Whether for therapeutics, diagnostics or for characterization, some proteins just don’t cooperate when it comes to expression. CHI’s 12th Annual “Difficult to Express Proteins” conference will present the most up-to-date strategies and technologies for successfully expressing the proteins that keep researchers up at night. Folding problems, toxicity to hosts, purification issues or any of a host of other problems make these proteins resist standard expression and purification methods. “Difficult to Express Proteins” presents solutions and strategies to tame these “finicky” proteins.
Final Agenda
Recommended Short Courses*
SC6: In-silico Protein Docking - Detailed Agenda
SC8: In silico Immunogenicity Predictions (Hands-On) Workshop - Detailed Agenda
*Separate registration required, please see page 5-6 for course details.
MONDAY, MAY 1
7:00 am Registration and Morning Coffee
8:30 Chairperson’s Remarks
Haruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.
8:40 Investigation of GPR34 Topogenesis and Cell Surface Trafficking by Monitoring a Unique Epitope That Is Simultaneously Sensitive to Topology, Conformation, and Redox Status
Haruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.
We identified and characterized an epitope that reports correct protein conformation, membrane topology, and cell surface trafficking competency of a GPCR family member GPR34. The epitope formation required the oxidation of four cysteine residues located individually in the four separate extracellular regions of GPR34. The underlying biochemical properties of the conformational epitope not only illustrated the challenges of raising mAbs against GPCRs, but also suggested preferred strategies for GPCR antigen design.
9:10 Glycoengineering of Chinese Hamster Ovary Cells for Enhanced Erythropoietin N-Glycan Branching and Sialylation
Bojiao Yin, Ph.D., Amgen Protein Technologies
In order to examine the impact of glycosyltransferase expression on the N-glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. In this way, coordinated overexpression of these three glycosyltransferases for the first time in model CHO-K1 cell lines provides a means for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future.
9:40 KEYNOTE PRESENTATION: Design of Water-Soluble Variants of Membrane Proteins to Facilitate “Soluble Expression”
Jeffrey G. Saven, Ph.D., Professor, Chemistry, University of Pennsylvania
Here we present the first study involving the computational design, expression and characterization of a water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.
10:10 Coffee Break
10:45 Chairperson’s Remarks
Haruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.
10:50 High-Throughput Baculovirus Expression System for Membrane Protein Production
Ravi C. Kalathur, Ph.D., New York Structural Biology Center, New York Consortium on Membrane Protein Structure (NYCOMPS)
The ease of use, robustness, cost-effectiveness, and post-translational machinery make the baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. The modified pFastBac vector with mammalian promoter called BacMam can be used to transduce mammalian cells like HEK, CHO and BHK and this is an attractive alternative for glycosylation and biosafety concerns.
11:20 Engineering High-Titer Heterologous Protein Secretion in Bacteria
Lisa Burdette, Laboratory for the Engineering of Membrane Proteins and Protein Membranes, Department of Chemical and Biomolecular Engineering, Northwestern University
Biomaterial production in bacteria would benefit greatly from a high-titer secretion strategy. We engineered the type III secretion system in Salmonella enterica for this purpose because it is non-essential for bacterial metabolism and allows for target proteins to cross both bacterial membranes in one step. Our platform enables the high-titer production of a variety of challenging biomaterial-forming proteins at titers >100 mg/l and >85% purity.
11:50 Improving Membrane Protein Overexpression in Mammalian Cells by Flow Cytometric Sorting
Juni Andréll, Ph.D., Researcher, Biochemistry and Biophysics, University of Stockholm
Many membrane proteins from higher eukaryotic organisms cannot be expressed in bacterial or yeast systems, but are favourably expressed in a functional state in mammalian cells. However, the expression levels can often be too low for protein structural studies requirements. Using an optimised protocol of flow cytometric sorting to generate stable-cell lines can significantly and sufficiently improve the inducible expression of different membrane proteins in mammalian cells.
