PEGS Europe Summit Protein Expression Stream

Cambridge Healthtech Institute’s Eighth Annual
Engineering Expression Systems

Genes, Codons, Vectors and Clones

2-3 November 2015


The demand for high-quality therapeutic recombinant proteins continues to grow. However, protein expression is never simple. Many variables must be considered when engineering an ideal system for protein expression, including verification and sequence analysis of the gene or protein of interest, codon optimisation, vector construction and clone/host selection. When bottlenecks arise, protein expression engineers must return to the drawing board and design new cloning schemes by altering the DNA sequence, moving a gene from one vector to another, transfecting the vector in an alternative host, re-selecting clones, re‐characterising the expressed protein or any of the above—a laborious, time‐consuming and expensive process.

The Engineering Expression Systems conference continues the tradition of applying effective engineering strategies for protein expression and production leading to functional biotherapeutic products. Learn from seasoned, savvy researchers as they share their real‐world experiences, applications and results.

Final Agenda

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Monday, 2 November

12:00 Conference Registration

 

COMBINED KEYNOTE SESSION

13:40 PEGS Europe Team Welcome & Chairperson’s Opening Remarks
Mary Ann Brown, Executive Director, Conferences, Cambridge Healthtech Institute
Mary Ruberry, Senior Conference Director, Cambridge Healthtech Institute

13:50 Streamlining Protein Expression & Production

Lorenz MayrLorenz M. Mayr, Ph.D., Vice President & Global Head, Reagents & Assay Development, Innovative Medicines/Discovery Sciences, AstraZeneca

Until recently, protein expression has been seen as a rather mature business with well-established technologies and highly standardized processes for the production of recombinant proteins. Due to significant technological advancements in the field of transient gene expression (TGE), we need to change our perception. The field of mammalian transient gene expression has now started to become one of the most dynamic areas of modern biotechnology with a steady flow of novel technologies and product innovations for the enhanced production of recombinant proteins in their native physiological environment, performed with high success rates and high yields.
Lorenz Mayr will discuss the comprehensive and contemporary activities for transient gene expression at AstraZeneca. He will describe new developments around these technologies for the generation of high-performance protein expression systems.

14:30 Mechanically Refolding Proteins beyond Unboiling an Egg

Gregory WeissGregory A. Weiss, Ph.D., Professor of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine

Traditionally, refolding proteins involves arduously optimising conditions to coax protein solution into thermodynamic equilibrium. Using a Vortex Fluid Device, my lab has applied shear stress during refolding to overcome barriers between protein folding states, rapidly driving refolding. For processes not dialysis dependent, this accelerated refolding allows a more thorough search for conditions favoring protein stability. We have demonstrated VFD-based protein refolding on recombinant proteins, plus refolded lysozyme from boiled egg whites.

15:10 INTERACTIVE PANEL DISCUSSION:
Emerging Technologies and Methods to Improve Yields

New technologies and approaches are leading to greater yields and greater efficiencies for analyzing quality. This panel discussion examines which technologies show the greatest promise, and discusses how these new methods will innovate the field of protein science. Discussion topics include:

  • High throughput
  • Parallel expression/purification
  • Purity
  • Automation
  • Protein folding
  • Assays
  • Cell line engineering

Moderator:

Gregory A. Weiss, Ph.D., Professor of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine

Panelists:

Ian Hodgson, Ph.D., BSc, Head, Molecular Biology, FUJIFILM Diosynth Biotechnologies

Kyle J. Lauersen, Ph.D., Faculty of Biology, Algae Biotechnology & Bioenergy, Center for Biotechnology, Bielefeld University

David O’ Connell, Ph.D., Lecturer & Director, MSc Programmes in Biotherapeutics, Biomolecular & Biomedical Research, University College Dublin

Saurabh Sen, Ph.D., Principal Scientist, Immune Modulation and NBE Discovery, Boehringer Ingelheim Pharmaceuticals, Inc.

