Cambridge Healthtech Institute’s 7th Annual
Optimizing Protein Expression
Solving Problems with Finicky Proteins
5-6 November 2014
This second part of the protein stream for PEGS Europe will focus on the methods and technologies for achieving the highest possible yield, with the best functionality.
Recommended Short Courses*
SC4: Solving Problems in Eukaryotic Expression Systems
SC5: Analytical Strategies for Comparability in Bioprocess Development
*Separate Registration Required.
Wednesday, 5 November
07:45 Registration and Morning Coffee
08:30 Chairperson’s Opening Remarks
08:35 KEYNOTE PRESENTATION
Playing Catch-Up with Escherichia coli: Using Yeast to Increase Success Rates in Recombinant Protein Production Experiments
Roslyn M. Bill, Ph.D., Professor, School of Life and Health Sciences, Aston University Birmingham
This presentation highlights the benefits of using yeast for more challenging targets such as membrane proteins. We’ve seen advances in understanding how a yeast cell responds to the stress of producing a recombinant protein and how this information can be used to identify improved host strains in order to increase functional yields. I argue that S. cerevisiae and P. pastoris should be considered at an early stage in any serious strategy to produce proteins.
09:05 Exploration of Expression Bottlenecks of Recombinant Polymeric Antibody Production
David Reinhart, Ph.D., Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences
We have previously generated a panel of recombinant CHO cell lines that recombinantly expressed several different polymeric antibodies. Some of these had remarkably low specific productivities. This encouraged us to broadly analyze the respective cell lines regarding their gene copy numbers, mRNA content, ER stress-associated chaperones, intra- and extracelllularly expressed polypeptides and more. Our results provided novel insights towards the underlying protein machinery to better understand potential expression limitations.
09:35 Proteomic Analysis of Bacillus Subtilis Strains Engineered for Improved Production of Heterologous Proteins
Collin R. Harwood, Ph.D., Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University
The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, the widely used bacterial expression systems based Escherichia coli, are not able to secrete soluble proteins directly into the culture medium and this limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein production. The strains were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen (PA), the productivity of which is extremely low in the unmodified strain.
10:05 Scalable Electroporation for the Rapid Production of Complex and Difficult to Express Proteins
Peer Heine, Ph.D., Field Application Scientist, MaxCyte, Inc.
Flow electroporation provides rapid, fully scalable transient (co)transfection. This universal technology is compatible with a range of cells including CHO, MDCK, BHK-21, Vero, NS0, CAP-T™, insect cells, and others used for large-scale production of proteins, including simple recombinant antigens, complex antibodies, antibody-like molecules, VLPs, and vaccines. High transfection efficiencies and cell viabilities enable production of gram quantities of antibodies within days of a single transfection. The ease of the scale-up/scale-down allows for aligning protein yields and resource usage with the stage of candidate development. Flow electroporation also can be used to generate stable pools and stable clones.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 Exploring the Protein Expression Space with pCoofy Vectors
Sabine Suppmann, Ph.D., Head, Recombinant Protein Production, Max-Planck Institute of Biochemistry
We previously reported the design of a “pCoofy” expression vector series that combines a variety of N- and C-terminal tags with a variety of backbones for several expression hosts. The parallel design allows to navigate easily between the systems in order to identify successful expression parameters. Cloning is based on SLIC plus ccdB counter selection. This presentation will highlight recent advances and new tools.
11:45 Periplasmic Chaperones Used to Enhance Functional Secretion of Proteins in E. coli
Martin Schlapschy, Ph.D., Lehrstuhl für Biologische Chemie, Technische Universität München
We have developed the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and/or folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC, which catalyze the formation and isomerization of disulfide bridges, and two peptidyl-prolylcis/trans isomerases with chaperone activity, FkpA and SurA. We present a detailed protocol how to use this system for the bacterial secretion of recombinant proteins, including human EGF as a new example, and we give hints on optimisation of the expression procedure.
