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
Plenary Keynote Session
8:30 Chairperson’s Opening Plenary Remarks
8:40 Cancer Stem Cells and Mechanisms of Malignant Progression
Robert A. Weinberg, Ph.D., Founding Member, Whitehead Institute for Biomedical Research; Professor, Biology, Massachusetts Institute of Technology
The cell-biological program termed the epithelial-mesenchymal transition (EMT) plays a role in conferring aggressive traits on carcinoma cells. In addition, it generates cancer stem cells (CSCs) that have the ability, following dissemination, to serve
as founders of new metastatic colonies. The relationship between these CSCs and the SCs residing in normal tissues, and the participation of the CSCs in metastatic dissemination will be described.
9:25 Building an Antibody Discovery Company in a Crowded Field – the Adimab Story
Tillman Gerngross, Ph.D., CEO, Co-Founder, Adimab
The presentation will cover the evolution of Adimab from its founding in 2007 to becoming one of the few privately held profitable biotech companies in the last decade. Industry trends and specific strategic decisions along the way will be discussed
and used to illustrate the importance of integrating finance and scientific information to build successful capital efficient biotech companies.
10:10 Coffee Break
10:45 Chairperson’s Remarks
Shahram Misaghi, Ph.D., Scientist, Early Stage Cell Culture, Genentech, Inc.
10:50 KEYNOTE PRESENTATIONS
Exploring Codon Optimization Strategies for Production of Membrane Proteins
Morton Nørholm, Ph.D., Center for Biomembrane Research, Biochemistry and Biophysics, Stockholm University
Using a library of GFP-tagged membrane proteins, we have compared different codon optimisation strategies including synonymous mutations in the 5´end, complete re-coding using multiparameter optimisation algorithms and complementing rare codon
usage with additional copies of the corresponding low-concentration tRNAs.
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.
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.
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.
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
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.Optimal vector and host combinationMethods for multi-parallel transfection and expression in HEK293 and CHO cellsQuantitation of protein yield and QC
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 BiosustainabilityCodon optimization choicesHost-related decisionsWhat to expect
Solving Problems in Expression, Purification and Sample Preparation for Structural Characterization of GPCRs
Moderator: Aditya Pandey, Ph.D., Biochemistry and Molecular Biology, Dalhousie UniversityChoosing the expression host depending on the downstream applicationFusion protein tags that can be used to enhance the expression yieldHow to tackle the hydrophobicity of the membrane proteins expressed in E. coli or by cell-free expression systemMembrane protein purification methodologies (using HPLC and FPLC)
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 PharmaceuticalsHow to improve protein solubility in E. coli expressionHow to eliminate protein degradation problemsHow to find a stable protein domain for crystallography
5:35 Welcome Reception in the Exhibit Hall with Poster Viewing
6:50 End of Day
TUESDAY, MAY 5
8:00 am Morning Coffee
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
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
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.
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
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.
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
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