CHI’s 10th annual Difficult to Express Proteins conference will present the most up-to-date strategies and technologies for successfully expressing and purifying the proteins that keep researchers up at night. Whether because of folding problems, toxicity to hosts, purification issues or any of a host of other problems, these proteins resist standard expression and purification methods.

MONDAY, MAY 4

7:00 am Registration and Morning Coffee

10:10 Coffee Break

EMERGING STRATEGIES FOR FINICKY PROTEINS

10:45 Chairperson’s Remarks

Shahram Misaghi, Ph.D., Scientist, Early Stage Cell Culture, Genentech, Inc.

11:20 It’s Time to Regulate: Coping with Product-Induced Nongenetic Clonal Instability in CHO Cell Lines via Regulated Protein Expression

Shahram Misaghi, Ph.D., Scientist, Early Stage Cell Culture, Genentech, Inc.

In some cases, clonal instability is due to the toxicity of the therapeutic protein(s) that clones express. To circumvent such product-induced clonal instability, we have developed a vector construct that utilizes a regulated protein expression system in which the constitutive expression of the target protein(s) is prevented unless doxycycline is added to the culture. Our findings suggest that a regulated expression system could be suitable for production of difficult proteins that trigger instability.

11:50 Expression of Difficult-to-Express Proteins via Novel in silico Software Coupled with Multi-Modal Expression Screen

Prabuddha K. Kundu, Ph.D., Co-Founder & Executive Director, Premas Biotech Pvt Ltd

Expression of difficult-to-express proteins is rigorous, fraught with failures and frequent delays. We have developed a multi-modal expression tool coupled with an in silico guidance software. We are able to express successfully multi-membrane pass proteins, immuno-modulatory proteins, rCRM197, viral vaccine candidates, etc in less than 8 weeks to generate the data.

12:05 pm New Tools for Protein Solubility

David Mead, Ph.D., Founder & CSO, Lucigen Corp

A panel of 24 solubility and expression enhancing fusion partners has been developed to simultaneously test multiple tags within the context of a single promoter, vector and host system. In addition, a novel yellow fluorescent protein significantly enhances solubility and expression while providing an instant visual report of the amount of soluble, active protein. This system permits rapid, simultaneous screening of multiple factors demonstrated to improve solubility and/or expression in a high-throughput format using a robust enzyme-free cloning platform. The utility of the panel was proven in expressing soluble, active LRRK2, a very challenging biomarker for Parkinson’s disease.

12:20 ESETEC® 2.0: New Generation of E. Coli Secretion Technology for the High-Yield Production of Fabs

Andreas Anton, Ph.D., Director, BioProcess Development, Wacker Biotech GmbH

WACKER has profoundly refined its patented ESETEC® E. coli based system for the manufacture of biopharmaceuticals. Targeted genetic modifications and process optimization measures led to the development of new, extremely productive cell lines and fermentation procedures. ESETEC® 2.0 is now able to produce several grams per liter of secreted Fabs.  

12:35 Luncheon Presentation: Comprehensive Engineering of Biological Systems

Claes Gustafsson, Ph.D., CCO, DNA2.0 Inc.

Gene synthesis and current molecular biology tools allow unprecedented ability to engineer biological systems at the protein, gene, vector, and host genome levels. We describe applications where engineering tools and machine learning are leveraged to systematically optimize protein production and function for a range of target proteins and hosts.

1:20 Session Break

TAGS, DETERGENTS, SOLUBILITY & PURIFICATION

1:50 Chairperson’s Remarks

Sotirios Koutsopoulos, Ph.D., Research Scientist, Center for Biomedical Engineering, Massachusetts Institute of Technology

1:55 Optimized E. coli Expression Strain LOBSTR Eliminates Common Contaminants from His-Tag Purification

Thomas U. Schwartz, Ph.D., Principal Investigator, Department of Biology, Massachusetts Institute of Technology

We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low-expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His-tag purifications.

2:25 Expression, Purification, and Micelle Reconstitution of Antimicrobial Piscidin 1 and Piscidin 3 for NMR Studies

Wen Chen, Ph.D., Biological Chemistry & Molecular Pharmacology, Harvard Medical School

The piscidin 1 and 3 genes were cloned into the TrpLE vector. The corresponding TrpLE-piscidin fusion partners were expressed in E. coli and recovered from inclusion bodies. Following steps that included Ni-NTA chromatography, cyanogen bromide cleavage of the fusion proteins, & reverse-phase HPLC, purified piscidins 1 & 3 were recovered in very good yield & characterized by NMR. High quality (15)N-(1)H HSQC spectra of piscidins 1 and 3 bound to SDS micelles were collected, demonstrating the feasibility of producing and purifying the isotopically-labeled piscidin peptides required to determine their full structures by multidimensional NMR spectroscopy.

