Cambridge Healthtech Instituteの第18回年次
Difficult-to-Express Proteins
(難発現タンパク質)
高難度タンパク質の発現、精製、生産の克服
2023年5月15〜16日 EDT(東部夏時間)
5月15日(月)
Registration and Morning Coffee7:00 am
PRODUCTION OF MEMBRANE PROTEINS
膜タンパク質の生産
Production of Human A2AAR in Lipid Nanodiscs for 19F-NMR and Single Molecule Fluorescence Spectroscopy
Matthew T. Eddy, PhD, Assistant Professor, Chemistry, University of Florida, Gainesville
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.
Applying Nanodisc Technologies for de novo Cell-Free Synthesis of Membrane Proteins
Matthew Coleman, PhD, Senior Scientist & Group Leader, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory
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.

Hayato Nagano, PhD, Lead Researcher, Research Institute for Bioscience Products & Fine Chemicals, AJINOMOTO Co. Inc.
Ajinomoto Bio-Pharma Services as a fully integrated contract development and manufacturing organization (CDMO) offers a broad range of innovative platform technologies and the End-to-End solutions for biopharmaceutical development and manufacturing. In this presentation, we will show our CDMO capability and CORYNEX® protein expression platform technology including the site-specific incorporation of non-canonical amino acid for biopharmaceuticals.
Networking Coffee Break10:00 am
FEATURED PRESENTATION: Molecular Engineering of Water-Soluble Integral Membrane Proteins and Their Application
Matthew DeLisa, PhD, Director, Cornell Institute of Biotechnology, Cornell University; Co-Founder, UbiquiTx, Inc.
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, access to these membrane-embedded proteins for basic and applied research is limited by technical difficulties associated with their recombinant expression. In this talk, I will describe a universal strategy called SIMPLEx (solubilization of IMPs with high levels of expression) for topologically converting IMPs into water-soluble proteins, which are expressed solubly and with retention of activity. A variety of biotechnological applications for water-soluble IMPs will be discussed, including as biocatalysts of post-translational protein modifications and as subunit vaccine antigens, among others.
The Simplicity and Complexity of T Cell Receptor Signal Spanning Transmembrane Domains
Kristine N. Brazin, PhD, Principal Scientist, Medical Oncology, Dana-Farber Cancer Institute
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.

Russell Coleman, Director, Strain Engineering, Ligand Pharmacueticals
The Pelican Expression Technology is a robust, validated, cost-effective and scalable platform for recombinant protein production, and is especially well-suited for complex, large-scale protein production where traditional systems are not suitable. An overview of how this Pseudomonas-based expression platform was developed specifically for recombinant protein production will be presented.

