Abstract
Abstract Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64 ± 0.35 μg mL-1 active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.
Original language | English (US) |
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Article number | 37201 |
Pages (from-to) | 1723-1727 |
Number of pages | 5 |
Journal | FEBS Letters |
Volume | 589 |
Issue number | 15 |
DOIs | |
State | Published - Jul 3 2015 |
Funding
We acknowledge Northwestern University and the DARPA Biomedicines on Demand program (N66001-13-C-4024) for support. M.C.J. is a Packard Fellow for Science and Engineering. J.C.S. is supported by the Northwestern Biotechnology Training Program ( NIH T32GM008449 ) and the Clare Boothe Luce Graduate Fellowship . The authors thank Jennifer Kay for performing and developing HPLC methods.
Keywords
- Cell-free biology
- Cell-free protein synthesis
- In vitro transcription and translation
- Natural energy metabolism
- Protein expression
- Saccharomyces cerevisiae
ASJC Scopus subject areas
- Biophysics
- Structural Biology
- Biochemistry
- Molecular Biology
- Genetics
- Cell Biology