Cell-free expression system having novel inorganic polyphosphate-based energy regeneration
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.
1. 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.
2. The cell-free expression system of claim 1 wherein said bacterial cell extract comprises a thermophilic bacterial cell extract or a thermostable bacterial cell extract.
3. The cell-free expression system of claim 2 wherein said thermophilic bacterial cell extract or said thermostable bacterial cell extract is a Geobacillus cell extract.
4. The cell-free expression system of claim 3 wherein said Geobacillus bacteria from which the cell extract is obtained is genetically modified for at least one of the following:
(a) knock-out expression of OmpT-homologue;
(b) knock-out expression of RNaseI;
(c) knock-out expression of DNA-methylation dependent DNase;
(d) reduce expression of culture density-dependent sporulation operon;
(e) overexpress sigma factor RpoD; or
(f) overexpress RNA polymerase (RNAP).
5. The cell-free expression system of claim 4 wherein said genetically modified Geobacillus comprises a genetically modified strain of Geobacillus stearothermophilus.
6. The cell-free expression system of claim 4 wherein said cell-free reaction components are selected from the group consisting of: amino acids; polyphosphate; Tris-Acetate; Mg(OAc) 2 ; K + -glutamate; amino-acetate; NaCl; KCl; MgCl 2 ; DTT; octyl-b-glycoside; NAD; NADP; sorbitol; FADH; ATP; GTP, UTP, CTP; CoA; PLP; and SAM.
7. The cell-free expression system of claim 4 wherein said cell-free reaction components are selected from the group consisting of: 2 mM of each natural amino acid; 1 mg/ml polyphosphate; 5 mM Tris-Acetate; 4 mM Mg(OAc) 2 ; 12 mM K + -glutamate; 1 mM amino-acetate; 100 mM NaCl; 10 mM KCl; 5 mM MgCl 2 ; 0.1 mM DTT; 0.2% octyl-b-glycoside; 0.8 mM NAD; 0.4 mM NADP; 200 mM sorbitol; 0.5 mM FADH; 1.5 mM ATP; 1 mM GTP, UTP, CTP each; 1 mM CoA; 2 mM PLP; and 0.2 mM SAM.
8. The cell-free expression system of claim 1 further comprising a quantity of thermostable RNA polymerase (RNAP).
9. The cell-free expression system of claim 8 wherein said quantity of thermostable RNAP comprises a quantity of RNAP isolated from G. stearothermophilus (Gst RNAP).
10. The cell-free expression system of claim 9 wherein said Gst RNAP isolated from G. stearothermophilus comprises RNAP subunits:
(a) subunit alpha having SEQ ID NO: 1;
(b) subunit beta′ having SEQ ID NO: 2;
(c) subunit beta′ having SEQ ID NO: 3 or subunit beta′ having SEQ ID NO: 4;
(d) subunit delta having SEQ ID NO: 5; and
(e) subunit omega having SEQ ID NO: 6.
11. The cell-free expression system of claim 3 further comprising a quantity of tRNAs isolated from a Geobacillus.
12. The cell-free expression system of claim 11 further comprising a supplemental quantity of tRNAs that correspond to one or more rare codons.
13. The cell-free expression system of claim 12 wherein said supplemental quantity of tRNAs that correspond to one or more rare codons are isolated from E. coli.
14. The cell-free expression system of claim 12 further comprising a quantity of Sorbitol-dehydrogenase (SDH).
15. The cell-free expression system of claim 14 wherein said SDH comprises a recombinant SDH isolated from Geobacillus stearothermophilus (Gst SDH).
16. The cell-free expression system of claim 15 further comprising a quantity of sorbitol.
17. The cell-free expression system of claim 1 wherein said at least one nucleic acid synthesis template comprises at least one linear DNA template.
18. The cell-free expression system of claim 17 wherein said linear DNA template comprises a codon optimized linear DNA template.
19. The cell-free expression system of claim 18 wherein said linear DNA template comprises:
(a) at least one target expression product gene operably linked to a promoter;
(b) at least one ribosome binding site (RBS);
(c) at least one expression product cleavage site; and
(d) at least one tag.
