Protein Expression System Engineering

 

蛋白質比現系統工程

CHI’s 5th annual Protein Expression System Engineering conference examines the functioning of the cellular machinery harnessed during protein biosynthesis and how to engineer hosts to efficiently express a protein of interest. The intricate steps required to achieve properly folded protein will be discussed, including verification and sequence analysis of the gene, codon optimization, vector construction, selecting and optimizing a clone and selecting a host system. In addition, engineering host cells to sustain expression for longer time periods will be discussed, along with overcoming cellular stress response to produce and secrete functionally active recombinant proteins.

Final Agenda

THURSDAY, APRIL 11

12:00 pm Registration

12:35 Luncheon in the Exhibit Hall with Poster Viewing

SYNTHETIC BIOLOGY & GENETIC ENGINEERING

1:40 Chairperson’s Opening Remarks

Susan Sharfstein, PhD, Professor, Nanobioscience, Nanoscale Science and Engineering, SUNY Polytechnic Institute


1:50 KEYNOTE PRESENTATION: Mammalian Synthetic Biology: Foundations and Application to Cell Line Engineering

Weiss_RonRon Weiss, PhD, Professor, Biological Engineering, Massachusetts Institute of Technology (MIT)

In this research, we appropriate from established engineering fields proven design principles such as abstraction, standardization, modularity, and computer aided design. But we also spend considerable effort towards understanding what makes synthetic biology different from all other existing engineering disciplines and discovering new design rules that are effective for the biological substrate. Building on this foundation, I will describe our recent application of synthetic biology tools and principles towards the improvement of cell line engineering and biomanufacturing.

2:20 Communicating with and Controlling Gene Expression via Redox-Linked Bioelectronics

Bentley_WilliamWilliam E. Bentley, PhD, Robert E. Fischell Distinguished Chair, Engineering; Inaugural Director, Robert E. Fischell Institute for Biomedical Devices, Chemical and Biomolecular Engineering, University of Maryland, College Park

We are developing tools of “biofabrication” that enable facile assembly of biological components within devices, including microelectronic devices, that preserve their native biological function. We have created redox-based synthetic biology to sample, interpret and report on biological information contained in molecular communications circuitry. We have also developed synthetic genetic circuits that enable electronic actuation of gene expression. These tools enable unparalleled means to control genetic circuits, creating new and exciting means to actuate and control biology.

2:50 Steering N-Glycosylation of Recombinant Proteins Using Systems Engineering

Smanski_MichaelMichael J. Smanski, PhD, Assistant Professor, Biochemistry, Molecular Biology & Biophysics, Biotechnology Institute, University of Minnesota

Chinese Hamster Ovary cells are used for industrial production of protein-based therapeutics (i.e. ‘biologics’), but systems-level genetic engineering of beneficial traits is slow, difficult, and empirically-guided. We exploit systems- and synthetic-biology approaches to design, build, and screen multi-gene constructs that rationally perturb the post-translational glycosylation of a secreted Immunoglobulin G (IgG) towards high galactose incorporation. Our approach allows for rapid hypothesis testing and quantification of synergistic behavior from genetic perturbations.

Pfenex-Strain-Engineering 3:20 Presentation to be Announced

3:50 Networking Refreshment Break

4:20 A Multi-Landing Pad DNA Integration Platform for Mammalian Cell Engineering

Wroblewska_LilianaLiliana Wroblewska, PhD, Principal Scientist, Biomedicine Design, Pfizer, Inc.

Reliable, large-scale engineering of CHO cells through precise insertion of large amounts of heterologous DNA into well-characterized genomic loci would have broad applications for mammalian synthetic biology, recombinant protein production, and biomanufacturing. Using multi-gene payload vectors, cell lines with multiple landing pads, and recombinase technology, we demonstrated controlled integration of up to nine copies of a monoclonal antibody (about 100 kb of heterologous DNA), and a corresponding linear increase in antibody expression.

4:50 Implementing Next-Generation Sequencing for DNA-Based Sequence Variant Analysis of Recombinant Proteins

Goepfert_UlrichUlrich Göpfert, PhD, Principal Scientist, Cell Line & Molecular Development, Roche Innovation Center Munich

Sequence variants are unintended amino acid substitutions in biopharmaceuticals, which can either be due to the manufacturing process or mutations of the transgene. Transgene mutations are permanent properties of affected cell lines and may give rise to critical quality attributes. Therefore, mutated cell lines need to be identified and excluded from development. We will share our experience with next-generation sequencing as an efficient and highly sensitive method to detect DNA-based sequence variants.

5:20 End of Day

5:20 Registration for Dinner Short Courses


5:45-8:45 pm Recommended Short Course*

SC15: Transient Protein Production in Mammalian Cells

Richard Altman, MS, Scientist, Protein Technologies, Amgen

Henry C. Chiou, PhD, Director, Cell Biology, Life Science Solutions, Thermo Fisher Scientific

Dominic Esposito, PhD, Director, Protein Expression Laboratory, Frederick National Laboratory for Cancer Research

 

*Separate registration required.

FRIDAY, APRIL 12

8:00 am Morning Coffee

ANALYZING & IMPROVING PRODUCTIVITY

8:30 Chairperson’s Remarks

Christopher H. Gray, PhD, Staff Scientist & Team Leader, Structural Biology, Drug Discovery Program, CRUK Beatson Institute


8:35 FEATURED PRESENTATION: Methylation Analysis of Cell Lines with Varying Productivities

Sharfstein_SusanSusan Sharfstein, PhD, Professor, Nanobioscience, Nanoscale Science and Engineering, SUNY Polytechnic Institute

DNA methylation plays a critical role in regulating gene expression, and it is well known that the CMV promoter contains a CpG island that is subject to silencing by methylation. Using a novel next-generation sequencing approach, we have analyzed the methylation status of the CMV promoter for cell lines with varying productivity to provide insight into the role of methylation in control of transgene expression.

