Difficult to Express Proteins April 30 - May 1
Membrane proteins, ion channels, toxic or insoluble proteins, low abundance protein complexes, vaccines and other “finicky” proteins often are the best choices for therapeutics, yet their successful expression is a minefield of failures and challenges. This conference will present innovative and imaginative methods to make those difficult to express proteins more cooperative and to increase yield and performance.
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SUNDAY, APRIL 29
4:00 - 6:00 pm Main Conference Registration
MONDAY, APRIL 30
7:00 am Registration and Morning Coffee
8:30 Chairperson’s Opening Remarks
» 8:40 Featured presentation
Overcoming Challenges of Difficult to Express Proteins
Jeff Culp, Ph.D., Associate Research Fellow, Primary Pharmacology Group, Pfizer Worldwide Research and Development (biography)
As our understanding of Biology and Disease evolves, challenges increase to deliver biologically relevant proteins for use with all Drug Discovery tools. Mammalian, insect and bacterial expression systems must be leveraged. Proper protein characterization is critical to eliminate potential mistakes. Successful examples will be presented for proteins intended for use in target screens, NMR, crystallization and biophysical characterization.
9:10 Peptide Surfactants for Cell-Free Production of Functional G Protein-Coupled Receptors
Shuguang Zhang, Ph.D., Associate Director, Center for Biomedical Engineering, Massachusetts Institute of Technology (biography)
We report using peptide surfactants in commercial E. coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs, or even other membrane proteins, for structure and function studies.
9:40 Expression of the Transmembrane Domain of the Human APP Binding Protein LR11 for “in Situ” NMR Structural Analysis
Fang Tian, Ph.D., Assistant Professor, Department of Biochemistry and Molecular Biology, College of Medicine, Pennsylvania State University (biography)
Information about protein structure in biological environment is scarce.To date, most membrane structure determinations have been carried out in detergent preparations and synthetic lipid bilayers. Using a new MBP expression vector, we successfully expressed the transmembrane domain of the human APP binding protein LR11 at high yields for a direct structural characterization in native Escherichia coli membranes.
10:10 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
11:10 Engineering Membrane Proteins for Better Expression in E. coli
Morten Nørholm, Ph.D., Senior Scientist, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark (biography)
In this study we show that fusion of an N-terminal peptide to a poorly-expressed membrane protein of E. coli or human origin, can significantly improve over-expression levels. Further, we could mimic the effect of the N-terminal peptide by re-engineered the 5’ mRNA with favorable synonymous mutations. These simple changes significantly improve over-expression of the native protein and provide a design principle for engineering better expressing membrane proteins.
11:40 Crystallization Chaperone Strategies for Membrane Proteins
Jennifer A. Maynard, Ph.D., Assistant Professor, Chemical Engineering, University of Texas Austin (biography)
From G protein-coupled receptors to ion channels, membrane proteins represent over half of known drug targets, yet structure-based drug discovery is hampered by the lack of available three-dimensional models. Here, we present a novel, generic solution: development of a toolbox of engineered antibodies recognizing short peptides to chaperone crystallization of membrane proteins presenting the peptide ligand in a permissive loop.
12:10 pm Maximizing Recombinant Protein Expression through Systematic Gene Design
Mark Welch, Ph.D., Director of Gene Designs, DNA2.0 Inc.
Advances in gene design and synthesis have enabled greater insight into the workings of the genetic code. Full control over features such as codon bias and mRNA structure allows systematic study of how gene sequence impacts expression of encoded proteins. We present studies on how gene design variables affect heterologous protein expression for a wide range of protein targets and host organisms, including mammalian, yeasts, bacteria, etc. We show predictive relationships between gene sequence features and expression that provide the basis for gene design algorithms that far outperform previous methods.
12:25 Biochemical and Pharmacological Interrogation of Membrane-Associated Enzymes using Template-Directed Assembly (TDATM)Norman Garceau, Ph.D., President & CSO, Blue Sky BioServices
Membrane proteins are difficult to study because they function within the hydrophobic environment of a lipid bilayer, so they are frequently studied as isolated domains in aqueous buffer lacking a physiological context that is important for proper function. We present studies using specially-formulated lipid vesicles (TDATM) to dock membrane proteins and accessory factors and demonstrate that TDA-enabled kinase assays exhibit improved biochemical activity and identify compounds with unique or altered potency.
12:55 Luncheon Presentations (Sponsorship Opportunities Available) or Lunch on Your Own
2:00 Chairperson’s Remarks
Paul Wengender, Founder & CEO, Blue Sky BioServices
2:05 Retention of Thrombin Inhibitory Activity by Recombinant Serpins Expressed as Integral Membrane Proteins Tethered to the Surface of Mammalian Cells
William P. Sheffield, Ph.D., Professor, Departments of Pathology and Molecular Medicine, McMaster University (biography)
Both TR- and AR-α(1) PI M358R were enriched in the integral membrane fraction of transfected COS-1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α(1) PI M358R.Two of three thrombin-inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.
2:35 A Novel Expression and Purification Platform for the Production of Soluble Human Lysozyme in E. coli
John Lamppa, Chemical and Biomolecular Engineering, Thayer School of Engineering, Dartmouth College (biography)
Pre-clinical assessment of novel lysozyme variants requires a robust, efficient, and scalable expression system. E. coli is accessible, efficient and a scalable platform, but expression of soluble lysozyme is toxic to these cells. To capitalize on the numerous benefits of this bacterial host, we have developed an anti-toxin co-expression system that yields a 1000-fold increase in soluble lysozyme relative to prior reports.