12:20 pm New Solutions for Production of Difficult-to-Express Proteins
Martina Huber, Ph.D., Local Site Head, Bioprocess Development, Wacker Biotech GmbH
Wacker Biotech will present highly competitive solutions for production of difficult-to-express proteins based on its proprietary E. coli expression systems ESETEC® and FOLDTEC®. Recent case studies will include secretion of functional antibody fragments and enzymes to the fermentation broth with up to 14 g/L. Together with its E. coli refolding platform FOLDTEC®, Wacker Biotech offers a novel and comprehensive approach to rapidly assess manufacturability of therapeutic proteins.
12:50 Luncheon Presentation I: Building Better Protein Pharmaceuticals at the Intersection of Machine Learning and Quantitative Biology
Claes Gustafsson, Ph.D., Co-Founder and Chief Commercial Officer, ATUM (formerly DNA2.0)
Gene synthesis and current molecular biology tools allow unprecedented ability to create any biology imaginable. Due to the high cost of commercially relevant functional measurements and the vast size of available sequence space it is not feasible to randomly search the space. However, utilizing Artificial Intelligence (AI) and systematic variance of the sequence space data we will describe how we engineer efficient optimization of genes, proteins, pathways, organisms without any mechanistic understanding of the system.
1:20 Luncheon Presentation II: Vmax™ – a Next-generation Microbial Workhorse for the Biotech Industry
Matthew Weinstock, Ph.D., Scientist II, DNA Technology, Synthetic Genomics, Inc.
This presentation will focus on Vmax™, a novel prokaryotic host with a rapid growth rate that promises to accelerate biotech R&D efforts on multiple fronts. We will describe the development of the platform, the advantages of using it in molecular cloning and protein expression applications, and ongoing large-scale genome engineering efforts to further enhance performance.
1:50 Session Break
2:20 Problem-Solving Breakout Discussions
These interactive discussion groups are open to all attendees, speakers, sponsors, & exhibitors. Participants choose a specific breakout discussion group to join. Each group has a moderator to ensure focused discussions around key issues within the topic. This format allows participants to meet potential collaborators, share examples from their work, vet ideas with peers, and be part of a group problem-solving endeavor. The discussions provide an informal exchange of ideas and are not meant to be a corporate or specific product discussion. Pre-registration to sign up for one of the topics will occur a week or two prior to the Event via the App.
Tools for Tackling Membrane Protein Characterization
Haruki Hasegawa, Ph.D., Principal Scientist, Amgen
Expression and Purification of Membrane Receptors: Challenges and Approaches
Alexei Yeliseev, Ph.D., Staff Scientist, NIAAA, NIH
4:00 Chairperson’s Remarks
4:10 Bicycles and Bicycle Drug Conjugates: Next Generation Therapeutics
Sir Gregory Winter, Ph.D., FRS, Master, Trinity College and Co-Founder and Director, Bicycle Therapeutics
Bicycles® are a novel therapeutic class of constrained bicyclic peptides that combine antibody-like affinity and selectivity with small molecule-like tissue penetration, tunable exposure and chemical synthesis. They have potential in many indications, including oncology, where Bicycles’ unique properties have been used to develop Bicycle Drug Conjugates™ (BDCs); a novel toxin delivery platform which greatly improves toxin loading into tumour tissues. This presentation will describe both the Bicycle® and BDC platforms.
4:55 Young Scientist Keynote: Programming Proteins by Deep Sequencing and Design
Tim Whitehead, Ph.D., Assistant Professor, Chemical Engineering and Materials Science, Michigan State University
Next-generation sequencing has presented protein scientists with the ability to observe entire populations of molecules before, during, and after a high-throughput screen or selection for function. My group leverages this unprecedented wealth of sequence-function information to design and engineer protein affinity, specificity, and function and to infer structural complexes of proteins. My talk will present an overview of the above and detail methodological improvements that enable the engineering work.
