Difficult-to-Express Proteins

The intrinsic nature of proteins tends to make them ‘difficult’ to produce. CHI’s 15th Annual Difficult-to-Express Proteins conference examines the challenges researchers encounter when striving for high-yield production of “difficult-to-express” proteins (DTEPs), and the strategies and technologies that have proven successful in overcoming those challenges. Besides exploring notoriously difficult proteins, such as membrane proteins, the conference will also look at the emerging tools that are proving helpful in determining structure, and protocols that are successful for handling proteins that are difficult to express. The Difficult-to-Express Proteins conference provides the latest developments in improving yield for DTEPs through case studies and breakthrough data.

Final Agenda


Recommended Short Course(s)*

SC4: LC-MS Method Validation for Small Molecules in Biological Matrix

SC6: Translational Biotherapeutic Development Strategies Part II: Analytical and Clinical Considerations

*Separate registration required.


7:00 am Registration and Morning Coffee


8:30 Chairperson’s Opening Remarks

Rana Sidhu, PhD, Protein Expression Lead, Early Solutions, UCB, Inc.

8:40 Building a Platform for Automated Implementation of Design-Build-Test-Learn Cycles to Improve Protein and Peptide Biologics

Love_JamesJames Love, PhD, Director, Applied Automation, Global Research Technologies, Novo Nordisk A/S

Protein production can be engineered for desirable properties using iterative cycles of improvements. This talk will highlight the groundwork for automated protein production and design of a platform for carrying out these cycles of improvement in a fully automated fashion, with maximal data capture and learning, at all stages of the processes.

9:10 Harnessing Synthetic Biology to Produce Difficult-to-Express-Proteins

Shlomo Zarzhitsky, PhD, Research Associate, Chemistry, Princeton University

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.

9:40 Strategies to Improve Single-Chain Fvs as Crystallization Chaperones

Susanne Gräslund, PhD, Principal Investigator, Structural Genomics Consortium, Karolinska Institute

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.

10:10 Networking Coffee Break


10:45 Chairperson’s Remarks

Rana Sidhu, PhD, Protein Expression Lead, Early Solutions, UCB, Inc.

10:50 KEYNOTE PRESENTATION: GPCR Expression for Structural Biology Using NMR in Solution

Kurt Wüthrich, PhD, Professor, Biophysics, Institute of Molecular Biology, ETH Zürich

Expression strategies for GPCRs to be used for structural biology by NMR in solution will be described. We have used insertion of “NMR probes” by expression of suitably mutated GPCRs for post-translational chemical modification in defined sequence positions, as well as an expression system that enables extensive labeling with stable isotopes for multi-dimensional high-resolution NMR experiments. The results obtained with the expression strategies used will be illustrated with NMR studies of β2AR and A2AAR.

11:20 Cryo-EM Guided Reconstitution System Optimization for Challenging Membrane Proteins towards High Conformational Stability

Xinchao Yu, PhD, Senior Scientist, Molecular Engineering, Amgen, Inc.

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.

11:50 Native Ion Mobility Mass Spectrometry of Intact Membrane Protein Complexes

Arthur Laganowsky, PhD, Assistant Professor, Chemistry, Texas A&M University

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.

12:20 pm Sponsored Presentation (Opportunity Available)

12:50 Session Break

12:55 Luncheon Presentation I to be Announced

Berkeley_Lights_Stacked 1:25 LUNCHEON PRESENTATION II: The Beacon Platform for Accelerated Discovery and Development of Therapeutic Antibodies to Difficult Targets

Anupam Singhal, PhD, Technology Development, Berkeley Lights, Inc.

I will demonstrate how Berkeley Lights’ tech enables rapid discovery & development of antibody therapeutics. I will show how Beacon Plasma B Cell Antibody Discovery enables target-to-lead candidate selection in 1 day. Once lead candidates have been selected, Beacon Cell-Line Development enables selection of production cell lines with >99% monoclonality assurance in less than 1 week. Case studies used demonstrate how our customers are using these products to accelerate the dev of next-generation antibody therapeutics.

1:55 Session Break

2:20 Problem-Solving Breakout Discussions View All Breakout Discussion Topics

TABLE: Experiences with Using Different Binder Types as Crystallization Chaperones or Cryo-EM Handles

Moderator: Susanne Gräslund, PhD, Principal Investigator, Structural Genomics Consortium, Karolinska Institute

  • What binder types/scaffolds have the participants worked with?
  • Has any comparisons of different binder types have been made?
  • What type of antigens, soluble proteins, complexes, integral membrane proteins?
  • Different methods of immobilizing the antigens during selections?

