A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.
ACS Synth Biol
; 9(1): 36-42, 2020 01 17.
Article
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| MEDLINE
| ID: mdl-31829622
ABSTRACT
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 (â¼530 µg/mL) beyond that of the three-kinase system (â¼400 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.
Palabras clave
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Biosíntesis de Proteínas
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Adenosina Trifosfato
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Fosfotransferasas (Aceptor del Grupo Fosfato)
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Cytophaga
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Guanosina Trifosfato
Límite:
Animals
Idioma:
En
Revista:
ACS Synth Biol
Año:
2020
Tipo del documento:
Article
País de afiliación:
Japón