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.
"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). "
"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). "
[Show abstract][Hide abstract] ABSTRACT: A new cost-effective metabolism providing an ATP-regeneration system for cell-free protein synthesis is presented. Hexametaphosphate, a polyphosphate molecule, is used as phosphate donor together with maltodextrin, a polysaccharide used as carbon source to stimulate glycolysis. Remarkably, addition of enzymes is not required for this metabolism, which is carried out by endogenous catalysts present in the Escherichia coli crude extract. This new ATP regeneration system allows efficient recycling of inorganic phosphate, a strong inhibitor of protein synthesis. We show that up to 1.34-1.65 mg/mL of active reporter protein is synthesized in batch-mode reaction after 5 h of incubation. Unlike typical hybrid in vitro protein synthesis systems based on bacteriophage transcription, expression is carried out through E. colt promoters using only the endogenous transcription-translation molecular machineries provided by the extract. We demonstrate that traditional expensive energy regeneration systems, such as creatine phosphate, phosphoenolpyruvate or phosphoglycerate, can be replaced by a cost-effective metabolic scheme suitable for cell-free protein synthesis applications. Our work also shows that cell-free systems are useful platforms for metabolic engineering.
"In vitro metabolic engineering or cell-free metabolic engineering has been used to understand complicated cellular metabolisms (Hodgman and Jewett, 2012; Jung and Stephanopoulos, 2004; Zhang, 2010). Recently, it is under investigation for its manufacturing potentials (Hodgman and Jewett, 2012; Rollin et al., 2013; Swartz, 2013), such as the synthesis of special proteins (Goerke et al., 2008; Hodgman and Jewett, 2012) and high-value polysaccharides ( "
[Show abstract][Hide abstract] ABSTRACT: Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to15 enzymes under mild reaction conditions (i.e. 37°C and 1atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12H2 per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmole of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.
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