TABLE: Cell-Free Approaches to Difficult Proteins

Moderator: Matthew Coleman, PhD, Senior Scientist & Group Leader, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory

  • Eukaryotic versus prokaryotic cell-free expression systems
  • Variants of cell free expression (batch versus dialysis)
  • Additives for screening and producing membrane bound proteins

TABLE: Optimising Protein Production Strategies for Multi-Protein Complexes Within Drug Discovery

Moderator: Saleha Patel, PhD, Senior Research Scientist, Protein Science, Discovery Biology, AstraZeneca

  • Obtaining high-quality homogeneous protein samples, at high yields, are often a pre-requisite for a successful drug discovery campaign
  • What are the current challenges for protein production in drug discovery?
  • What are the best protein production strategies for recombinant multi-protein complexes?
  • Are there any new technologies available to help with the increasing complexity of protein targets/antigens?
  • How do we ensure protein reagents are fit-for-purpose for downstream applications?

3:20 Networking Refreshment Break


4:00 Chairperson’s Remarks

K. Dane Wittrup, PhD, J.R. Mares Professor, Chemical Engineering & Bioengineering, Massachusetts Institute of Technology

4:10 From Energy to Machine Learning

George-ChurchGeorge Church, PhD, Professor of Genetics, Harvard Medical School; Professor of Health Sciences and Technology, Harvard and the Massachusetts Institute of Technology (MIT)

In 1974, I adapted energy optimization methods for use in models of nucleic acids, protein and their interactions, and then for use in crystallographic refinement. In the last days of the second millennium, David Baker's team won the Critical Assessment of Structure Prediction (CASP) by an unbelievable margin. Since then, our labs exchanged 3 PhD students (Dantas, Raman, Lajoie), for Wannier from Mayo's group, Stranges from Kuhlman, and Mandell from Kortemme. We engineered new sensor proteins for metabolic engineering, essential proteins with non-standard amino acids for biocontainment, and polymerase-pore fusions for nanopore sequencing. None of this prepared us for the revolution following Gleb Kuznetsov joining our lab in 2012, joined soon by Surge Biswas, Pierce Ogden, Ethan Alley, and Sam Sinai. Together we abruptly moved to "sequence only" deep machine learning for protein design – ranging from fluorescent proteins to AAV capsids to antibodies. When combined with libraries of millions of designed gene segments from chip-synthesis and rapid testing, each design cycle can take large leaps in sequence space and function space.

4:55 The Case for Intelligent Design in Protein Engineering

spangler-jaimeJamie Spangler, PhD, Assistant Professor, Biomedical Engineering and Chemical & Biomolecular Engineering, Johns Hopkins University

Directed evolution is in its prime, and it is deepening our understanding of biological systems and empowering therapeutic design. Recent breakthroughs in structural biology, computational design, and high-dimensional data analytics afford us the unprecedented opportunity to apply molecular, structural, and computational principles to guide protein engineering, employing a so-called “intelligent design” approach. This talk will highlight how my lab harnesses this interfacial approach to overcome the deficiencies of natural proteins.

5:40 Welcome Reception in the Exhibit Hall with Poster Viewing (Co-Sponsorship Opportunity Available)

7:15 End of Day


8:00 am Registration and Morning Coffee


8:25 Chairperson’s Remarks

James Love, PhD, Director, Applied Automation, Global Research Technologies, Novo Nordisk A/S

8:30 Large Nanodiscs Going Viral

Nasr_MahmoudMahmoud Nasr, PhD, RPh, Group Leader, Medicine, Brigham and Women’s Hospital, Harvard Medical School

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.

9:00 Cell-Free Co-Translational Approaches for Producing Mammalian Receptors Using Nanolipoproteins (Nanodisc)

Matthew Coleman, PhD, Senior Scientist & Group Leader, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory

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.

9:30 Recombinant Production of a G-Protein Coupled Receptor Using an Escherichia coli Cell-Free Expression System

Ho Leung Ng, PhD, Associate Professor, Biochemistry & Molecular Biophysics, Kansas State University

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.