3:05 Daedalus: A Robust, Turnkey Platform for Rapid Production of Decigram Quantities of Active Recombinant Proteins in Human Cell Lines Using Novel Lentiviral Vectors
Ashok Bandaranayake, Ph.D., Associate, Department of Biochemistry, Albert Einstein College of Medicine (biography)
We describe a novel system for the rapid production of recombinant mam-malian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression.
3:35 Expression of a Self Assembling Immune Adjuvant and Antigen Targeting Fusion Protein to Accelerate the Development of New Vaccines for Emerging Infectious DiseasesMark C. Poznansky, M.D., Ph.D., Director, Vaccine and Immunotherapy Center, Massachusetts General HospitalThis case study will describe the production of a difficult to express MTBHsp70-avidin fusion protein using the Pfenex Expression Technology platform. Successful production of this protein is critical in the quest for a platform technology that allows accelerated development and preclinical testing of new vaccines for emerging infectious diseases.
4:05 Refreshment Break in the Exhibit Hall with Poster Viewing
4:45 Problem Solving Breakout Discussions
Concurrent problem solving breakout discussions, open to all attendees, speakers, sponsors, and exhibitors, provide a forum for discussing key issues and meeting potential collaborators. Plan to take part and explore these topics in-depth. Please pick a topic of your choice, find your table and join in.
Delivery of Expressed Proteins to the Central Nervous System
Moderator: Girish Kotwal, Ph.D., Professor, Biochemistry and Microbiology, University of Medicine and Health Sciences-St. Kitts
• Possibilities to pass the blood brain barrier for small protein delivery..
• Immunomodulation in brain and spinal cord injury and Alzheimer's Disease.
• Enhancing the quantities to be delivered: nanotechnology, pressure devices, direct delivery.
• State-of-the-art in delivery to the CNS and areas of future research.
Platforms for Fast Screening of Non-Antibody Protein Therapeutic Candidates
Moderator: Liang Tang, Ph.D., Senior Scientist, Molecular Biology & Protein Expression, Bayer HealthCare LLC
• Unmet needs in Protein Expression from Discovery to Production
o Fast Turnaround Time
o PTM Consistency and Protein Integrity in Process
• Pros & Cons of Different Expression Systems
o Cell types
o Episomal vs chromosomal
• Non-Mammalian Expression Systems
o E. coli, yeast and baculovirus
o Transgenic, non-mammalian systems
Considerations for Expressing Integral Membrane Proteins in E. coli
Moderator: Brian Kloss Ph.D., Senior Research Associate, NYCOMPS, New York Structural Biology Center
• How many proteins do you want to express?
• From what organism is the protein you are trying to express?
• For what purpose are you expressing your protein?
• How much protein do you need?
Strategies to Tackle Aggregation Problems During Purification
Moderator: Mario Lebendiker, Ph.D., Manager, Protein Purification Facility, Hebrew University
• How do you check soluble aggregates
• At what purification step is better to eliminate the soluble aggregates
• What additives you prefer to use
• What about the use of low concentration of unfolding agents as Urea or Guanidine HCl
• Buffer Screening for Protein Solubility: thermofluor, commercial kits, others
• Use of reducing agents for proteins with free Cis plus disulfide bridges
Tuning the Baculovirus System for Best Outcomes
Moderator: Arnaud Poterzsman, Ph.D., Research Director, Structural Biology and Genomis, IGBMC
• Time and cost considerations
• Reproducibility and quality control
• Virus storage and infection/co-infection protocols
• Engineering the baculoviral genome
• Multi-gene expression
• Baculovirus versus Mammalian expression
Why are Some Antibodies Difficult to Express?
Moderator: Haruki Hasegawa, Ph.D., Senior Scientist, Protein Science, Amgen Inc.
• Cell biological or biochemical basis for the various difficulties
• Roles of physicochemical properties
• Expression diagnostic assays
• Case studies for tested approaches
Enhancing Cell Resistance to Proteolytic Attack via Protein Engineering
Moderator: William Sheffield, Ph.D., Associate Professor, Pathology, McMaster University
• Why "armor" cells? Applications in transplantation
• Avoiding making engineered cells immunogenic
• Do we have to? Gene therapy vs other cell modification approaches
Can the Daedalus System Solve your Protein Expression Problems?
Moderator: Ashok Bandaranayake, Ph.D., Senior Fellow, Pediatrics and Immunology, Seattle Children’s Research Institute
• Use of the Daedalus platform in expressing a wide variety of proteins
• Limitations of the Daedalus system
• Further optimization and cross-platform uses
Improving Cell Free Expression of GPCRs
Moderator: Shuguang Zhang, Ph.D., Associate Director, Center for Biomedical Engineering, MIT
Data Mining to Improve Choices and Outcomes
Moderator: John F. Hunt, Ph.D., Professor, Biological Sciences, Cornell University
Improving Finicky Protein Expression in E. coli
Moderator: Morten Norholm, Ph.D., Senior Scientist/Project Leader, Technical University of Denmark
5:45-6:45 Welcome Reception in the Exhibit Hall with Poster Viewing
Poster Awards Sponsored by
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