9:05 Improving Cytidine and Adenine Base Editors by Expression Optimization and Ancestral Reconstruction

Koblan_LukeLuke Koblan, PhD, Scientist, Chemical Biology, Chemistry & Chemical Biology, Harvard University

Base editors enable targeted single-nucleotide conversions in genomic DNA. The usefulness of base editors for research and therapeutic applications strongly depends on the efficiency with which they modify target nucleotides. Optimizations to improve editor expression, nuclear localization, and the component deaminase domain enable substantially improved editing by both cytidine and adenine base editors in a variety of mammalian cell types. BE4max, AncBE4max, and ABEmax represent the current state-of-the-art base editors.

CEVEC 9:35 Presentation to be Announced 

10:05 Networking Coffee Break

10:35 Synonymous Codon Selection for Enhanced Yield of Functional Proteins

Clark_PatriciaPatricia Clark, PhD, John Cardinal O’Hara, C.S.C. Professor, Chemistry & Biochemistry, University of Notre Dame

Historically, “optimizing” a gene for heterologous expression consisted of substituting rare codons with synonymous common codons. This strategy can increase the amount of protein produced but at the expense of adversely affecting the yield of active, functional protein. This talk will focus on our recent discoveries regarding rare codon distribution in naturally occurring coding sequences and rational strategies for rare codon placement to enhance folding yield.

11:05 Cell-Free Synthetic Biology for Therapeutics, Sensing, and Remediation

Karig_DavidDavid Karig, PhD, Associate Professor, Systems and Synthetic Biology, Bioengineering, Clemson University

Cell-free protein expression systems offer a number of advantages for implementing synthetic biology applications. They simplify system composition and tuning, avoid evolution away from the intended function, and alleviate safety concerns associated with the spread of engineered living cells. Key developments in the preservation and ruggedization of cell-free reagents will enable therapeutics production in the field as well as environmental sensing and remediation.

11:35 Identification of ADP-Ribosylation by Tandem Mass Spectrometry

Poirier_GuyGuy Poirier, PhD, Professor, Faculty of Medicine, Scientific Advisor, Proteomics Platform CHU of Quebec, Université Laval

We have developed a new method to identify all the ADP-ribosylation sites independent of the acceptor amino acid. This method uses tandem mass spectrometry amenable to CID or other modes of fragmentation. The mass spectrometry signature is very unique and is stable in any mode of fragmentation.

12:05 pm Production of Antimicrobial Peptides Using Protein-Cage Carrier Proteins

Yung_Mimi_ChoMimi Cho Yung, PhD, Staff Scientist, Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory (LLNL)

The bioproduction of antimicrobial peptides (AMPs) in bacterial expression systems remains a challenging problem due to host toxicity and proteolysis. We will discuss our recent efforts to engineer encapsulin nanocompartment systems to enhance expression of AMPs in Escherichia coli.

12:35 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:05 Networking Refreshment Break

IMPROVING EXPRESSION SYSTEMS

1:35 Chairperson’s Remarks

David Karig, PhD, Associate Professor, Systems and Synthetic Biology, Bioengineering, Clemson University

1:40 Molecular Approaches that Improve Soluble Protein Yields from Bacterial Expression Systems

Gray_ChristopherChristopher H. Gray, PhD, Staff Scientist & Team Leader, Structural Biology, Drug Discovery Program, CRUK Beatson Institute

Expression systems targeting well folded products often employ contradictory strategies, pushing production with strong promoters and codon-enhanced cDNAs, while simultaneously slowing the process by titrating back inducing reagents or culture temperature. Often, elevated total expression levels aren’t matched by a similar increase in the recovery of soluble protein. We compare a series of alterations to key codons and expression-vector sequence elements that attenuate protein production rates and maximise soluble recovery.

2:10 Engineering CHO Metabolic Essential Gene for Efficient and High Expressing Clones Suited for Perfusion Processes

Hatim Motiwala, PhD, Head, Cell Line Engineering and Bio-Analytical Sciences, Enzene Biosciences Limited

A double knock-out cell line for two important metabolic genes was created using CRISPR/Cas. This allows for a strong clonal selection of a monomeric or dimeric protein. The cell line is also tested in continuous upstream process for significantly higher expression.

2:40 Recombinant (Membrane) Protein Production in Yeast

Bill_RoslynRoslyn M. Bill, DPhil, Professor, Biotechnology; Associate Dean, Research, Aston University

My lecture will focus on methods that are available for protein synthesis in yeasts, which are an important source of recombinant eukaryotic membrane proteins. I will provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for further study.

3:10 Development of the Filamentous Fungus Myceliophthora thermophila C1 into a Next-Generation Therapeutic Protein Production System

Anne HuuskonenAnne Huuskonen, MSc, Research Scientist, VTT Technical Research Centre of Finland, Ltd.

We are utilizing the vast protein production capability of the filamentous fungus Myceliophthora thermophila to construct a highly potent therapeutic protein production platform. Superb productivities of full-length antibodies, up to 2.5 g/l/day, have been reached. We have also successfully produced several difficult-to-express proteins such as bispecific antibodies and vaccine proteins in titers superior to other expression systems. Our work also aims at humanizing the glycosylation pathway of this fungus, and the first steps in this research line have been successful.

3:40 End of Conference

* 活動內容有可能不事先告知作更動及調整。

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