Cell-free expression system having novel inorganic polyphosphate-based energy regeneration
AssigneeNTxBio, LLC
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.
Claims45
A cell-free expression system comprising:
- (A) a reaction mixture containing
- (a) a bacterial cell extract having all cell-free reaction components necessary for in vitro polypeptide and nucleotide synthesis;
- (b) at least one nucleic acid synthesis template;
- (c) a cellular adenosine triphosphate (ATP) energy regeneration system comprising:
- (i) a quantity of purified Adenosyl Kinase (AdK) enzyme according to SEQ ID NO:
- 8;
- and
- (ii) a quantity of purified Polyphosphate Kinase (PPK) enzyme according to SEQ ID NO:
- 11, wherein the quantity of AdK is higher than the quantity of PPK;
- (iii) a supplemental quantity of isolated inorganic polyphosphate (PPi);
- and
- (iv) a supplemental quantity of isolated adenosine monophosphate (AMP);
- (d) wherein said AdK and PPK enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP.
An inorganic polyphosphate-based energy regeneration system comprising:
- (a) a quantity of purified Adenosyl Kinase (AdK) enzyme according to SEQ ID NO:
- 8;
- and
- (b) a quantity of purified Polyphosphate Kinase (PPK) enzyme according to SEQ ID NO:
- 11, wherein the quantity of AdK is higher than the quantity of PPK;
- (c) wherein said AdK and PPK enzymes work synergistically to regenerate adenosine triphosphate (ATP) energy in a reaction requiring ATP energy from inorganic polyphosphate (PPi) and adenosine monophosphate (AMP).
A cell-free reaction mixture comprising:
- (a) a bacterial cell extract having all cell-free reaction components necessary for in vitro polypeptide synthesis;
- (b) a nucleic acid synthesis template;
- (c) a quantity of RNA polymerase (RNAP) isolated from G. stearothermophilus (Gst RNAP) having the following subunits:
- (i) subunit alpha having SEQ ID NO:
- 1;
- (ii) subunit beta′ having SEQ ID NO:
- 2;
- (iii) subunit beta′ having SEQ ID NO:
- 3, or subunit beta′ having SEQ ID NO:
- 4;
- (iv) subunit delta having SEQ ID NO:
- 5;
- and
- (v) subunit omega having SEQ ID NO:
- 6.
A cell-free expression system for producing a tentoxilysin comprising:
- (a) a bacterial cell extract having all cell-free reaction components necessary for in vitro polypeptide synthesis;
- (b) at least one nucleic acid synthesis template encoding an amino acid product according to SEQ ID NO:
- 17.
Dependencies are inferred from the claim text.