Recent advances in the production of proteins in insect and mammalian cells for structural biology

The Oxford Protein Production Facility and Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford, UK.
Journal of Structural Biology (Impact Factor: 3.23). 02/2010; 172(1):55-65. DOI: 10.1016/j.jsb.2010.02.006
Source: PubMed


The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.

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Available from: Joanne E Nettleship
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    • "Recombinant protein production in heterologous expression systems represents a major bottleneck in the development of biopharmaceutical, industrial and research applications (Liras 2008). Even though high throughput techniques for cloning and protein production have been already described (Barnard et al. 2010; Nettleship et al. 2010; Savitsky et al. 2010; Xiao et al. 2010b), purification of challenging proteins is still a matter of trial-and-error approaches. As expression system, E. coli offers many advantages over eukaryotic systems Fig. 5 Detection of GST-GLA- GFP protein in Rosetta-gami B(DE3)/pGEX4T2-GLA-GFP induced cell cultures by Western blot analysis. "
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    ABSTRACT: Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.
    Full-text · Article · Jan 2015 · Applied Microbiology and Biotechnology
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    • "Eukaryotic expression systems including yeasts, insect cells and mammalian cells have been established to produce such proteins (reviewed in refs. [158] [159] [160]). The well-studied baker's yeast, Saccharomyces cerevisiae and the methylotroph Pichia pastoris are frequently used as alternatives to E. coli, owing to their ability to grow in simple and inexpensive media.[161] "
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    ABSTRACT: Early on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Röntgen discovered X-rays in 1895, and in 1912, Max von Laue and his associates discovered that X-ray irradiated salt crystals would produce diffraction patterns that could reveal the internal atomic periodicity of the crystals. In the same year, the father-and-son team, Henry and Lawrence Bragg successfully solved the first crystal structure of sodium chloride and the era of modern crystallography began. Protein crystallography (PX) started some 20 years later with the pioneering work of British crystallographers. In the past 50–60 years, the achievements of modern crystallography and particularly those in PX have been due to breakthroughs in theoretical and technical advancements such as phasing and direct methods; to more powerful X-ray sources such as synchrotron radiation; to more sensitive and efficient X-ray detectors; to ever faster computers and to improvements in software. The exponential development of PX has been accelerated by the invention and applications of recombinant DNA technology that can yield nearly any protein of interest in large amounts and with relative ease. Novel methods, informatics platforms and technologies for automation and high-throughput have allowed the development of large-scale, high-efficiency macromolecular crystallography efforts in the field of structural genomics. Very recently, the X-ray free-electron laser sources and its applications in PX have shown great potential for revolutionizing the whole field again in the near future.
    Full-text · Article · Jan 2015 · Crystallography Reviews
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    • "The technique relies on NMR observable nuclei such as 1 H, 15 N, and 13 C. Due to the low natural abundance of 15 N and 13 C, these isotopes must be incorporated into the protein . Metabolic labeling using bacterial hosts and recombinant DNA is the most common approach to produce fully 15 N-and 13 C-labeled proteins [24]. This labeling technique is not amenable to all proteins, especially those requiring post-translational modifications . "
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    ABSTRACT: Reductive methylation of lysyl side-chain amines has been a successful tool in the advancement of high-resolution structural biology. The utility of this method has continuously gained ground as a protein chemical modification, first as a tool to aid protein crystallization and later as a probe in protein nuclear magnetic resonance (NMR) spectroscopy. As an isotope-labeling strategy for NMR studies, reductive methylation has contributed to the study of protein–protein interactions and global conformational changes. Although more detailed structural studies using this labeling strategy are possible, the hurdle of assigning the NMR peaks to the corresponding reductively methylated amine hinders its use. In this review, we discuss and compare strategies used to assign the NMR peaks of reductively methylated protein amines.
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