ExpressionWorld 2018

Presentations


Introduction to the LabCoat Live SmartStart Training Courses for Expi Protein Expression Systems

Abstract

Getting started with an unfamiliar life science research application or a new product can always present challenges. LabCoat Live SmartStart Training is a professional interactive education experience designed to help you through this process. Our experienced application scientists will guide you step-by-step, via a series of live online lectures, as you complete a self-paced hands-on experiment in your lab using provided reagents and protocols. In this session we will review agendas of the three courses offered for our Expi Expression systems and how we can support your lab with detailed discussions about the unique system components and the key workflow steps where optimal techniques will maximize results.

Biography

Stephanie is a Technical Training Specialist with the Global Services & Support team at Thermo Fisher Scientific. She has more than 12 years of laboratory research and commercial manufacturing experience in Molecular and Cell Biology applications, including protein production, and 10 years experience helping other scientists through Technical Support Scientist and Training roles. She works at our Carlsbad, CA office and loves to spend time outdoors in the beautiful Southern California weather. Stephanie obtained a BSc in Microbiology from the University of Arizona in 1998.

Stephanie Culbertson

Technical Training Specialist
Thermo Fisher Scientific


High capacity magnetic supports for automated antibody and epitope-tagged protein purifications

Abstract

For academic, biotech, and pharmaceutical scientists who are screening clones or performing high throughput protein purification, the goal is to automate the sample processing without sacrificing binding capacity. Traditionally, magnetic beads have facilitated process automation, but they lack the high binding capacity of agarose resins. On the other hand, agarose or sepharose resins have high binding capacity, but are not amenable to automation. Magnetic agarose resins combine the best of both formats by enabling high throughput sample processing with high protein binding capacity. Here we demonstrate the advantages of using magnetic agarose resins in simple benchtop protein purifications as well as their utility in 1) the screening of recombinant antibodies using the InvitrogenExpiCHOExpression Systems and 2) the purification of recombinant proteins expressed by in vitro translation (cell-free) systems using the Thermo Scientific KingFisherFlex magnetic particle processor. Both the Protein AG and anti-DYKDDDDK magnetic agarose supports result in isolation of >0.5mg protein per sample at >90% purity.

Biography

Barbara received her Ph.D. in biochemistry from the Medical College of Wisconsin. Her post-doctoral fellowship was performed at The Pennsylvania State University under the direction of Stephen Benkovic. She has over twenty years of industry experience, and she currently leads a team of R&D scientists in new product development in the areas of protein interactions, affinity chromatography, and protein sample preparation.

Barbara Kaboord, PhD

Sr. Manager R&D
Thermo Fisher Scientific


Ultra high titers comparable to stable Expression System within two weeks (presented in Chinese)

Abstract

ExpiCHO is a highly efficient transient expression system that provides an expression level comparable to stable cell lines within two weeks. While CHO cells have been widely used in the industry for a long time, stable cell lines must be constructed to obtain CHO-expressed proteins, a time-consuming and labor-intensive procedure that requires experienced R&D personnel. In the drug development phase, it is necessary to produce a large quantity of proteins in a relatively short period of time, which makes the 293-transient expression system the only choice in most cases. This system, however, expresses at low levels with inconsistencies in the quality of the yielded proteins compared with the CHO system. In contrast, ExpiCHO, as an efficient transient expression system, can reach a yield of at least 1 g/L in just two weeks. In 2017, we used this system for the production of multiple fusion proteins and monoclonal antibodies. The expression levels were maintained at 1 g/L or more, exceeding 2 g/L and up to 3 g/L in some cases. When comparing the expression levels of the transient expression platform with those of stable CHO-S cell lines, we found that the two systems were similar. In addition, while expressing a large amount of a protein, the transient system could also evaluate the expression level of the molecule in stable cell lines. ExpiCHO therefore provides an excellent tool for users who need to prepare a high volume of proteins rapidly.