20. The cell-free expression system of claim 19 wherein said promotor comprises either a RhIII promotor according to SEQ ID NO: 18, or a T7 promotor according to SEQ ID NO: 19.
21. The cell-free expression system of claim 19 wherein said at least one target expression product gene operably linked to a promoter comprises a tentoxilysin (TetNT) gene operably linked to a promoter.
22. The cell-free expression system of claim 21 wherein said TetNT gene comprises a recombinant first construct coding for a TetNT light chain according to the amino acid sequence SEQ ID NO: 12, with an autocatalytic protease site according to the amino acid sequence SEQ ID NO: 13, and disulfide bridge forming cysteines, and a second construct coding for a TetNT heavy chain according to the amino acid sequence SEQ ID NO: 14, containing a TEV site according to the amino acid sequence SEQ ID NO. 15, and mCherry-His6 according to the amino acid sequence SEQ ID NO: 16.
23. The cell-free expression system of claim 21 wherein said TetNT gene comprises a recombinant TetNT gene identified in SEQ ID NO: 21.
24. The cell-free expression system of claim 19 wherein said RBS comprises an RBS identified in SEQ ID NO: 20.
25. The cell-free expression system of claim 19 wherein said expression product cleavage site comprises a TEV site identified in SEQ ID NO: 15.
26. The cell-free expression system of claim 19 wherein said a target expression gene encodes an amino acid product identified as SEQ ID NO: 17.
27. The cell-free expression system of claim 1 wherein said quantity of purified AdK enzyme comprises a quantity of purified thermostable AdK enzyme.
28. The cell-free expression system of claim 27 wherein said quantity of purified thermostable AdK enzyme comprises a quantity of purified thermostable AdK enzyme isolated from G. stearothermophilus (Gst Adk).
29. The cell-free expression system of claim 1 wherein said quantity of purified PPK enzyme comprises a quantity of purified thermostable PPK enzyme.
30. The cell-free expression system of claim 29 wherein said quantity of purified thermostable PPK enzyme comprises a quantity of purified thermostable PPK enzyme isolated from Thermus aquaticus (TaqPPK).
31. The cell-free expression system of claim 30 wherein said quantity of purified AdK enzyme and said quantity of purified PPK enzyme comprises a quantity of purified Gst AdK enzyme identified as SEQ ID NO: 8, and a quantity of purified Taq PPK enzyme identified as SEQ ID NO: 11.
32. The cell-free expression system of claim 31 wherein said quantity of Gst Adk enzyme is greater than said quantity of TaqPPK enzyme.
33. The cell-free expression system of claim 32 wherein the molar ratio of Gst Adk:TaqPPK is 3:1.
34. The cell-free expression system of claim 1 wherein said quantity of PPi maintains the equilibrium of the ATP regeneration reaction.
35. The cell-free expression system of claim 34 wherein said quantity of PPi comprises a concentration range that maintains the equilibrium of the ATP regeneration reaction comprises a concentration range of 0.2-2 mg/ml PPi in a 100 μl volume ATP regenerating reaction.
36. The cell-free expression system of claim 1 wherein the reaction mixture is added to said cell-free expression system as batch, continuous flow, or semi-continuous flow.
37. The cell-free expression system of claim 1 wherein said cell-free reaction components are added to said cell-free expression system as batch, continuous flow, or semi-continuous flow.
38. 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).
39. The inorganic polyphosphate-based energy regeneration system of claim 38 wherein said quantity of AdK enzyme comprises a quantity of AdK enzyme isolated from G. stearothermophilus (Gst Adk).
40. The inorganic polyphosphate-based energy regeneration system of claim 39 wherein said quantity of PPK enzyme comprises a quantity of purified thermostable isolated from Thermus aquaticus (TaqPPK).
41. The inorganic polyphosphate-based energy regeneration system of claim 40 wherein the quantity of Gst Adk enzyme is greater than the quantity of TaqPPK enzyme.
42. The inorganic polyphosphate-based energy regeneration system of claim 41 wherein the molar ratio of Gst Adk:TaqPPK enzyme is 3:1.
43. 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.
44. The cell-free reaction mixture of claim 43 wherein said quantity of RNAP comprises a quantity of thermostable RNAP isolated from G. stearothermophilus (Gst RNAP).
45. 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.