 

15:50 Refreshment Break in the Exhibit Hall with Poster Viewing

 

VECTOR ENGINEERING

16:30 Chairperson’s Remarks

Kirill Alexandrov, Ph.D., Professor, Institute for Molecular Bioscience, Australian Institute of Bioengineering and Nanotechnology, University of Queensland

16:35 A Synthetic Optimised Vector System for Chlamydomonas reinhardtii

Kyle LauersenKyle J. Lauersen, Ph.D., Faculty of Biology, Algae Biotechnology & Bioenergy, Center for Biotechnology, Bielefeld University

Microalgae are interesting hosts for numerous natural products and hold the capacity for light-driven photo-bioproduction. However, complicated genetics and limited molecular tools have hindered significant development in transgenic algal technologies that could further their biotechnological potential. This work presents the development of a modular versatile vector for the model microalgae Chlamydomonas reinhardtii. This system serves as a standardised platform for future genetic engineering of this valuable algal species.

17:05 In vitro Reconstitution and Analysis of Protein Interaction Networks

Kirill AlexandrovKirill Alexandrov, Ph.D., Professor, Institute for Molecular Bioscience, Australian Institute of Bioengineering and Nanotechnology, University of Queensland

The exponential increase sequenced genomes has focused attention on how best to produce, study and modify the encoded gene products. We created a suite of in vitro expression Gateway vectors that allow us to rapidly express genes from human ORFeome libraries in Leishmania tarentolae cell-free system. We combine single molecule fluorescence spectroscopy and Amplified Luminescent Proximity Homogeneous Assay Screen to analyse the interactions of subunits of several in vitro reconstituted multisubunit eukaryotic protein complexes.

17:35 An Efficient Plant Virus Vector Able to Target Proteins to Different Subcellular Compartments in Plants

Eszter_MajerEszter Majer, Research Scientist, Plant Virus Biotechnology Research Group, Instituto de Biología Molecular y Celular de Plantas, CSIC-Universidad Politécnica de Valencia

A plant virus-based expression system derived from tobacco etch virus (TEV; genus Potyvirus) permits the co-expression of several recombinant proteins from the same viral backbone in tobacco. In a recent work, protein targeting to chloroplasts, nuclei and mitochondria was successfully achieved from the amino terminus of the viral polyprotein and simultaneous expression of several proteins targeted to distinct subcellular locations was also demonstrated. The TEV-based system is highly promising for manipulation of plant endogenous metabolic pathways through expression of regulatory transcription factors and biosynthetic enzymes.

18:05 Welcome Reception in the Exhibit Hall with Poster Viewing

19:05 End of Day One

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Tuesday, 3 November

07:45 Registration and Morning Coffee

 

GENE ENGINEERING

08:30 Chairperson’s Remarks

Helene Faustrup Kildegaard, Ph.D., Co-Principal Investigator, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark

08:40 Tailoring CHO Cell Metabolism via MicroRNA Manipulation to Boost Recombinant Protein Productivity

Niall BarronNiall Barron, Ph.D., Program Leader, Mammalian Cell Engineering, National Institute for Cellular Biotechnology, Dublin City University

MicroRNA engineering has already proved successful in enhancing various CHO cell phenotypes relevant to production of recombinant products. miR-23 has previously been demonstrated to play a role in glutamate metabolism providing an alternative source of critical metabolites for the TCA cycle, ultimately strengthening oxidative metabolism. We consider how reprogramming cellular bioenergetics through miR-23 may allow mammalian production cells to be more productive by favouring metabolic channeling into oxidative metabolism.

09:10 Bicistronic mRNAs to Enhance Membrane Protein Overexpression

Jacopo MarinoJacopo Marino, Ph.D., Research Scientist, Chemistry and Biochemistry, Gene Center, University of Munich

We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. Transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein.

09:40 PROBLEM SOLVING ROUNDTABLE DISCUSSIONS

Table 10: Advantages and Limitations of Cell-Free Expression Systems

Moderator: Kirill Alexandrov, Ph.D., Professor, Institute for Molecular Bioscience, Australian Institute of Bioengineering and Nanotechnology, University of Queensland

  • There are a lot of cell-free systems to choose from – which one will prevail?
  • What are the most suitable applications for cell-free systems?
  • Analytical vs. preparative applications of cell-free systems

Table 11: CRISPR/Cas9-Mediated Cell Factory Engineering: Opportunities and Challenges

Moderator: Helene Faustrup Kildegaard, Ph.D., Co-Principal Investigator, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark

  • How can we apply the CRISPR/Cas9 system in cell factory engineering (editing, engineering, imaging, etc.)?
  • Which challenges do we encounter when applying the CRISPR/Cas9 system?
  • What are the future perspectives of the CRISPR/Cas9 system?