Biography

Ms. Kong is a research and development engineer at Shanghai Bai Ze Medical Equipment Co., Ltd., where she is responsible for the development of cell lines in the company, including the construction of the transient platform and the stable cell plant construction, as well as the process optimization, cell plant domestication and other work. In 2017, the Expicho instantaneous platform expressed close to 3g/l where she completed nearly 10 projects in a short time; as well as the development of stable cell strains which made the expression more than 2g/l. She also participated in the development of antibody application, including coupling, testing and so on.

Yuedan Kong

Research and development engineer
Shanghai Bai Ze Medical Equipment Co., Ltd.

A comprehensive workflow solution from gene synthesis to purified proteins

Abstract

Reliable expression of recombinant proteins in human or CHO cells is essential for many aspects of biomedical research and drug development but is often hampered by low expression yields that limit subsequent structural and functional analyses. The full portfolio of Thermo Fisher Scientific, from GeneArt gene synthesis to optimized mammalian expression systems, offers researchers the unique opportunity to perfect their gene expression results. Our proprietary sequence optimization algorithm GeneOptimizer enhances the sequence through a parallel multiparameter approach. To further maximize protein yield, you can choose from our best serum-free Gibco FreeStyle or Invitrogen Expi293 and ExpiCHO expression systems. This holistic approach - from electronic gene sequence or accession number to the purified protein - is the most efficient way to exploit your gene-to-protein workflow for highest performance, either by purchasing reagents for internal work or outsourcing the entire workflow as a highly customized service to the GeneArt protein team. The presentation explains the basics of gene optimization and gene synthesis and how the combination with the best expression system produces unsurpassed expression reliability and protein yield.

Biography

Michael Liss, Sr. Manager R&D, received his PhD in 2000 from University of Regensburg & Boulder gaining expertise in virology, molecular biology and directed evolution. 2000-2002: Postdoctoral Researcher in Biosensoring with aptamers. 2002-2012: Scientist in R&D at GeneArt GmbH. 2012-2014: Sr. Manager R&D at Life Technologies, since 2014: Sr. Manager R&D at Thermo Fisher Scientific.

During his work in synthetic biology and gene synthesis Michael has setup and managed the directed evolution unit for more than five years. He further advanced the progress of economic and reliable gene synthesis and installed the gene-to-protein service. Today, he is responsible for R&D projects including process development, portfolio accretion and novel applications of synthetic biology.

Michael Liss

Sr. Manager R&D
University of Regensburg & Boulder


Basic concepts in transient expression using 293 and CHO cells (presented in Chinese)

Abstract

HEK293 and CHO cells are the primary cell lines used in rapid, scalable suspension culture systems for transient protein expression. This presentation provides a primer on fundamental concepts and materials used for transient expression and key differences to consider when using 293 or CHO cells.

Biography

Jian Liu works as a Scientist III at Cell Biology of the Life Sciences Solutions Group (now part of Thermo Fisher Scientific) in Frederick, Maryland, USA. He received his Bachelor’s degree and Master’s degree in Biochemistry from East China University of Science and Technology in Shanghai, China. Mr. Liu worked in University of Southern California for six years to develop AIDS vaccine. In 2000, he joined Invitrogen in Carlsbad, California. During his tenure at Invitrogen, he worked in different departments and played different roles from developing amplifiable gene jumper kit to purifying various enzymes including MMLV, SuperScriptII and III, Taq DNA Polymerase, RNaseOUT, E. coli DNA Polymerases and DNaseI in large scale. In 2006, he moved to Bioproduction division located in Frederick, Maryland, and started working on stable cell line development. He generated both DG44 and CHO-S stable cell lines expressing over 1 g/L monoclonal antibody in fed-batch mode. In 2014, he joined cell culture essential group and focused on scalability of novel transient transfection system -- ExpiCHO Expression System.

