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: 8.26). 05/2008; 10(3-4):187-200. DOI: 10.1016/j.ymben.2008.04.001
Source: PubMed

ABSTRACT 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.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: The proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins (GPs). Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B'/B followed by the herein newly identified C'/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other PCs, all immature intermediates of SKI-1/S1P showed full catalytic activity towards cellular substrates, whereas optimal cleavage of viral GPs depended on B'/B processing. Immature forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1-P8) and P1' are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity towards cellular and pathogen-derived substrates.
    Journal of Biological Chemistry 11/2014; 289(52). DOI:10.1074/jbc.M114.588525 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In present study, CdSe quantum dots (QDs) were prepared with a novel but simple, effective and exercisable method. Nine different types of carbohydrate molecules were used to modify CdSe QDs. D-mannose (Man)-coated quantum dots were prepared for labeling human hepatoma (HepG2) cells, because of the high expression of mannose receptor (MR) on HepG2 cells. The uptake characteristics of CdSe QDs-Man were investigated in HepG2 cells. The absorption rate result of MTT assay in 48 h suggested the extremely low cytotoxicity of CdSe QDs-Man. The presence of quantum dots was confirmed with fluorescence microscopy. These results were encouraging regarding the application of QDs molecules for early detection of HepG2 cells.
    Bulletin- Korean Chemical Society 02/2012; 33(2). DOI:10.5012/bkcs.2012.33.2.571 · 0.84 Impact Factor
  • Source
    [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.
    Metabolic Engineering 10/2014; 27. DOI:10.1016/j.ymben.2014.10.007 · 8.26 Impact Factor