Deborah Moore-Lai, Senior Director of Protein Development, R&D Leadership, abcam
More than ever, speed and quality are of utmost importance in reagent generation for early stage research and drug discovery. Generating high-quality reagents requires time, resources and an array of protein production methods. Scientists at Abcam have developed a large toolbox of techniques, including multiple expression systems and a complimentary suite of bioanalytical characterization methods. The talk will highlight the robustness of the platforms generating commercial reagents meeting stringent release criteria.
INTERACTIVE DISCUSSIONS
インタラクティブディスカッション
Interactive Discussions are informal, moderated discussions, allowing participants to exchange ideas and experiences and develop future collaborations around a focused topic. Each discussion will be led by a facilitator who keeps the discussion on track and the group engaged. To get the most out of this format, please come prepared to share examples from your work, be a part of a collective, problem-solving session, and participate in active idea sharing. Please visit the Interactive Discussions page on the conference website for a complete listing of topics and descriptions.
Production and Stabilization Membrane Proteins
Matthew Coleman, PhD, Senior Scientist & Group Leader, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory
Matthew DeLisa, PhD, Director, Cornell Institute of Biotechnology, Cornell University; Co-Founder, UbiquiTx, Inc.
- What are the current major limitations of obtaining intact and stable membrane protein complexes?
- What would we like to see developed in terms scaffold/reagent supports for assessing membrane proteins?
- Are there ideal techniques/additives for long term storage of functional membrane proteins and the complexes they form?
- How do we assess the biological compatibility of nanodisc technologies for invitro and in vivo experimentation?
- What keeps cell-free expression synthesis from playing a bigger role in membrane protein production?
Session Break1:30 pm
MEMBRANE PROTEIN TARGETS
膜タンパク質のターゲット
Expression, Purification, and Characterization of Human Membrane Proteins for Structure-Based Drug Discovery by cryoEM
Scott Jackson, PhD, Senior Research Associate, Molecular Sciences, Astex Pharmaceuticals Ltd.
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.
Novel Salipro-CXCR Complexes Enable Development of Next Generation Therapeutics
Sara Bonetti, PhD, Scientist, Salipro Biotech AB, Sweden
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.
Sponsored Presentation (Opportunity Available)2:50 pm
Networking Refreshment Break3:20 pm
Transition to Plenary Keynote Session3:50 pm
PLENARY KEYNOTE SESSION
プレナリーセッション・基調講演
Advances in CAR T Therapy
Carl H. June, MD, Richard W. Vague Professor in Immunotherapy; Professor of Medicine; Director, Center for Cellular Immunotherapies; Director, Parker Institute for Cancer Immunotherapy, University of Pennsylvania Perelman School of Medicine
Advances in the understanding of basic immunology have ushered in two major approaches for cancer therapy over the past 10 years. The first is checkpoint therapy to augment the function of the natural immune system. The second uses the emerging discipline of synthetic biology and the tools of molecular biology and genome engineering to create new forms of engineered cells with enhanced functionalities. The emergence of synthetic biology approaches for cellular engineering provides a broadly expanded set of tools for programming immune cells for enhanced function. Barriers to therapy of solid tumors will be discussed.
The Next Frontier in Machine Learning and Biologics: "Lab in a Loop" Large Molecule Drug Discovery, From Optimization to de novo Discovery
John Marioni, PhD, Senior Vice President and Head of Computation, Research and Early Development, Genentech
A key opportunity in applying machine learning to augment biologic drug discovery and development is through constant iteration - a process we call "lab in a loop." By developing integrated methods for optimizing affinity and multiple developability parameters, as well as a close integration of antibody engineering, machine learning, and structural biology, we have the potential to more rapidly identify and test novel candidate molecules. Sophisticated machine learning frameworks allow us to integrate later stages of optimization into the earliest stages of discovery, while high-throughput experimental systems allow rapid improvement of all methods and molecules. This process starts with the integration of people and scientific culture and ends with tightly integrated computational and experimental systems.
Welcome Reception in the Exhibit Hall with Poster Viewing5:40 pm
Close of Day7:00 pm
5月16日(火)
Registration and Morning Coffee8:00 am
EXPRESSION AND PRODUCTION OF CHALLENGING PROTEINS
高難度タンパク質の発現と生産
Development of mRNA Vaccines and Therapeutics Requires Small- to Mid-Scale Production of Difficult-to-Produce Recombinant Proteins
Ethan Dunn, Manager, Protein Sciences, Moderna, Inc.
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.
End-to-End Multi-Host Screening Platform for Difficult-to-Express Proteins and Protein Complexes
Inna Zilberleyb, Scientist 4, Biomolecular Resources, Genentech, Inc.
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.
A Semi-Automated DoE-Based Approach to Accelerate Discovery and Engineering
Eric R Sterner, PhD, Associate Principal Scientist, Biologics Discovery, Merck Research Labs
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.

Chun-Nan Chen, Ph.D., CEO, Single Cell Technology
It’s now possible to screen all antibodies in parallel for an expanding list of properties down to the antibodies that meet your wish list, and to sequence all antibodies in parallel, digitizing all the data. With Single Cell’s parallel screen-and-sequence workflow, nothing gets missed-including deadlines. We will be showcasing our approach applied to key industry challenges: screening for broadly neutralizing anti-influenza antibodies and cell-based screening for membrane-bound targets.
Coffee Break in the Exhibit Hall with Poster Viewing10:30 am
TECHNOLOGY MEETS MEMBRANE TARGET DISCOVERY
テクノロジーと膜ターゲット探索の融合
Close of Difficult-to-Express Proteins Conference1:40 pm
Recommended Dinner Short Course6:30 pm
SC7: Use and Troubleshooting of Eukaryotic Expression Systems
*Separate registration required. See short courses page for details.
* 不測の事態により、事前の予告なしにプログラムが変更される場合があります。
2023年 プログラム
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