Jian Liu

Scientist III
Thermo Fisher Scientific


Strategies for High-Titer Protein Expression Using the ExpiCHO and Expi293 Transient Expression Systems

Abstract

The ExpiCHO and Expi293 transient expression systems offer all-in-one solutions for generating high-titer recombinant proteins for a broad range of research applications including candidate drug identification, reagent production, structural biology and vaccine research and membrane protein biology. While both Expi systems offer the ability to generate high levels of recombinant proteins, the inherent differences between HEK293 cells and CHO cells make these systems differentially applicable for various protein expression needs. Here, we present the latest data on the ExpiCHO and Expi293 Expression systems as well as suggested paradigms for instances where either Expi293 or ExpiCHO would be most applicable for a given research requirement.

Biography

Jonathan Zmuda will present this talk in English

Jonathan Zmuda is a Director of Cell Biology at Thermo Fisher Scientific in Frederick, Maryland, USA. Jon leads a team dedicated to discovering and developing new technologies and products useful for cell biology applications, including transient protein expression, advanced cell culture, and rare-cell analysis. He received his Ph.D. in Cell Biology from the University of Maryland, College Park.

Chao Yan Liu will present this talk in Chinese

Chao Yan Liu, is a Senior Staff Scientist, at Cell Biology of the Life Sciences Solutions Group – now part of Thermo Fisher Scientific - at the Frederick (MD - USA) location. She received the M.D. degree from the Hebei Medical University (Hebei, China) and the Master degree in Pathology from the Academy of Military Medical Sciences in Beijing, China. After being very active for more than ten years in academic research in the field of cellular biology and immunology, in 2005 she joined the Research and Development department of the former Life Technologies. She has made great contributions to more than ten of the Gibco brand Cell Culture Essential products including AlgiMatrix 3D Culture System, OpTmizer CTS T-Cell Expansion SFM, TrypLE Select Enzyme, ES Cell Qualified FBS, GlutaMAX media supplement, FreeStyle F17 Expression Medium and CD FortiCHO Medium. She has extensive experiences in mammalian cell culture, serum and protein free, chemical defined medium formation as well as protein expression. In the past five years, she served as leading scientist to focus on the development of two novel transient transfection systems, Expi293™ and ExpiCHO Expression Systems.

Jonathan Zmuda, PhD

Director, Cell Biology
Thermo Fisher Scientific

Chao Yan Liu

Sr. Staff Scientist
Thermo Fisher Scientific


Accelerating Bio-Production Using ExpiCHO Stable Production Medium

Abstract

Many research laboratories use the ExpiCHO Expression System to transiently produce candidate biomolecules for biologics development. However, after the lead candidate is found, a stable cell clone expressing the protein of interest is in many instances desired. Thermo Fisher Scientific has recently developed tools to help researchers that are interested in generating ExpiCHO-S-derived stable clones capable of producing high titers and scaling up into a large bio-production process. The high costs and time involved to create and adapt stable cell lines into a suitable production medium can be laborious. ExpiCHO Stable Production Medium provides a seamless transition from transient expression to ExpiCHO stable production, requiring minimal to no cell adaptation. We have generated ExpiCHO-S stable cell lines that produce up to 3.5 more protein than stable clones from traditional CHO-S cells. With our new ExpiCHO Stable Production Medium, Thermo Fisher Scientific provides a clear path toward creating higher-producing stable ExpiCHO-S cell lines while supporting the scalability and product quality attributes important for cell line development.

Biography

Shreya received her Masters of Science degree in Pharmaceutical Sciences from the School of Pharmacy and Pharmaceutical Sciences at the University at Buffalo. Her research focused on disease progression in multiple sclerosis patients with a particular emphasis in lipid and cholesterol derivative analysis. She has previous experience as a Research Technician at the University at Buffalo, Department of Microbiology and Immunology where she gained a vast knowledge in molecular biology techniques. She joined as a Scientist in Gibco Bioproduction Services in 2016 with her current research efforts focusing on mammalian cell culture optimization, New Product Introduction, cell line and process development.