12:15 PUREfrex®: A Next-Generation of Reconstituted Cell-Free Protein Synthesis System
Takashi Kanamori, Ph.D., Director, Business Development, GeneFrontier Corporation
PUREfrex® is a reconstituted cell-free protein synthesis system which can provide highly pure proteins in a fast and easy way. We will present our recent developments regarding the increase of the productivity and also show our application to the expression of some difficult targets and Ribosome Display.
12:30 Corynex®: A Gram-Positive Microbial Protein Secretion System that Delivers Better Results
Yoshimi Kikuchi, Ph.D., Principal Researcher, AJINOMOTO CO., Inc.
Corynex® is a powerful protein expression system based on the gram-positive bacteria C. glutamicum that can secrete various active proteins directly into media with high purity. These advantages enable less downstream time and costs compared to traditional methods. Our newly-developed enzymatic protein modification technologies will also be introduced.
12:45 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own
14:00 Chairperson’s Remarks
14:05 The Relevance of the Kinome on the Optimisation of a Human Production Cell Line
Andreas Wagner, Researcher, Institute for Applied Biotechnology, University of Applied Science Biberach
We identified genes and pathways of the kinome that are important for recombinant protein expression bioprocess performing a high-throughput screening. Using RNAi technology employing a novel transfection agent CAP®-SEAP cells were transfected in complex production media and evaluated for modulated proliferation, productivity and apoptosis. Inhibition of 128 relevant genes was further characterized using an antibody-producing CAP® cell line. Genetically engineered cell lines with enhanced phenotypically characteristics for a bioprocess were established.
14:35 Optimizing the Solubility of a Recalcitrant Protein for Structural Studies: Lessons from the Papillomavirus Oncoprotein E6
Gilles Trave, Ph.D., Group Leader, Ecole Supérieure de Biotechnologie de Strasbourg
The papillomavirus E6 oncoprotein is aggregation-prone and has resisted structural analysis for almost 30 years. Through the years, we analyzed the multiple causes of E6 aggregation and developed strategies against them, to finally obtain its 3D structure by NMR and crystallography (Zanier, Charbonnier et al., Science 2013). I will summarize the project's history and the lessons we learnt, notably about the use of solubilizing tags and of solubilizing mutations.
15:05 High-Throughput Cell Line and Process Optimisation of Mammalian Cell Culture Processes: About Small (50 ml) Tubes and Large Bioreactors
Maria De Jesus, Ph.D., COO, ExcellGene SA
Process and cell line optimisations will continue to be necessary requiring study of thousands of interacting parameters - a true dilemma. Small tubes as bioreactors have proven to be true predictors of cell line performance - even for large scale operation. My talk will indicate key insights and will show how multi-gram/l processes can be developed with minimal resources.
15:35 Refreshment Break in the Exhibit Hall with Poster Viewing
16:15 The Impact of microRNA Expression on Growth and Productivity in CHO Cells
Nicole Borth, Ph.D., Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria; Austrian Centre of Industrial Biotechnology GmBH
Overall biogenesis of miRNAs is dependent on growth rate and many miRNAs are differentially regulated depending on growth and productivity in a variety of CHO cell lines. Compared to other engineering strategies, the manipulation of miRNA expression does not burden the translational machinery of cells and provides the capability of global phenotypic control. The recent availability of genome sequence information has enhanced our ability to rapidly and efficiently optimise and fine-tune the properties of CHO cells.
16:45 Recombinant Production of Human Aquaporin-1 to an Exceptionally High Membrane Density in Saccharomyces cerevisiae
Julie Bomholt, MSc, Researcher, Aquaporin A/S
We explore the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of humanAquaporin-1.Aquaporin-1 cDNA was expressed from a galactose-inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol.
17:15 Problem Solving Roundtable Discussions
MicroRNA Expression to Improve CHO Cell Expression
Moderator(s): Nicole Borth, Ph.D., Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria; Austrian Centre of Industrial Biotechnology GmBH
Challenges for Determining and Improving Catalytic Activities of Therapeutic Enzymes
Moderator: Manfred Konrad, Ph.D., Head, Enzyme Biochemistry Research Group, Max-Planck-Institute for Biophysical Chemistry; Goettingen University Ph.D. Program Faculty Member
- What are critical quality attributes for the intactness of recombinantly produced enzymes as compared to the one from the natural source?
