ENGINEERED PROTEINS WITH A PROTEASE CLEAVAGE SITE

• US 20150037868A1
• Filed: 08/05/2014
• Published: 02/05/2015
• Est. Priority Date: 08/05/2013
• Status: Active Grant

• Current Assignee: GreenLight Biosciences, Inc.
• Original Assignee: GreenLight Biosciences, Inc.
• Inventors: Blake, William Jeremy, Cunningham, Drew S.
• Granted Patent: US 9,688,977 B2
• Time in Patent Office: Days
• US Class Current: 435/193
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12Y 202/01001 Transketolase (2.2.1.1)
• CPC Class Codes: C12Y 203/01008 Phosphate acetyltransferase (2.3.1.8)
• CPC Class Codes: C12Y 503/01009 Glucose-6-phosphate isomerase (5.3.1.9)

Abstract

Various aspects and embodiments herein relate to recombinant proteins with at least one protease recognition sequence that can be inactivated by a cognate protease and methods of preparing such proteins. In some embodiments, recombinant phosphoglucose isomerase (Pgi) proteins are provided. In other embodiments, recombinant phosphotransacetylase (Pta) proteins are provided. In yet other embodiments, recombinant transketolase A (TktA) proteins are provided.

Claims

1. A recombinant phosphoglucose isomerase protein with a protease recognition sequence located between two amino acids of the wild-type protein.

2. The recombinant protein of claim 1, wherein the protease recognition sequence is the protease recognition sequence recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E, picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, and Xaa-pro aminopeptidase.

4. The recombinant protein of claim 2, wherein the protease recognition sequence is the human rhinovirus 3C protease recognition sequence and comprises the sequence of SEQ ID NO:

38.

5. The recombinant protein of claim 1, wherein the protease recognition sequence is located in a C terminal region of the protein and/or in a solvent-exposed loop region of the protein.

7. The recombinant protein of claim 1, wherein the protease recognition sequence is located after amino acid 410, 524, 525, 526, 527, 528, 529, 530, 531, 532, or 545 of the sequence of SEQ ID NO:

17.

9. The recombinant protein of claim 1, wherein the recombinant protein comprises the sequence of SEQ ID NO:

25.

10. A nucleic acid encoding the recombinant protein of claim 1.

11. A vector comprising the nucleic acid of claim 10.

12. The vector of claim 11, wherein the vector is a cloning vector or an expression vector.

13. The vector of claim 12, wherein the vector is a plasmid, a fosmid, a phagemid, a virus genome or an artificial chromosome.

15. A cell comprising the vector of claim 11.

16. The cell of claim 15, wherein the cell is a bacterial cell, a fungal cell, a mammalian cell or a plant cell.

19. A cell that expresses the recombinant protein of claim 1.

20. A lysate of the cell of claim 15.

3. (canceled)

6. (canceled)

8. (canceled)

14. (canceled)

17-18. -18. (canceled)

21-40. -40. (canceled)

41. A recombinant protein with at least one protease recognition sequence located between two amino acids of the wild-type protein in a solvent-exposed loop region of the protein, wherein the protease recognition sequence is cleaved by a cognate protease with single recognition sequence specificity, and wherein the recombinant protein activity in the presence of the cognate protease is reduced by at least 30% of the recombinant protein activity in the absence of the cognate protease.

42. The recombinant protein of claim 41, wherein the recombinant protein is a recombinant phosphoglucose isomerase protein.

43. The recombinant protein of claim 41, wherein the protease recognition sequence is the protease recognition sequence recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria mellea, astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E, picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, Factor X protease, HIV protease, and Xaa-pro aminopeptidase.

44-45. -45. (canceled)

46. A method of engineering a recombinant protein with a protease recognition sequence, the method comprising the steps of:

(a) transforming cells with

a plurality of nucleic acid variants, wherein each nucleic acid variant contains a nucleotide sequence that encodes a recombinant protein with at least one protease recognition sequence located between two codons of the wild-type nucleic acid, and

a nucleic acid with an inducible promoter sequence operably linked to a nucleotide sequence encoding a cognate protease;

(b) culturing the cells under non-inducing conditions on media that prohibits cell growth of inactive recombinant proteins and collecting cells with a growth rate comparable to wild-type control cells; and

(c) culturing the cells under conditions that induce expression of the cognate protease and collecting cells that do not grow or that have a reduced growth rate in comparison to wild-type control cells.

47-64. -64. (canceled)

65. A recombinant phosphotransacetylase protein with a protease recognition sequence located between two amino acids of the wild-type protein.

66-100. -100. (canceled)

101. A recombinant transketolase A protein with a protease recognition sequence located between two amino acids of the wild-type protein.

102-136. -136. (canceled)