Shreya Lowmaster

Scientist II, Cell Biology
Thermo Fisher Scientific

Efficient purification of recombinant Strep-tag® fusion proteins from Expi supernatants using Strep-Tactin®XT

Abstract

Mammalian transient expression has become an effective method to provide recombinant proteins in a short time. Therefore, mammalian transient expression systems are applied to a great extend in R&D departments of pharmaceutical companies and academia to screen drug candidates and proteins. The Expi Expression System enables high transient protein titers and reduces the expression scale as well as the expression time. Affinity tags simplify protein purification and support the high throughput process by using the same purification strategy for many different proteins. However, many affinity tags have remarkable drawbacks like inconsistent purity, low binding capacity or large sizes. IBA´s Strep-tag® purification system (Strep-Tactin®XT:Twin-Strep-tag®) enables protein purification at high yields and purity under physiological conditions from several hosts following a simple one-step purification protocol. Providing the highest binding affinity among all affinity tag systems on the market, it is also well suited for many downstream applications like e.g. SPR. Due to its performance it complies well with the demands of a high throughput process.

The webinar introduces the Strep-Tactin®XT technology and demonstrates the efficient and easy purification of Strep-tag® proteins from Expi supernatants. Additionally, it delivers an insight into the use of Strep-Tactin®XT for SPR analysis.

Biography

Dennis Niermeier received his master’s degree in biotechnology from the University of Applied Sciences in Bremerhaven. During his master’s study at the department of Pharmaceutical Biotechnology at the Fraunhofer Institute for Toxicology and Experimental Medicine, he worked on the development of a protein purification platform and in cell line development. In 2012, Dennis Niermeier joined IBA GmbH. He is responsible for the mammalian custom protein expression and purification service and the product development in this field.

Dennis Niermeier

Scientist
IBA Life Sciences


Optimizing G Protein-Coupled Receptor Expression in Mammalian Cells

Abstract

G protein-coupled receptors (GPCRs) regulate virtually all known physiological processes and are a premier class of drug targets. However, a detailed mechanistic understanding of their complex activation mechanisms has been hindered by the difficulty of expressing and purifying these membrane proteins. We will describe how mammalian expression provides a versatile platform for high-level GPCR expression. In addition, we will discuss how new expression technologies can address the challenges that have previously limited the scope of mammalian expression systems for biochemical and biophysical studies of GPCRs.

Biography

Dean Staus received his Bachelor of Science degree in biology from the University of Wisconsin, Whitewater and his PhD in pathology from the University of North Carolina, Chapel Hill. He is currently a postdoctoral researcher in Dr. Robert Lefkowitz’s laboratory at Duke University.

Laura Wingler received her Bachelor of Science degrees in chemistry and biochemistry from North Carolina State University and her PhD in chemistry from Columbia University. She is currently a postdoctoral researcher in Dr. Robert Lefkowitz’s laboratory at Duke University.

Dean Staus, PhD

Postdoctoral Researcher
Duke University

Laura Wingler, PhD

Postdoctoral Researcher
Duke University


Cryo Electron Microscopy: Revolutionizing the world of structural biology and healthcare

Abstract

Studying the molecular machinery of cells from atomic detail to the cellular context and beyond is a great challenge for cell biology. It is hugely important to zoom into the atom-by-atom arrangement of a biomolecule that gives researchers a grasp of how proteins work. Structural biologists have made great progress in unraveling the structures of individual proteins and protein complexes. Much of the recent results have been obtained using Cryo Electron Microscopy (CryoEM) Single Particle Analysis (SPA), which has turned the generation of 3D reconstructions of protein (complexes) into an accessible and well recognized application. As CryoEM SPA works best on samples of isolated and purified proteins, the expression, isolation and purification is of extreme importance. A purified protein fraction is a key prerequisite for successful data acquisition and 3D reconstruction of a structure in its natural state. Ultimately, the goal is to understand the structure - functional relationship of molecular machineries of all living systems within their natural, functional contexts.

During this talk the concept of CryoEM SPA will be introduced, the most recent developments around the unique CryoEM SPA workflow as well as the applicatory medical breakthrough’s that have been obtained with CryoEM resulting in the 2017 Nobel Prize for Chemistry. Special attention will be paid to dependencies on sample preparation and associated biochemical and vitrification requirements.