5:40 Welcome Reception in the Exhibit Hall with Poster Viewing
6:55 End of Day
TUESDAY, MAY 2
8:00 am Registration and Morning Coffee
8:25 Chairperson’s Remarks
Alexei Yeliseev, Ph.D., Staff Scientist, LMBB, NIH/ NIAAA
8:30 Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations
Sharon Rosovsky, Ph.D., Assistant Professor, Chemistry and Biochemistry, University of Delaware
Selenoproteins are enzymes that contain the rare amino acid selenocysteine. They play a cardinal role in the management and regulation of reactive oxygen species. Selenoproteins remain understudied because of the technical challenges associated with their production since the selenocysteine codon UGA also encodes a stop codon. We have developed a new method to prepare selenoproteins using expressed chemical ligation. Selenocysteine high reactivity can be used to introduce site-specific chemistry into the protein scaffold.
9:00 The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology
Vincent Noireaux, Ph.D., School of Physics and Astronomy, University of Minnesota
In vitro transcription-translation (TX-TL) is becoming a highly versatile technology for applications in synthetic biology and quantitative biology. I will present a unique TXTL system that my laboratory has developed. We implemented novel metabolisms to stimulate reporter protein synthesis up to 2 mg/ml in batch mode reactions and 6 mg/ml in semi-continuous mode. We use this experimental platform for synthetic biology, from gene circuit prototyping to the cell-free synthesis of entire bacteriophages.
9:30 Applications of panARS-Based Vectors for High Throughput Screens and Structural Studies in Pichia pastoris
Damien B. Wilburn, Ph.D. Post-Doctoral Senior Researcher, Genome Sciences, University of Washington
Pichia pastoris is widely considered an industrial workhorse for expression of recombinant proteins, but a limitation of the system has been the requirement for stable integration of heterologous genes that can result in high clonal variability and limit high throughput screening. Here we describe expression of multiple protein constructs using episomal vectors that include panARS: an autonomous replicating sequence that enables stable plasmid maintenance and excellent protein yield in Pichia expression systems.
10:00 Coffee Break in the Exhibit Hall with Poster Viewing
10:50 High Yield, Large-Scale Affinity Purification of the Functional G Protein-Coupled Receptors
Alexei Yeliseev, Ph.D., Staff Scientist, LMBB, NIH/ NIAAA
We purified the recombinant CB2 expressed in E. coli cells as a fusion with maltose-binding protein and small affinity tags, with yields up to 2-3 mg/ L. We demonstrate an efficient isolation of functional CB2 receptor labeled with stable isotopes in minimal medium. The protocols developed in our laboratory can be applied to expression and purification of other membrane receptors for structural and functional studies.
11:20 A Systematic Approach to Improve the Expression and Purification of Membrane Proteins in E. coli
Luis G. Cuello, Ph.D., Associate Professor, Cell Physiology and Molecular Biophysics and Center for Membrane Protein Research, Texas Tech University Health Sciences Center
We developed a method for the production of membrane proteins in E. coli. The method consists in the systematic pipeline evaluation of different: E. coli strains, chemical chaperones, growth media, blockers or inhibitors, temperature, inexpensive detergents for solubilizing the target membrane protein alongside optimizing the ionic strength, pH, and temperature.
11:50 Lessons from an α-Helical Membrane Enzyme: Expression, Purification, and Detergent Optimization for Biophysical and Structural Characterization
Raquel Lieberman, Ph.D., School of Chemistry & Biochemistry, Georgia Institute of Technology
We outline the protocol developed in our lab to produce a multipass α-helical membrane protein. We present our work flow, from ortholog selection to protein purification, including molecular biology for plasmid construction, protein expression in E. coli, membrane isolation and detergent solubilization, protein purification and tag removal, biophysical assessment of protein stability in different detergents, and detergent concentration determination using thin-layer chromatography. We focus on results from our ongoing work with intramembrane aspartyl proteases from archaeal organisms.
12:20 pm Luncheon Presentation I: High-Resolution Epitope Mapping and Specificity Profiling of mAbs Targeting Complex Proteins
Duncan Huston-Paterson, Project Leader, Integral Molecular
Integral Molecular specializes in characterizing antibodies against complex targets, including GPCRs, ion channels, and transporters. Our Shotgun Mutagenesis technology rapidly maps conformational antibody epitopes at single-amino acid resolution using comprehensive mutagenesis and cellular-expression with >95% success, generating critical IP and detailed mechanistic insights. Our Membrane Proteome Array enables safety analysis of antibodies by testing each antibody against an expression array of 5,304 structurally-intact membrane proteins, providing a comprehensive assessment of off-target antibody interactions.
