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.

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.

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.

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.

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.

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.

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.

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.

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.

摘要

杆状病毒表达载体系统（BEVS）是重组蛋白生产的主要平台之一。与哺乳动物表达系统早已转换为无血清，化学成分限定的培养基不同，昆虫表达系统的创新相对较少。昆虫细胞继续依赖成分不限定的含酵母提取物的培养基，而这些培养基可以显示出明显的细胞生长和蛋白质表达的批次差异。在这里，我们介绍一种新型的Sf9细胞杆状病毒表达系统的开发。此系统包括支持高细胞密度、化学成分限定的培养基，高表达Sf9细胞系，以及优化的培养辅料。此表达系统提供一致的重组蛋白生产，且比传统的BEVS提高了两倍或更多的蛋白滴度。

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.

HEK293和CHO细胞是快速、规模可调的悬浮培养系统中常用的细胞系，可用于瞬时蛋白表达。此次演讲介绍了瞬时表达的基本概念和使用材料，以及使用293或CHO细胞时需要考虑的主要差异。

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.

ExpiCHO是一种高效的瞬时表达系统，可以在2周内获得与稳定细胞株相当的表达量。 CHO细胞长期以来一直在工业界广泛使用，获得CHO表达蛋白必须构建稳定的细胞株，这一过程不仅耗时耗力，而且需要经验丰富的研发人员。在药物开发阶段需要在较短时间获得大量蛋白，往往只能采用293细胞的瞬转表达系统，但该系统产量低，表达蛋白与CHO系统存在质量差异。ExpiCHO作为高效的瞬时表达系统能够在2周时间内获得克级每升表达量。2017年，我们采用该系统进行了多种融合蛋白及单抗的生产，表达量均维持在1g/L以上，部分超过2g/L，接近3g/L水平。同时，我们对比了瞬转表达水平与基于CHO-S的稳定细胞株表达水平，发现瞬转表达系统的产量与稳转细胞株的产量类似，瞬转系统在制备大量蛋白的同时也能评估该分子在稳定细胞株的表达水平。因此，ExpiCHO为需要快速制备大量蛋白的用户提供了一种极好的工具。

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

Gibco™ ExpiCHO 和Expi293系统是两款全新优化的瞬时重组蛋白高表达系统，目前已广泛应用于治疗性候选药物的早期鉴定，诊断性试剂的快速生产，以及疫苗研发、结构生物学和膜蛋白生物学等研究领域，成为可以在瞬转中获得极高的蛋白产量的一站式有效途径和工具。虽然多种重组蛋白均可在这两款瞬时表达系统中获得高水平表达，但HEK293 细胞和 CHO 细胞之间存在的内在差异，决定了它们对应各种不同的蛋白质表达需求有各自的适应性。在今天的报告中我们将与同道们分享ExpiCHO和Expi293两大系统研发和应用的最新进展，以及一些具有建设性意义的范例，以期帮助广大用户在特定的研究需求下知晓如何从ExpiCHO 和Expi293中选择最适用的瞬时重组蛋白表达系统。

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 proteinsynthesis

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.