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Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase

Paper ID Volume ID Publish Year Pages File Format Full-Text
31867 44846 2008 14 PDF Available
Title
Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase
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 412 μg/mL of purified GLuc, relative to 5 μg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2×1024 photons/s/mol, the highest reported activity for any characterized luciferase.

Keywords
Gaussia princeps; Luciferase; Cell-free protein synthesis; In vivo protein expression; Post-translational activation
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Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 10, Issues 3–4, May–July 2008, Pages 187–200
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us