Energizing eukaryotic cell-free protein synthesis with glucose metabolism

Mark J. Anderson; Jessica C. Stark; C. Eric Hodgman; Michael C. Jewett

doi:10.1016/j.febslet.2015.05.045

Energizing eukaryotic cell-free protein synthesis with glucose metabolism

Mark J. Anderson, Jessica C. Stark, C. Eric Hodgman, Michael C. Jewett^*

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

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)
Article number	37201
Pages (from-to)	1723-1727
Number of pages	5
Journal	FEBS Letters
Volume	589
Issue number	15
DOIs	https://doi.org/10.1016/j.febslet.2015.05.045
State	Published - Jul 3 2015

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

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title = "Energizing eukaryotic cell-free protein synthesis with glucose metabolism",

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.",

keywords = "Cell-free biology, Cell-free protein synthesis, In vitro transcription and translation, Natural energy metabolism, Protein expression, Saccharomyces cerevisiae",

author = "Anderson, {Mark J.} and Stark, {Jessica C.} and Hodgman, {C. Eric} and Jewett, {Michael C.}",

note = "Funding Information: 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. Publisher Copyright: {\textcopyright} 2015 Federation of European Biochemical Societies.",

year = "2015",

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doi = "10.1016/j.febslet.2015.05.045",

language = "English (US)",

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T1 - Energizing eukaryotic cell-free protein synthesis with glucose metabolism

AU - Anderson, Mark J.

AU - Stark, Jessica C.

AU - Hodgman, C. Eric

AU - Jewett, Michael C.

N1 - Funding Information: 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. Publisher Copyright: © 2015 Federation of European Biochemical Societies.

PY - 2015/7/3

Y1 - 2015/7/3

N2 - 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.

AB - 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.

KW - Cell-free biology

KW - Cell-free protein synthesis

KW - In vitro transcription and translation

KW - Natural energy metabolism

KW - Protein expression

KW - Saccharomyces cerevisiae

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JF - FEBS Letters

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IS - 15

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ER -

Energizing eukaryotic cell-free protein synthesis with glucose metabolism

Abstract

Keywords

ASJC Scopus subject areas

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