Table 12: Integrative Approaches for Improving Heterologous Membrane Protein Expression in E. coli  

Moderator: Jacopo Marino, Ph.D., Research Scientist, Chemistry and Biochemistry, Gene Center, University of Munich 

  • Codon bias and mRNA structure at the translation initiation region: What did we learn so far?
  • The use of N-terminal fusion proteins: A trial and error approach
  • Promoters, plasmids and E. coli strains: How important are these variables?
  • High-throughput screenings: From expression yields to biophysical experiments
 

10:40 Coffee Break in the Exhibit Hall with Poster Viewing

 

GENE ENGINEERING (CONT’D)

11:20 Improved CHO Cell Factories Using CRISPR Cas9 Genome Editing Technologies

Helene Faustrup KildegaardHelene Faustrup Kildegaard, Ph.D., Co-Principal Investigator, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark

The high efficiency of the CRISPR Cas9 genome editing technology has enabled fast and easy construction of engineered cell lines. Here, our efforts on generating precise gene disruptions and gene insertions using CRISPR Cas9 will be presented together with a fast and high-throughput platform for constructing CRISPR Cas9 reagents. In addition, examples of improved CHO cell factories generated using these technologies will be presented.

11:50 Inactivation of GDP-Fucose Transporter Gene (Slc35c1) in CHO Cells by ZFNs, TALENs and CRISPR-Cas9 for the Production of Fucose-Free Antibodies

Zhiwei SongZhiwei Song, Ph.D., Principal Scientist, Lead PI for GlycoSing Programme, Bioprocessing Technology Institute, A*STAR

GDP-fucose transporter gene (Slc35c1) in CHO-K1 cells was inactivated by ZFNs, TALENs and CRISPRs for the production of fucose-free antibodies. The Slc35c1 gene was also inactivated in a pre-existing antibody-producing CHO line that produces the anti-Her2 antibody. Inactivation of the GDP-fucose transporter did not affect cell growth or antibody productivity.

12:20 Enjoy or Lunch on Your Own

14:00 Dessert Break in the Exhibit Hall with Poster Viewing

 

ENGINEERING HIGHER EXPRESSION

14:30 Chairperson’s Remarks

Diethard Mattanovich, Ph.D., Professor, Microbial Cell Design, University of Natural Resources and Life Sciences, Vienna and Director, Area Systems Biotechnology and Microbial Cell Engineering, Austrian Centre of Biotechnology


FEATURED PRESENTATION

14:35 Streamlining the Expression Screening of Membrane Proteins

Ray OwensRay Owens, Ph.D., Head, Oxford Protein Production Facility-UK, Research Complex at Harwell and Professor, Molecular Biology, University of Oxford

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilised in detergents. One approach to overcoming these issues is to evaluate multiple membrane protein/variants. Combining ligation independent cloning of membrane proteins as GFP fusions with expression detected by GFP fluorescence enables this to be carried out rapidly and efficiently.
August 2015 Speaker Interview

15:05 Tiny but Mighty – Functional High-Content MicroRNA Screening Provides a Versatile Toolbox for Next-Generation CHO Cell Engineering

Simon FischerSimon Fischer, Ph.D., Scientist, BP Process Development Germany, Boehringer Ingelheim Pharma GmbH & Co. KG

MicroRNAs constitute an important class of non-coding RNAs in mammals. Individual miRNAs control entire cellular pathways without adding translational burden to cells and thus have received growing attention in biotechnology. A functional high-content miRNA screening in CHO cells discovered hundreds of miRNAs to substantially improve bioprocess relevant cell functions. Our comprehensive miRNA research led to the generation of a “miRNA target catalog” providing an avenue for next-generation CHO cell engineering.

