Sabine Geisse

X-Ray Structure of the hRORα LBD at 1.63 Å: Structural and Functional Data that Cholesterol or a Cholesterol Derivative Is the Natural Ligand of RORα

Abstract The retinoic acid-related orphan receptor α (RORα) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORα has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORα, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3β-ol (cholesterol). Moreover, RORα transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORα could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

Reflections on more than 10 years of TGE approaches.

The introduction of foreign genetic material into cultured eukaryotic cells on a transient basis, that is, by transfection, without selection for stable integration of plasmids into the host genome, is a standard experimental procedure. For decades, transient transfections have been performed in cell biology labs on a small scale basis to analyze the effect of genes/proteins on overexpression applicable to, for example, pathway analyses. It has allowed the production of small amounts of recombinant protein, often in crude supernatants and without subsequent purification. This approach offers many advantages to research: it is rapid, simple and, with a plethora of different transfection reagents and protocols available, can be applied to a multitude of cell lines of choice. Early attempts to employ the same protocols for production of recombinant proteins in milligram to gram quantities on a larger scale focused initially on the use of COS cell lines [1–4], but were of limited success due to the restricted scalability of this naturally very adherent cell line. Furthermore, the relatively short life-span of the transfected culture—thought to be caused by the high copy number of replicating plasmids per cell resulting in cytopathic effects—rendered the approach less valuable. A major breakthrough in transient gene expression (TGE) technologies was achieved by switching to other host cell lines, such as HEK293 and CHO lines, and the development of large scale transfection protocols using calcium-phosphate and polyethylenimine (PEI) as a carrier of plasmid DNA. Since then many groups in academia and industry have reported on successful production of large quantities of recombinant proteins in publications or at relevant meetings. In addition, quite a large number of modifications and ameliorations have reportedly been introduced to the original protocols. The first TGE approaches employed predominantly HEK293 cells as the host cell line. More recently, large scale transient transfection protocols have also been established for CHO cells. As CHO cells represent the most commonly used expression host for production of biopharmaceuticals, an alignment of early research activities with cell line development and characterization of product for production purposes, without change of host cell line, is clearly desirable.

Advances in recombinant protein expression for use in pharmaceutical research

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells.

Optimisation of protein expression and establishment of the Wave Bioreactor for Baculovirus/insect cell culture.

As the interest of research is beginning to shift from genomicsto proteomics the number of proteins to be expressed is rapidlyincreasing. To do so, well-established, high-level expressionsystems and rapid, cost-effective production means are needed. For addressing the latter, a novel cultivation system for recombinant cells, the Wave Bioreactor™ has recently becomeavailable. We describe the set-up and the optimisation of parameters essential for successful operation and growth of insect cells to high cell densities in the Wave Bioreactor. According to our experience, the Cellbag™ system comparesvery favorably to conventional cultivation vessels such as bioreactors and roller cultures with respect to simplicity ofoperation and cost. Additionally, we developed a rapid and simple protocol for assessing expression and production conditions for the Baculovirus/insect cell system applicable to many different genes/proteins. Important parameters like MOI,TOI, peak cell density (PCD) and expression levels are determinedin pre-experiments on small scale to achieve optimal expressionof a given protein. These conditions are subsequently transformedand applied to large scale cultures grown in nutrient-supplemented medium in the Wave Bioreactor.

Large-scale transient transfection of mammalian cells: A newly emerging attractive option for recombinant protein production

Mammalian expression systems have an undisputed long-standing and very successful history for the generation of recombinant proteins, mainly as biopharmaceuticals. However, for use as ‘tool proteins’ in, e.g. assay development and screening, for structure elucidation and as antigens these expression systems were generally regarded as being cumbersome, tedious and expensive. This bias has largely been overcome with the very recent development of large-scale transient transfection (LST) approaches. Especially the HEK.EBNA expression system described here has contributed significantly to this success. The simplicity and speed of this approach compares well with expression trials using the widely applied Baculovirus/insect cell system. In addition, proteins generated in mammalian cells are usually correctly folded, fully processed and functionally active.

RECOMBINANT PROTEIN PRODUCTION BY TRANSIENT GENE TRANSFER INTO MAMMALIAN CELLS

The timely availability of recombinant proteins in sufficient quantity and of validated quality is of utmost importance in driving drug discovery and the development of low molecular weight compounds, as well as for biotherapeutics. Transient gene expression (TGE) in mammalian cells has emerged as a promising technology for protein generation over the past decade as TGE meets all the prerequisites with respect to quantity and quality of the product as well as cost-effectiveness and speed of the process. Optimized protocols have been developed for both HEK293 and CHO cell lines which allow protein production at any desired scale up to >100 l and in milligram to gram quantities. Along with an overview on current scientific and technological knowledge, detailed protocols for expression of recombinant proteins on small, medium, and large scale are discussed in the following chapter.

Large-scale transient expression of therapeutic proteins in mammalian cells

Keywords: Animals ; Calcium Phosphates ; Cell Line ; Gene Expression/*genetics ; Humans ; Mammals ; Plasmids ; Polyethyleneimine ; Recombinant Proteins/*genetics/metabolism/*therapeutic use ; Time Factors ; Transfection/instrumentation/*methods ; Transformation ; Genetic/genetics ; Protocols Note: Novartis Pharma Research CT/BMP, Basel, Switzerland. Reference LBTC-CHAPTER-2005-006doi:10.1385/1-59259-922-2:087 Record created on 2007-06-05, modified on 2016-08-08

Transient Expression Technologies: Past, Present, and Future

The first protocols describing transient gene expression in mammalian cells for the rapid generation of recombinant proteins emerged more than 10 years ago as an alternative to the establishment of stable, often amplified clonal cell lines, and relieved somewhat the bias against mammalian cell systems as being too complicated, labor intensive, and tedious to serve as a source for tool proteins in industrial research and academia. Over the past decade, these attempts have been refined and optimized, giving rise to expression protocols applicable in every lab in dependence on available tools, equipment, and envisaged outcome. This chapter summarizes the development of transient expression technologies over the past decade up to its current status and provides an outlook into what may be the future of transient technology development.

Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system.

The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult‐to‐express proteins. For this reason, we evaluated the more recently established novel CAP‐T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP‐T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. Biotechnol. Bioeng. 2012;109: 2250–2261.

Automated baculovirus titration assay based on viable cell growth monitoring using a colorimetric indicator

The fastest methods reported are based on viral DNA quantitation using either flow cytometry (10) or real-time PCR (11), which allow titer determination within two hours. The drawbacks of these approaches are the costs of equipment and staining reagents, and the fact that the total number of particles and not the number of infectious particles is determined. An alternative approach is based on the lytic nature of the viral system (12), and more specifically on the fact that cell growth is attenuated upon virus infection. This growth reduction is dose-dependent and can be estimated by measuring the viable cell concentration and subsequently correlating this to the virus titer. Indeed, a new method was recently developed for virus titration by spectrophotometrically monitoring the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) (4). The accuracy of the method was clearly demonstrated; however, the number of preparation steps and the overall duration (6 days) are not compatible with a fast and automated high-throughput process. In this work, we demonstrate that