Biography

Marc Storms has studied biology / plant pathology at Wageningen University and UC Davis. He obtained his PhD in Virology studying the intercellular trafficking of Bunyaviruses. After his PhD he joined Philips / FEI Electron Optics / Thermo Fisher Scientific, first as applications specialist TEM and later as Product Manager where he has been involved in the (hardware and software) development and release of various Transmission Electron Microscopes (e.g. Morgagni and Tecnai Spirit) as well as instruments for sample preparation (Vitrobot Mk I-IV generations).

Currently, Marc is responsible for the product marketing of Structural Biology related products and workflow solutions for single particle analysis (SPA). As such, he has been involved in new key initiatives for structural biology Cryo-EM solutions such as the Titan Krios, the Talos Arctica, Talos L120C, applications software developments for SPA (i.e. EPU SW) as well as methods to enhance contrast and detector performance such as the Volta phase plate and several generations of the Falcon direct electron detectors.

Dr. Marc M.H. Storms

Product Marketing Manager
Thermo Fisher Scientific


In vitro research method for screening inhibitors of protein translation

Abstract

In vitro translation (IVT), or cell-free expression, offers a unique and powerful research tool to screen for translational inhibitors that regulate both cellular and viral protein expression. Here we discuss a simple, quick method to identify inhibitors of both cap-dependent and cap-independent protein translation, using the Thermo Scientific1-Step Human Coupled IVT Kit. Rapid assay readout (6090 min), amenability for miniaturization, and insensitivity to compound toxicity make in vitro translation an attractive alternative to cell-based screens for high-throughput screening (HTS) of novel inhibitors of protein synthesis.

Biography

Krishna's PhD and postdoctural work focused on understanding control mechanisms regulating the initiation of protein translation in eukaryotes. Following his academic training, he joined Thermo Fisher Scientific and has led multiple projects including the development of the HeLa and CHO lysate based cell-free translation systems, also known as Thermo Scientific1-Step In VitroTranslation (IVT) Systems, which contain all the necessary reagents to express proteins of interest in as little as one hour.

Krishna Vattem, Ph.D.

Senior R&D Scientist
Thermo Fisher Scientific


A chemically defined baculovirus-based expression system for enhanced protein production in Sf9 cells

Abstract

The baculovirus expression vector system (BEVS) is one of the major platforms for recombinant protein production. Unlike mammalian expression systems that have long since transitioned to serum-free, chemically-defined culture media, relatively little innovation has taken place in insect expression systems, with insect cells continuing to rely on undefined, yeastolate-containing culture media that can exhibit significant lot-to-lot variability in terms of both cell growth and protein expression. Here, we present the development of a novel Sf9-based baculovirus expression system based on a high-density, chemically-defined culture medium, a high-expressing Sf9 cell line and expression enhancers that allow for the consistent production of recombinant proteins with two-fold or greater improvements in protein titers compared to traditional BEVS workflows.

Biography

Maya Yovcheva will present this talk in English

Maya has been an R&D Scientist with Thermo Fisher Scientific for about three years, focusing primarily on the development of novel tools for protein expression in insect cells. She has a strong background in cell culture, molecular biology, and protein expression. Maya has obtained a BSc in Molecular Biology from Sofia University, St. Kliment Ohridski, Bulgaria and an MSc in Medical Biotechnologies from Wageningen University, The Netherlands.

Nan Yang will present this talk in Chinese

Nan Yang is the Field Application Scientist for transient protein expression in Europe. She obtained her Bachelor’s degree from Nanyang Technological University, Singapore in 2006 and her PhD from Institut National des Sciences Appliquees (INSA) de Lyon on biofilm in 2011.

Maya Yovcheva, MSc

Scientist III
Thermo Fisher Scientific

Nan Yang, PhD

Field Application Scientist
Thermo Fisher Scientific