Engineering genes for predictable protein expression

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Protein Expression and Purification (Impact Factor: 1.51). 03/2012; 83(1):37-46. DOI: 10.1016/j.pep.2012.02.013
Source: PubMed

ABSTRACT The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

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Available from: Sridhar Govindarajan, Aug 27, 2015
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    • "Achieving increased productivity of therapeutically important protein pharmaceuticals is a continuous process involving improvements in gene engineering, DNA delivery systems, host cell engineering and culture conditions (Baldi et al., 2005; Gustafsson et al., 2012; Ng et al., 2010; Peng et al., 2010; Wurm, 2004). Other than Chinese hamster ovary (CHO) cells, human embryonic kidney derived HEK293 cells are adaptable in serum free suspension culture and is thus an attractive platform for the transient or stable expression of recombinant proteins requiring proper post-translational modifications (Henry and Durocher, 2011; Loignon et al., 2008; Thomas and Smart, 2005; Walsh and Jefferis, 2006). "
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    ABSTRACT: Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through anin silicoapproach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.
    Plasmid 12/2013; 71(1). DOI:10.1016/j.plasmid.2013.11.001 · 1.76 Impact Factor
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    • "Many groups exploit this fact by substituting synonymous codons in the coding sequence. In these exercises, codons that, based on statistics, are used less frequently or whose corresponding tRNA abundance is low are classified as rare, and are substituted by synonymous codons that are considered to be more abundant (for reviews on the topic of codon optimization see [5] [7] [8]). Re-designed coding sequences can be both designed and synthesised by commercial vendors. "
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    ABSTRACT: Membrane proteins are extremely challenging to produce in sufficient quantities for biochemical and structural analysis and there is a growing demand for solutions to this problem. In this study we attempted to improve expression of two difficult-to-express coding sequences (araH and narK) for membrane transporters. For both coding sequences, synonymous codon substitutions in the region adjacent to the AUG start led to significant improvements in expression, whereas multi-parameter sequence optimization of codons throughout the coding sequence failed. We conclude that coding sequences can be re-wired for high-level protein expression by selective engineering of the 5' coding sequence with synonymous codons, thus circumventing the need to consider whole sequence optimization.
    FEBS letters 06/2013; 587(15). DOI:10.1016/j.febslet.2013.05.063 · 3.34 Impact Factor
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    ABSTRACT: Escherichia coli has been widely used for the production of recombinant proteins. However, the unbalances between host metabolism and recombinant biosynthesis continue to hamper the efficiency of these recombinant bioprocesses. The additional drainage of biosynthetic precursors toward recombinant processes burdens severely the metabolism of cells that, ultimately, elicits a series of stress responses, reducing biomass growth and recombinant protein production. Several strategies to overcome these metabolic limitations have been implemented; however, in most cases, improvements in recombinant protein expression were achieved at the expense of biomass growth arrest, which significantly hampers the efficiency of recombinant bioprocesses. With the advent of high throughput techniques and modelling approaches that provide a system-level understanding of the cellular systems, it is now expected that new advances in recombinant bioprocesses are achieved. By providing means to deal with these systems, our understanding on the metabolic behaviour of recombinant cells will advance and can be further explored to the design of suitable hosts and more efficient and cost-effective bioprocesses. Here, we review the major metabolic responses associated with recombinant processes and the engineering strategies relevant to overcome these stresses. Moreover, the advantages of applying systems levels engineering strategies to enhance recombinant protein production in E. coli cells are discussed and future perspectives on the advances of mathematical modelling approaches to study these systems are exposed.
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