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.

Monday, August 31

OVERCOMING PRODUCTION CHALLENGES

9:05 am

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.

9:25 am

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.

Alison Porter, Head, Expression Systems Sciences, Lonza Pharma & Biotech

-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®

 

10:10 am LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Rana Sidhu, PhD, Protein Expression Lead, Early Solutions, UCB, Inc.
Panelists:
Shlomo Zarzhitsky, PhD, Research Associate, Chemistry, Princeton University
Susanne Gräslund, PhD, Principal Investigator, Structural Genomics Consortium, Karolinska Institute
10:30 am Coffee Break - View our Virtual Exhibit Hall

BREAKTHROUGH TECHNOLOGIES TO ANALYZE & EXPRESS DTEPs

10:50 am

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.

11:10 am

Native Ion Mobility Mass Spectrometry of Intact Membrane Protein Complexes

Arthur Laganowsky, PhD, Associate 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.

Natalia Herrera, Graduate Student/ PhD Candidate, Albert Einstein College of Medicine

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.

 

Nicholas Morano, Graduate Student, Albert Einstein College of Medicine
11:55 am LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Rana Sidhu, PhD, Protein Expression Lead, Early Solutions, UCB, Inc.
Panelists:
Xinchao Yu, PhD, Senior Scientist, Molecular Engineering, Amgen, Inc.
Arthur Laganowsky, PhD, Associate Professor, Chemistry, Texas A&M University
Natalia Herrera, Graduate Student/ PhD Candidate, Albert Einstein College of Medicine
Nicholas Morano, Graduate Student, Albert Einstein College of Medicine
12:15 pm Lunch Break - View our Virtual Exhibit Hall

INNOVATING SOLUTIONS

12:45 pm

Tailoring HDL Mimetics to Deliver mRNA for in vivo Protein Expression

Wei He, PhD, Biomedical Scientist, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory

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.

1:05 pm

Driving Biological Discovery: An Expanding Toolkit for Affinity Proteomics

John LaCava, PhD, Group Leader, Laboratory of Macromolecules and Interactomes, European Research Institute for the Biology of Aging, University Medical Center Groningen

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.

Rob Burgess, Chief Business Officer, Sino Biological Inc

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.

1:50 pm LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Wei He, PhD, Biomedical Scientist, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory
Panelists:
John LaCava, PhD, Group Leader, Laboratory of Macromolecules and Interactomes, European Research Institute for the Biology of Aging, University Medical Center Groningen
Rob Burgess, Chief Business Officer, Sino Biological Inc
2:10 pm Refresh Break - View Our Virtual Exhibit Hall
2:30 pm Problem Solving Breakout Discussions - Part A

This session provides the opportunity to discuss a focused topic with peers from around the world in an open, collegial setting. Select from the list of topics available and join the moderated discussion to share ideas, gain insights, establish collaborations or commiserate about persistent challenges.

TABLE 7: Experiences with Using Different Binder Types as Crystallization Chaperones or Cryo-EM Handles

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?
3:00 pm Refresh Break - View Our Virtual Exhibit Hall
3:20 pm Problem Solving Breakout Discussions - Part B

TABLE 8: Cell-Free Approaches to Difficult Proteins

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
3:50 pm Refresh Break - View our Virtual Exhibit Hall

PLENARY KEYNOTE SESSION

4:10 pm

Chairperson's Remarks

K. Dane Wittrup, PhD, CP Dubbs Professor, Chemical Engineering & Bioengineering, Massachusetts Institute of Technology
4:15 pm

From Energy to Machine Learning

George M. Church, PhD, Robert Winthrop Professor, Genetics, Harvard Medical School

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 Ph.D. students (Dantas, Raman, Lajoie), 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.

4:40 pm

The Case for Intelligent Design in Protein Engineering

Jamie B. 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.

5:15 pm LIVE Q&A:

Session Wrap-Up

Panel Moderator:
K. Dane Wittrup, PhD, CP Dubbs Professor, Chemical Engineering & Bioengineering, Massachusetts Institute of Technology
Panelists:
George M. Church, PhD, Robert Winthrop Professor, Genetics, Harvard Medical School
Jamie B. Spangler, PhD, Assistant Professor, Biomedical Engineering and Chemical & Biomolecular Engineering, Johns Hopkins University
5:35 pm Happy Hour - View our Virtual Exhibit Hall
6:10 pm Close of Day

Tuesday, September 1

EXPRESSING MEMBRANE PROTEINS & UTILIZING NANODISCS

9:05 am

Large Nanodiscs Going Viral

Mahmoud Nasr, PhD, RPh, Assistant Professor, 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.

9:25 am

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.

10:10 am LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Matthew Coleman, PhD, Senior Scientist & Group Leader, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory
Panelist:
Mahmoud Nasr, PhD, RPh, Assistant Professor, Medicine, Brigham and Women’s Hospital, Harvard Medical School
10:30 am Coffee Break - View our Virtual Exhibit Hall

EXPRESSING MEMBRANE PROTEINS

10:50 am

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.

11:10 am

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.


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.

11:55 am LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Ho Leung Ng, PhD, Associate Professor, Biochemistry & Molecular Biophysics, Kansas State University
Panelists:
Alexei Yeliseev, PhD, Staff Scientist, Group Leader, LMBB, NIH/NIAAA
Thariska Tharmakulasingam, Specialist, Orbital Bioreactors, Kuhner Shaker AG
12:15 pm Lunch Break - View Our Virtual Exhibit Hall

STRATEGIES FOR OPTIMIZING DTEP EXPRESSION

12:50 pm

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.

1:10 pm

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

Shahram Misaghi, PhD, Senior Scientist, Cell Culture and Bioprocess Operations (CCBO), 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.

1:55 pm LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Mahmoud Nasr, PhD, RPh, Assistant Professor, Medicine, Brigham and Women’s Hospital, Harvard Medical School
Panelists:
Rana Sidhu, PhD, Protein Expression Lead, Early Solutions, UCB, Inc.
Shahram Misaghi, PhD, Senior Scientist, Cell Culture and Bioprocess Operations (CCBO), Genentech, Inc.
2:15 pm Close of Difficult-to-Express Proteins





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