15:35 Construct Engineering Provides a Platform for High-Quality GPCR Generation

Oliver SchlenkerOliver Schlenker, Ph.D., Senior Research Scientist, Protein Engineering, Heptares Therapeutics

Protein engineering allows achieving high-quality protein from difficult-to-tackle G protein-coupled receptors (GPCRs). Our stabilised receptors (StaRs) enable us to express and purify material suitable for biophysical characterisation. Optimisation of protein termini and utilisation of fusion proteins result in significant improvements in expression, quality and crystallisation. We provide a powerful solution for high-end quality protein delivery to find drug candidates for diseases such as Alzheimer’s and diabetes.

Polyplus small logo16:05 Superior Protein Yields in CHO and HEK-293 cells using a Novel, Highly Efficient Transfection Reagent - FectoPRO®

Vjetrovic_JelenaJelena Vjetrovic, Ph.D., Bioproduction Technical Support Specialist, Polyplus-transfection, France

Low transfection efficiency of CHO cells is a major bottleneck hampering Transient Gene Expression (TGE). Polyplus-transfection®, with its 10+ year expertise in transfection, has developed a novel technologically advanced transfection solution specifically designed for bioproduction. FectoPRO® outperforms currently available PEI-based and lipid-based transfection reagents in both CHO and HEK-293 cells in variety of commercially-available media. We will present data and protocols leading to unmatched protein and antibody yields in CHO and HEK-293 cells as well as customer data that substantiate our findings.


16:35 Refreshment Break in the Exhibit Hall with Poster Viewing

 

ENGINEERING HIGHER EXPRESSION (CONT’D)

17:15 Metabolic Engineering of NADPH Supply Enhances Recombinant Protein Production in Pichia pastoris

Diethard MattanovichDiethard Mattanovich, Ph.D., Professor, Microbial Cell Design, University of Natural Resources and Life Sciences, Vienna and Director, Area Systems Biotechnology and Microbial Cell Engineering, Austrian Centre of Biotechnology

Based on a genome-scale metabolic model of the yeast Pichia pastoris we predicted gene knockouts and gene overexpressions in the central metabolism to improve recombinant protein production. Experimental validation showed that the best effect was achieved by enhancing the pentose phosphate pathway, which is the major supplier of reduced NADPH. The optimum metabolic interventions will be discussed based on detailed analysis of gene regulation and metabolic fluxes.

17:45 Enhanced Protein Production by Transient Transfection of HEK293 by Extended Gene Expression

Francesc GòdiaFrancesc Gòdia, Ph.D., Professor, Chemical Engineering, Universitat Autònoma de Barcelona

Extended gene expression (EGE) is a strategy proposed to prolong the production period of recombinant proteins by HEK293 cells transient transfection by repeatedly transecting cell cultures, together with performing a medium exchange. The benefits of this method are discussed for the production of three model products including two recombinant proteins (intracellular and secreted GFP) and a complex VLP (Gag-based VLPs).

18:15 INTERACTIVE PANEL DISCUSSION:
Enabling Protein Expression through Intelligently Engineered Platforms

Technical challenges for accelerating recombinant protein production persist from genes to vectors to constructs to clones, and these multiple variables are required when developing reliable expression systems. This panel gathers experts to debate the benefits and tradeoffs of traditional and novel engineering strategies that lead to greater, faster high-quality biotherapeutics.

Moderator: Diethard Mattanovich, Ph.D., Professor, Microbial Cell Design, University of Natural Resources and Life Sciences, Vienna and Director, Area Systems Biotechnology and Microbial Cell Engineering, Austrian Centre of Biotechnology

 

Panelists:

Niall Barron, Ph.D., Program Leader, Mammalian Cell Engineering, National Institute for Cellular Biotechnology, Dublin City University

Simon Fischer, Ph.D., Scientist, BP Process Development Germany, Boehringer Ingelheim Pharma GmbH & Co. KG

Francesc Gòdia, Ph.D., Professor, Chemical Engineering, Universitat Autònoma de Barcelona

Ray Owens, Ph.D., Head, Oxford Protein Production Facility-UK, Research Complex at Harwell and Professor, Molecular Biology, University of Oxford

Oliver Schlenker, Ph.D., Senior Research Scientist, Protein Engineering, Heptares Therapeutics

18:45 End of Engineering Expression Systems

Day 1 | Day 2 | Speaker Biographies | Download Brochure