TY - JOUR

T1 - A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis

AU - Wang, Po-Hsiang

AU - Fujishima, Kosuke

AU - Berhanu, Samuel

AU - Kuruma, Yutetsu

AU - Jia, Tony Z

AU - Khusnutdinova, Anna N

AU - Yakunin, Alexander F

AU - McGlynn, Shawn E

PY - 2020/1/17

Y1 - 2020/1/17

N2 - Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 μg/mL) beyond that of the three-kinase system (∼400 μg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

AB - Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 μg/mL) beyond that of the three-kinase system (∼400 μg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

KW - Adenosine Triphosphate/metabolism

KW - Amino Acyl-tRNA Synthetases/metabolism

KW - Animals

KW - Cell-Free System/metabolism

KW - Cytophaga/enzymology

KW - Fireflies/enzymology

KW - Green Fluorescent Proteins/metabolism

KW - Guanosine Triphosphate/metabolism

KW - Luciferases, Firefly/metabolism

KW - Phosphorylation

KW - Phosphotransferases (Phosphate Group Acceptor)/metabolism

KW - Polyphosphates/metabolism

KW - Protein Biosynthesis

KW - RNA, Messenger/metabolism

KW - RNA, Transfer, Amino Acid-Specific/metabolism

U2 - 10.1021/acssynbio.9b00456

DO - 10.1021/acssynbio.9b00456

M3 - Article

C2 - 31829622

VL - 9

SP - 36

EP - 42

JO - ACS synthetic biology

JF - ACS synthetic biology

SN - 2161-5063

IS - 1

ER -