Cell-Free Metabolic Engineering Promotes High-Level Production of Bioactive Gaussia Princeps Luciferase

Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA.
Metabolic Engineering (Impact Factor: 6.77). 05/2008; 10(3-4):187-200. DOI: 10.1016/j.ymben.2008.04.001
Source: PubMed


Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.

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    • "We also tested the expression efficiency of Gaussia luciferase (Gluc) in BL21 cells. Gluc contains five pairs of disulfide bonds, which are essential for its full luminescent activity (Goerke et al., 2008). The results showed that co-expression of Q6-PDI fusion significantly enhanced the Gluc activity (Supplementary Fig. S10A). "
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    • "Cell-free protein synthesis platforms are open systems that provide direct access to complex biochemical networks, and controlled variations of the system's parameters. This type of approach allows quantitative characterization of biological networks and prototyping of metabolic pathways for production of valuable compounds (Goerke et al., 2008; Hodgman and Jewett, 2012; Krutsakorn et al., 2013; Liu et al., 2014). "
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