Granted Patent B2

US 12,227,778 · App. 17/039,575 · Granted Feb 18, 2025

Inventors: Alexander Koglin (Santa Fe, NM); Michael Humbert (Santa Fe, NM)

Assignee: NATURE'S TOOLBOX, INC.

C12N9/52C12N9/0006C12N9/1205C12N9/1229C12N9/1247C12Y101/01014C12Y207/0102C12Y207/04001C12Y207/07006C12Y304/24068

Abstract

Claims (90)

1. 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.

2. The system of claim 1 , wherein said isolated 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.

3. The system of claim 1 , wherein said isolated 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.

4. The system of claim 1 , further comprising purified thermostable RNA polymerase (RNAP).

5. The system of claim 4 , wherein RNAP comprises RNAP isolated from Geobacillus stearothermophilus (Gst RNAP).

6. The system of claim 5 , wherein said Gst RNAP comprises RNAP subunits:

subunit alpha according to SEQ ID NO: 1;

subunit beta according to SEQ ID NO: 2;

subunit beta′ according to SEQ ID NO: 3 or subunit beta′ according to SEQ ID NO: 4;

subunit delta according to SEQ ID NO: 5; and

subunit omega according to SEQ ID NO: 6.

7. The system of claim 1 , further comprising tRNAs isolated from Geobacillus.

8. The system of claim 1 , further comprising tRNAs isolated from E. coli mre600.

9. The system of claim 1 , further comprising a quantity of isolated Sorbitol-dehydrogenase (SDH).

10. The system of claim 9 , wherein said SDH is isolated from Geobacillus stearothermophilus (Gst SDH).

11. The system of claim 10 , further comprising a quantity of isolated sorbitol.

12. The system of claim 1 , wherein said macromolecule comprises a polypeptide.

13. The system of claim 1 , wherein said at least one isolated nucleic acid synthesis template comprises at least one isolated linear DNA template.

14. The system of claim 13 , wherein said at least one isolated nucleic acid synthesis template comprises at least one isolated codon optimized nucleic acid synthesis template.

15. The system of claim 1 , wherein said at least one isolated nucleic acid synthesis template comprises at least one isolated nucleic acid synthesis template having:

at least one target expression product gene operably linked to a promoter;

at least one ribosome binding site (RBS);

at least one expression product cleavage site; and

at least one tag.

16. The system of claim 15 , wherein said promoter comprises either an RhIII promoter according to SEQ ID NO: 18, or a T7 promoter according to SEQ ID NO: 19.

17. The system of claim 15 , wherein said at least one target expression product gene operably linked to a promoter comprises a tentoxilysin (TetNT) gene operably linked to a promoter.

18. The system of claim 17 , wherein said TetNT gene comprises a recombinant a first construct coding for a TetNT light chain according to SEQ ID NO: 12, with an autocatalytic protease site according to SEQ ID NO: 13 and disulfide bridge forming cysteines, and a second construct coding for a TetNT heavy chain according to SEQ ID NO: 14 and containing a partially integrated TEV site according to SEQ ID NO: 15, and mCherry-His6 according to SEQ ID NO: 16.

19. The system of claim 17 , wherein said TetNT gene comprises a recombinant TetNT gene according to SEQ ID NO: 21.

20. The system of claim 15 , wherein said RBS comprises an RBS according to SEQ ID NO: 20.

21. The system of claim 15 , wherein said at least one expression product cleavage site comprises a TEV site according to SEQ ID NO: 15.

22. The system of claim 15 , wherein said at least one target expression product is a peptide according to SEQ ID NO: 17.

23. The system of claim 1 , wherein said purified AdK enzyme comprises a purified thermostable AdK enzyme, or fragment thereof.

24. The system of claim 23 , wherein said purified thermostable AdK enzyme comprises purified thermostable Geobacillus stearothermophilus AdK enzyme (Gst Adk), or fragment thereof.

25. The system of claim 1 , wherein said purified PPK enzyme comprises purified thermostable PPK enzyme, or fragment thereof.

26. The system of claim 25 , wherein said purified thermostable PPK enzyme comprises purified thermostable Thermus aquaticus PPK enzyme (TaqPPK), or fragment thereof.

27. The system of claim 1 , wherein the quantity of purified Gst Adk enzyme is greater than the quantity of purified TaqPPK enzyme.

28. The system of claim 27 , wherein the molar ratio of purified Gst Adk:purified TaqPPK is 3:1.

29. The system of claim 1 , wherein said isolated PPi comprises a PPi concentration range that maintains the equilibrium of the ATP regeneration reaction.

30. The system of claim 29 , wherein said PPi 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.

Assignments (4)

Continuity (3)

References Cited (43)