2:55 Designer Surfactant-Like Peptides for Membrane Protein Purification and Stabilization

Sotirios Koutsopoulos, Ph.D., Research Scientist, Center for Biomedical Engineering, Massachusetts Institute of Technology

Membrane proteins are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of membrane proteins is limited due to difficulties in producing sufficient quantities of soluble, functional, and stable receptors. Designer, surfactant-like peptides may be used to extract the protein from the cell membrane and stabilize the protein outside the membrane bilayer for further studies.

3:25 The Saga of T3SS Translocator Protein Purification

Wendy L. Picking, Ph.D., Professor, Pharmaceutical Chemistry, University of Kansas

The type III secretion apparatus (T3SA) resembles a syringe embedded in the bacterial membranes with an external needle and needle tip complex that senses target cell contact. The translocators associate with host cell membranes. The adventures of the purification of these proteins and their vaccine formulations with various detergents will be discussed.

3:55 Refreshment Break in the Exhibit Hall with Poster Viewing

4:35 Problem-Solving Breakout Discussions

Protein Expression Screening in Mammalian Cells

Moderator: Michael Dyson, Ph.D., Group Leader, IONTAS Ltd.

Using Codon Optimization to Facilitate Expression of Membrane Proteins

Moderator: Morton Nørholm, Ph.D., Academic Entrepreneurial Research Group Leader, DTU Biosustain; Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability

Solving Problems in Expression, Purification and Sample Preparation for Structural Characterization of GPCRs

Moderator: Aditya Pandey, Ph.D., Biochemistry and Molecular Biology, Dalhousie University

Improving Folding and Expression of Proteins in S. cerevisiae

Moderator: Eric Shusta, Ph.D., Professor, Department of Chemical and Biological Engineering, University of Wisconsin, Madison

Solving Over-Expression Problems in E. coli

Moderator: Ning Gao, Associate Principal Scientist, Discovery Science, Innovative Medicine Unit, AstraZeneca Pharmaceuticals

5:35 Welcome Reception in the Exhibit Hall with Poster Viewing

6:50 End of Day

TUESDAY, MAY 5

8:00 am Morning Coffee

MAMMALIAN EXPRESSION SOLUTIONS

8:25 Chairperson’s Remarks

Michael R. Dyson, Ph.D., Group Leader, IONTAS Ltd.

8:30 Protein Expression Screening in Mammalian Suspension Cells

Michael R. Dyson, Ph.D., Group Leader, IONTAS Ltd.

Multi-domain and membrane proteins can often be expressed by a combination of domain truncation and screening in mammalian suspension cells. However proteins can exist in different conformations when in their natural environment, in complex with binding partners. Here methods are presented to identify antibodies binding to components of the EGFR and FGFR signalling pathways by traditional and phenotypic screening.

9:00 Enhanced Transient Recombinant Protein Production in CHO Cells through the Co-Transfection of the Product Gene with Bcl-xL

Matthew Zustiak, Ph.D., Researcher, Gallus Biopharmaceuticals

We examine an alternative method of using the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, namely, through the co-transfection of Bcl-xL and the product-coding gene. Cells co-transfected with Bcl-xL showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a stable cell line and subsequent screening, which are both time- and resource-consuming.

9:30 TAPBOOST Technology: Enhanced Production for Hard-to-Produce Proteins

Akinori Hishiya, Ph.D., Director, Biology, Strategia Therapeutics, Inc.

Therapeutic recombinant proteins produced in mammalian expression systems might have folding issues and are confined in the endoplasmic reticulum by cellular quality control system, resulting in poor expression and yields. We have developed a novel technology called TAPBOOST technology, which controls protein folding and cellular quality control systems specifically for a targeted protein. A proprietary protein (TAPBOOSTER) is expressed together with a therapeutic protein (targeted protein), followed by the interaction between TAPBOOOSTER and the targeted protein, resulting in enhanced production of the targeted protein.

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

Solving Expression Problems for Structural Studies

10:50 Overproduction and Biophysical Characterization of Human HSP70 Proteins

Rebba Boswell-Casteel, Ph.D., Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center

Functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5in either E. coli or S. cerevisiae. This work provides the basis for future biochemical studies of human HSP70 protein function and structure.

11:20 Overcoming Barriers to Expression, Purification and Sample Preparation for Structural Characterization of GPCRs using NMR Spectroscopy

Aditya Pandey, Ph.D., Biochemistry & Molecular Biology, Dalhousie University

G-protein coupled receptors are inherently dynamic membrane proteins that have remained elusive to structural characterization using NMR spectroscopy. Due to the challenges involved in production of large quantities of isotope enriched GPCRs, we have employed a “divide and conquer” approach. Here, we discuss various strategies that we have used to express, purify and biophysically characterize large fragments of the apelin receptor.