- How to select an appropriate expression system for enzymes of human origin
- Which posttranslational modifications may be required for a particular enzyme?
- Which biochemical parameters are essential for comparison of enzymatic activities from different sources?
- What types of non-radioactive assays are suitable for low- and high-throughput catalytic activity measurements?
- Should we put efforts into in vitro-evolution of catalytically poor enzymes of human origin, rather than developing strategies for mitigating immunogenicity of catalytically efficient microbial enzymes?
- Can tools of material science and nanotechnology offer platforms for improving therapeutically established protein drugs?
Issues with Transient Expression
Moderator: Peer Heine, Field Application Scientist, MaxCyte
- Gram-level antibody production
- Difficult-to-transfect cells
- Shorten Development Timelines via Large-Scale Transient Transfection in CHO Cells
18:15 Networking Reception in the Exhibit Hall with Poster Viewing
19:15 End of Day One
Thursday, 6 November
08:00 Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee
08:30 Chairperson’s Remarks
08:35 Nitrogen Catabolite Repressible GAP1 Promoter: A New Tool for Efficient Recombinant Protein Production in S. cerevisiae
Fabien Debailleul, Ph.D., Université Libre de Bruxelles
Our work shows that the nitrogen catabolite repressible GAP1 promoter can be used to obtain high levels of recombinant protein while allowing for large biomass production in S. cerevisiae. This approach can be used to express membrane and soluble proteins from higher eukaryotes (from yeast to human). Therefore, this system stands as a promising alternative to commonly used expression procedures in yeasts.
09:05 Solving Problems of Recombinant Production of Two Human L-asparaginases
Manfred Konrad, Ph.D., Head, Enzyme Biochemistry Research Group, Max-Planck-Institute for Biophysical Chemistry; Goettingen University Ph.D. Program Faculty Member
Asparaginases hydrolyzes L-asparagine in the blood, thus depriving leukemic cells from the external source of this natural amino acid. To replace therapeutically used bacterial enzymes, we pursue the design of human asparaginases. We developed a co-expression system to expose the catalytically critical threonine residue at the N-terminus of the b-subunit.
09:35 Protein Expression Screening in Mammalian Suspension Cells
Michael R. Dyson, Ph.D., Group Leader, IONTAS Ltd.
High throughput protein expression screening in mammalian suspension cells is an important step in the process of recombinant antibody selection and optimisation. This is both to select an optimal antigen expression construct and to screen IgG and Fab clones that can be expressed in high yield to provide antibodies for cell-based functional assays. Methods will be presented for high-throughput antibody expression in HEK293, CHO and stem cells including case studies for the selection of functionally active antibodies.
10:05 Sponsored Presentation (Opportunity Available)
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 HTP Affinity-Based Identification and Ranking of the Human PDZ Domains Targeted by HPV E6 Oncoprotein
Renaud, Vincentelli, Ph.D., Coordinator of the IBiSA Structural Genomics Facility, Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Marseille University
Using our E. coli HTP protein production pipeline (Saez et al, jove 2014), we over-expressed most of the 266 human PDZ domains and analyzed their binding profile for the viral oncoprotein E6 using the holdup assay, a novel automated quantitative approach for HTP measurement of domain-peptide affinities. The protocols and results of this study will be exposed during the seminar.
11:45 Cell Line Engineering Using the Potential of microRNAs
Kerstin Otte, Ph.D., Professor, Pharmaceutical Biotechnology, University of Applied Sciences Biberach
miRNA technology is a highly innovative and novel engineering tool available to the optimise production cells. miRNAs are involved in virtually all cellular processes, and thus have recently gained much attention as valuable tools for cell engineering. The revelation of the CHO genome and transcriptome as well as the CHO miRnome substantially accelerated miRNA research in this industrially relevant cell type.
12:15 End of Optimizing Protein Expression
Download Brochure | Register