12:50 Luncheon Presentation II to be Announced
Ian Hodgson, Ph.D., Head, Molecular Biology, FUJIFILM Diosynth Biotechnologies
1:20 Ice Cream Break in the Exhibit Hall with Poster Viewing
2:00 Chairperson’s Remarks
Vinodh Kurella, Ph.D., Senior Scientist, Protein Engineering, ImmunoOncology
Division, Intrexon Corporation
2:05 Making Spider Silk: How Hard Can It Be, Spiders Do It?
Randolph P. Lewis, Ph.D., USTAR Professor, Biology / Synthetic Bio-Manufacturing, Utah State University
Spider silk proteins are unique in being highly repetitive, very large and composed of very few amino acids. These each lead to complications when trying to express these proteins in quantities needed to develop commercial products. With a major focus on expression in E.coli solutions to some of these unique problems will be discussed as well as identifying difficulties still to be overcome. Brief discussions of other expression systems will be presented.
2:35 Development of a Dual Fluorescent Protein Reporter System for Detection of Unfolded Protein Response Pathway Activation in Mammalian (CHO) Cells and its Application in Therapeutic Proteins Production
Gargi Roy, MSc., Scientist, MedImmune
We developed a reporter system that detects IRE1α mediated XBP1 splicing for monitoring the UPR activation in IgG expressing cells. Using this reporter we show ER stress activation in stable IgG expressing cells during fed-batch. We demonstrate that it can be used to isolate high expresser clonal hosts. This reporter system with its ability to monitor the stress has a potential for devising strategies for the selection of high expressers and improved yields of bio-therapeutics.
3:05 Oral Insulin: Challenges, Learnings and Pitfalls: Premas Biotech
Prabuddha Kundu, Ph.D., Executive Director & Co-Founder, Premas Biotech Pvt Ltd
The ability to Translate scientific concepts created at research scale to a viable commercial scale, is of significant importance to clinical medicine; an area of specialization for Premas over a decade. Oral Insulin, a revolutionizing concept for Diabetics, conceptualized by Oramed, where Premas has partnered actively in the journey to make it a success through Clinical trials from lab scale, technology development, scale up, to manufacturing (grams to kilograms per batch) and into Phase III.
3:35 Refreshment Break in the Exhibit Hall with Poster Viewing
4:25 IQGAP1 Protein Purification and X-Ray Crystallography
Vinodh Kurella, Ph.D., Senior Scientist, Protein Engineering, ImmunoOncology Division, Intrexon Corporation
Protein purification process is a prelude to X-ray crystallography for certain studies. A case study will be presented on a scaffold protein - IQGAP1, overexpressed in colon cancer. IQGAP1- Calponin Homology domain was purified but precipitated before crystallization trials. Construct redesign lead to stabilization and also yielded high-quality crystals. However, in-house diffraction pattern revealed a fragile crystal. Finally, a synchrotron beam was utilized to solve the structure to 2.4 Å.
4:55 Recombinant Protein Expression in Stable Drosophila S2Cell Lines
Thomas Krey, Ph.D., Institute of Virology, Structural Virology Group, Hannover Medical School
Production of recombinant proteins has become indispensable for basic and applied research. Among the various expression systems, insect cells allow the production of complex proteins that require posttranslational modifications. Expression in stable Drosophila Schneider2 (S2) cell transfectants offers important advantages in particular for difficult-to-express proteins like membrane proteins or heavily glycosylated multi-domain proteins containing a complex disulfide connectivity pattern. S2 cells therefore constitute a cost-efficient and easy-to-use alternative to the widely used baculovirus expression system.
5:25 End of Difficult to Express Proteins
5:30 Registration for Dinner Short Courses