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US 12,227,778 B2Granted
Cell-free expression system having novel inorganic polyphosphate-based energy regeneration
AssigneeNature's Toolbox, Inc.
InventorsAlexander Koglin, Michael Humbert
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Abstract
The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.
Claims30
A cell-free protein expression system comprising:
- a bioreactor having:
- a reaction mixture containing isolated cell-free reaction components necessary for in vitro macromolecule synthesis;
- at least one isolated nucleic acid synthesis template;
- and
- a cellular adenosine triphosphate (ATP) energy regeneration system comprising:
- purified Adenosyl Kinase (AdK) enzyme according to SEQ ID NO:
- 8, or fragment thereof, or an Adk enzyme having a sequence having at least 95% sequence homology with SEQ ID NO:
- 8, that catalyzes the conversion of adenosine diphosphate into adenosine triphosphate (ATP) and adenosine monophosphate (AMP);
- purified Polyphosphate Kinase (PPK) enzyme according to SEQ ID NO:
- 11, or fragment thereof, or a PPK enzyme having a sequence having at least 95% sequence homology with SEQ ID NO:
- 11, that catalyzes the reversible formation of ATP from ADP and inorganic polyphosphate (PPi);
- isolated PPi;
- and
- isolated adenosine monophosphate (AMP);
- wherein said AdK and PPK enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP.
12 more elements
Dependencies are inferred from the claim text.