11:50 Recombinant Expression, Purification, and Biophysical Characterization of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41

Tsafrir Mor, Ph.D., Associate Professor, The Biodesign Institute, Infectious Diseases and Vaccinology, Arizona State University

While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His ), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41.

12:20 pm Luncheon Presentation I: Overcoming the Challenges Associated with the Production of Bone Morphogenetic Proteins in CHO Cells

Christopher T. Brown, Program Manager – Early Stage Protein Manufacturing, Research & Development, Bioventus LLC

BMP production for preclinical/clinical studies offers unique challenges not present with more conventional biologics. BMPs, expressed as large precursor proteins, undergo proteolytic processing by furin-like proteases to remove the N-terminal propeptide which releases the mature cytokine. CHO cells produce low amounts of endogenous furin which leads to N-terminal heterogeneity and the presence of unprocessed/partially processed forms that must be removed during purification. This presentation will focus on process development and implementation to overcome BMP production challenges in mammalian systems.

12:50 Luncheon Presentation II (Sponsorship Opportunity Available)

1:20 Ice Cream Break in the Exhibit Hall with Poster Viewing

Conquering Overexpression

2:00 Chairperson’s Remarks

Ning Gao, Associate Principal Scientist, Discovery Sciences Innovative Medicine Unit, AstraZeneca R&D

2:05 Development of an Improved Mammalian Overexpression Method for Human CD62L

Peter D. Sun, Ph.D., Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health

Like other stable mammalian over-expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6 months to produce. To shorten the time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.

2:35 Engineering Strategies for Improving Yeast Production of Brain-derived Neurotrophic Factor

Eric V. Shusta, Ph.D., Professor, Department of Chemical and Biological Engineering, University of Wisconsin, Madison

Brain-derived neurotrophic factor (BDNF) is one of a family of difficult-to-produce cysteine knot proteins. Here we describe protein and cellular engineering approaches to optimize the display and secretion of BDNF from yeast. Engineered proteins exhibit better per molecule folding as demonstrated by improved receptor binding in addition to elevated display and secretion levels.

3:05 Discovery of MAbs Against Difficult GPCRs, Ion Channels, and Transporters

Benjamin Doranz, Ph.D., MBA, President & CEO, Integral Molecular

To enable the isolation, characterization, and engineering of MAbs against challenging membrane protein targets, Integral Molecular has developed the MPS Discovery Engine™ platform, encompassing Lipoparticles for concentrating native membrane proteins and Shotgun Mutagenesis for membrane protein engineering and epitope mapping. Using the MPS platform, we have generated inhibitory MAbs against the ion channel P2X3 for treating neuropathic and inflammatory pain, and have ongoing discovery programs against additional GPCR, ion channel, and transporter targets.

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

Looking Towards a Therapeutic

4:25 Overexpression of Pseudomonas aeruginosa LpxC with Its Inhibitors in an acrB-Deficient Escherichia coli Strain

Ning Gao, Associate Principal Scientist, Discovery Sciences, AstraZeneca Pharmaceuticals

LpxCprotein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxCin the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump.

4:55 Rapid Production of High-Quality, Functional Membrane Proteins using ACM Technology

Sourabh Banerjee, Ph.D., Director of Technology, ACM Biolabs, Singapore

Artificial Cell Membrane (ACM) technology dramatically simplifies the production of different classes of challenging membrane proteins using a combination of cell-free synthesis and specialized block copolymer membranes. We discuss how ACMs allow rapid access to ‘hard targets’, and demonstrate flexibility and scalability for downstream assay development, finding broad applicability for the discovery of new therapeutics.

5:25 End of Conference

5:30 Registration for Dinner Short Courses

2025 PROGRAMS

View By:

• Display of Biologics
• Engineering Antibodies
• Machine Learning for Protein Engineering

• Antibodies for Cancer Therapy
• Emerging Targets for Oncology & Beyond
• Driving Clinical Success in Antibody-Drug Conjugates

• TS: Intro to Multispecific Antibodies
• Advancing Multispecific Antibodies
• Engineering Bispecific and Multifunctional Antibodies

• Advances in Immunotherapy
• Engineering Cell Therapies
• Next-Generation Immunotherapies

• Difficult-to-Express Proteins
• Optimizing Protein Expression
• Maximizing Protein Production Workflows

• ML and Digital Integration in Biotherapeutic Analytics
• Biophysical Methods
• Characterization for Novel Biotherapeutics

• TS: Intro to Immunogenicity
• Predicting Immunogenicity with AI/ML Tools
• TS: Introduction to Bioassay Development and Analysis

• Biologics for Immunology Indications
• Radiopharmaceutical Therapies
• Next-Generation Immunotherapies

• ML and Digital Integration in Biotherapeutic Analytics
• Predicting Immunogenicity with AI/ML Tools
• Machine Learning for Protein Engineering