coupled estrogen receptor, for the first published biochemical binding assays.

Our work focuses on human cannabinoid receptor CB2, an important regulator of inflammatory pathways. To obtain thermostable variants of this receptor, we expressed it in suspension expi293 and GNTI- cells and purified using protocols developed in our laboratory. The mammalian cell-expressed receptor was functionally active and homogenous. The detergent-solubilized receptor is stable at 15 oC for several days which enables its characterization by solution-state NMR; post-translational modifications of this protein likely determine its structural stability.

Off-gas measurement from shake flasks will facilitate a fundamental understanding of upstream processes and enable reliable scale-up into larger-scale bioreactors. Combining this powerful technology with the proven scale-up benefits of orbital-shaken bioreactors offers fast and reproducible results. Here we introduce the new Kuhner TOM instrument for off-gas analysis online in parallel shake flasks and highlight its use to scale into large-scale shaken bioreactors - focusing on recent successes at the 10L scale.

The Protein Expression group within the Early Solutions team at UCB generates protein targets for gene to structure analysis and biochemical assays. Case studies will be presented describing the expression and process optimization of challenging-to-express proteins.

Randomized configuration targeted integration (RCTI) CLD approach allows simultaneous transfection of multiple configurations of transgenes encoding a complex protein to generate a plurality of clones each with a unique transgene configuration, specific folding, and product quality. Screening RCTI single cell clones allows seamless isolation of clones with comparable titers and product quality attributes to that of several parallel standard CLDs, significantly reducing resources needed to express difficult or complex molecules.