10:00 Coffee Break in the Exhibit Hall with Poster Viewing


10:45 Chairperson’s Remarks

James Love, PhD, Director, Applied Automation, Global Research Technologies, Novo Nordisk A/S

10:50 Adenosine Receptor Chimeras for Improved Receptor Expression in Yeast

Anne Skaja Robinson, PhD, Trustee Professor and Head, Chemical Engineering, Carnegie Mellon University

The adenosine A2A receptor (A2AR) has a much longer C-terminus than the other adenosine receptor subtypes within the G-protein coupled receptor superfamily, which may contribute to its exceptional trafficking. To test the possibility to improve trafficking of A1R and A3R, chimeric receptors with A2AR were constructed. The chimeric receptors showed improved localization to the plasma membrane and were functionally active, with greater than three-fold higher yields than previously reported from other heterologous expression systems.

11:20 Expression of Challenging Heterodimeric Extracellular Domain of Immune Receptors

Anirban Adhikari, PhD, Associate Director, Biologics Research, Parvus Therapeutics, Inc.

Expression of heterodimeric membrane bound immune receptors (e.g., MHC, TCR) has been challenging. This presentation will describe a novel approach of stabilizing the heterodimer and expressing them as a secreted soluble protein in large scale.

11:50 Expression of Thermostable Human Cannabinoid Receptor CB2 in Mammalian Cell Cultures and Its Biophysical Characterization

Alexei Yeliseev, PhD, Staff Scientist, Group Leader, LMBB, NIH/NIAAA

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.

12:20 pm Session Break

12:25 Luncheon Presentation I to be Announced

12:55 LUNCHEON PRESENTATION II: Shake Flask Off-Gas Measurement and Scale-Up to Single Use Orbital Bioreactors

Thariska Tharmakulasingam, Specialist, Orbital Bioreactors, Kuhner Shaker AG

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.

1:25 Ice Cream Break in the Exhibit Hall with Poster Viewing and Poster Award


2:00 Chairperson’s Remarks

Mahmoud Nasr, PhD, RPh, Group Leader, Medicine, Brigham and Women’s Hospital, Harvard Medical School

2:05 Strategies for Optimizing Challenging-to-Express Protein Targets

Rana Sidhu, PhD, Protein Expression Lead, Early Solutions, UCB, Inc.

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.

2:35 Utilizing Randomized Configuration Targeted Integration (RCTI) Cell Line Development (CLD) Approach for Expression of Difficult or Complex Therapeutic Proteins

Shahram Misaghi, PhD, Senior Scientist, Early Stage Cell Culture (ESCC), Genentech, Inc.

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.

3:05 Scaling Up and Scaling Out: Pushing the Boundaries of Transient Protein Production

Ian Wilkinson, CSO, Absolute Antibody

Whilst transient yields have improved drastically in the last decade, scalable systems are time-consuming and costly to implement. Absolute Antibody has developed systems which scale up and scale out protein expression and purification, enabling the rapid and cost effective production of milligram to gram quantities of large panels of proteins.

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing


4:25 Sequence-Based Prediction of Protein Solubility Using State-of-the-Art Machine Learning Approaches

Reda Rawi, PhD, Staff Scientist, Structural Bioinformatics Core Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID/NIH)

Protein solubility is an important physiochemical property associated with protein expression, and thus is a critical determinant of the manufacturability of therapeutic proteins. Given that the primary determinant of protein solubility is its amino acid sequence, in silico sequence-based machine learning (ML) tools predicting solubility propensity of proteins are highly sought after. Here, we present PaRSnIP and DeepSol, the two most accurate sequence-based ML protein solubility predictors, enabling in silico screening for protein variants with enhanced manufacturability.

4:55 Hacking Heterologous Protein Expression with High-Throughput Omics & Machine Learning

Elizabeth Brunk, PhD, Scientist, Bioengineering, Moores Cancer Center, University of California, San Diego (UCSD)

Standardized, multi-omics datasets are becoming increasingly available in the biomedical sciences. Extracting knowledge from big data will enable overcoming current bottlenecks that hinder high-throughput protein expression systems. Modern machine learning brings the promise of leveraging large-scale omics data to make accurate predictions and improve biotechnological processes. Here, I present recent efforts on the development of appropriate in silico tools to advance heterologous protein expression, solubility and biosynthesis of target compounds.

5:25 End of Difficult-to-Express Proteins

5:30 Registration for Dinner Short Courses

6:00-8:30 pm Recommended Dinner Short Course*

SC12: Emerging Applications for Gene Editing, Base Editing and RNA Editing

*Separate registration required.

* The program is subject to change without notice, due to unforeseen reason.

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