Engineering genes for predictable protein expression

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Protein Expression and Purification (Impact Factor: 1.7). 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, Sep 28, 2015
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    • "Given a target protein, its coding sequence can affect both transcription and translation processes [15, 88]. As described above, mRNA secondary structures could affect mRNA degradation and limit RBS accessibility to ribosomes and, in addition, AT-rich sequences can cause premature transcriptional termination [89]. Codon usage has been reported to affect the translation process [90]. "
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    ABSTRACT: The design process of complex systems in all the fields of engineering requires a set of quantitatively characterized components and a method to predict the output of systems composed by such elements. This strategy relies on the modularity of the used components or the prediction of their context-dependent behaviour, when parts functioning depends on the specific context. Mathematical models usually support the whole process by guiding the selection of parts and by predicting the output of interconnected systems. Such bottom-up design process cannot be trivially adopted for biological systems engineering, since parts function is hard to predict when components are reused in different contexts. This issue and the intrinsic complexity of living systems limit the capability of synthetic biologists to predict the quantitative behaviour of biological systems. The high potential of synthetic biology strongly depends on the capability of mastering this issue. This review discusses the predictability issues of basic biological parts (promoters, ribosome binding sites, coding sequences, transcriptional terminators, and plasmids) when used to engineer simple and complex gene expression systems in Escherichia coli. A comparison between bottom-up and trial-and-error approaches is performed for all the discussed elements and mathematical models supporting the prediction of parts behaviour are illustrated.
    Computational and Mathematical Methods in Medicine 08/2014; 2014:369681. DOI:10.1155/2014/369681 · 0.77 Impact Factor
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    • "Over the past decade, synthetic biology has contributed to significantly reduce the cost of many products manufactured in microbial systems where only one gene needs to be over-expressed. In many cases, the production of a target protein can be boosted by several orders of magnitude by replacing a native sequence with its optimized counterpart (Gustafsson et al., 2004, 2012). This seemingly simple adjustment is of remarkable importance, since many of these products are now traded as commodities and thus there is a continuous need to reduce manufacturing costs in order to remain competitive in the global markets (Menzella, 2011). "
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    ABSTRACT: The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent "silent" nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.
    Frontiers in Microbiology 02/2014; 5:21. DOI:10.3389/fmicb.2014.00021 · 3.99 Impact Factor
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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 · 1.58 Impact Factor
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