Genetically encoded system for constructing and detecting biologically active agents
This invention relates to the field of genetic engineering. Specifically, the invention relates to the construction of operons to produce biologically active agents. For example, operons may be constructed to produce agents that control the function of biochemical pathway proteins (e.g., protein phosphatases, kinases and/or proteases). Such agents may include inhibitors and modulators that may be used in studying or controlling phosphatase function associated with abnormalities in a phosphatase pathway or expression level. Fusion proteins, such as light activated protein phosphatases, may be genetically encoded and expressed as photoswitchable phosphatases. Systems are provided for use in controlling phosphatase function within living cells or in identifying small molecule inhibitors/activator/modulator molecules of protein phosphatases associated with cell signaling.
1. A method of using a genetically encoded detection system, said method comprising:
a. providing:
i. an inhibitor detection system comprising a first region of DNA and a second region of DNA, wherein:
(A) the first region of DNA encodes:
1. a first promoter;
2. a first fusion protein comprising a substrate recognition domain linked to a DNA-binding protein;
3. a second fusion protein comprising a substrate domain linked to a protein capable of recruiting RNA polymerase to DNA;
4. a second promoter;
5. a protein kinase;
6. a molecular chaperone; and
7. a protein phosphatase; and
(B) the second region of DNA encodes:
8. a third promoter;
9. an operator for said DNA-binding protein;
10. a binding site for said RNA polymerase; and
11. a reporter protein; and
ii. an isoprenoid pathway DNA sequence encoding one or more components of an isoprenoid pathway under control of a fourth promoter;
iii. a synthase DNA sequence encoding a synthase enzyme; and
iv. a plurality of cells;
b. introducing into each cell of said plurality of cells said inhibitor detection system under conditions sufficient to express said reporter protein;
c. introducing into each cell of said plurality of cells said isoprenoid pathway DNA sequence under conditions sufficient to express said one or more components of said isoprenoid pathway;
d. introducing into each cell of said plurality of cells said synthase DNA sequence under conditions sufficient to express said synthase enzyme; and
e. identifying a cell of said plurality of cells expressing said reporter protein, wherein expression of said reporter protein in said cell is indicative that said cell comprises an inhibitor of said protein phosphatase that is synthesized by said synthase enzyme.
2. The method of claim 1 , further comprising:
f. isolating said inhibitor of said protein phosphatase; and
g. treating a mammalian cell culture with said inhibitor for reducing activity of said protein phosphatase.
3. The method of claim 2 , wherein said reducing said activity of said protein phosphatase reduces growth of mammalian cells of said mammalian cell culture.
4. The method of claim 1 , wherein said protein phosphatase is human protein tyrosine phosphatase 1B (PTP1B).
5. The method of claim 1 , wherein said protein phosphatase is a wild-type form of said protein phosphatase.
6. The method of claim 1 , wherein said protein phosphatase has at least one mutation.
7. The method of claim 1 , wherein said one or more components of said isoprenoid pathway comprises mevalonate kinase (ERG12), phosphomevalonate kinase (ERGS), mevalonate pyrophosphate decarboxylatse (MVD 1), Isopentenyl pyrophosphate isomerase (IDI gene), or Farnesyl pyrophosphate (FPP) synthase (ispA), or any combination thereof.
8. The method of claim 1 , wherein said synthase enzyme comprises a terpene synthase.
9. The method of claim 8 , wherein said terpene synthase comprises amorphadiene synthase (ADS) or y-humulene synthase (GHS).
10. The method of claim 8 , wherein said synthase DNA sequence further encodes geranylgeranyl diphosphate synthase (GPPS) and said terpene synthase comprises abietadiene synthase (ABS) or taxadiene synthase (TXS), or any combination thereof.
11. The method of claim 8 , wherein said terpene synthase is a wild-type form of said terpene synthase.
12. The method of claim 8 , wherein said terpene synthase has at least one mutation.
13. The method of claim 12 , wherein said inhibitor comprises a terpenoid or a structural variant of said terpenoid.
14. The method of claim 1 , wherein said reporter protein confers antibiotic resistance to said cell.
15. The method of claim 14 , wherein said reporter protein confers antibiotic resistance to said cell to two different antibiotics.
16. The method of claim 1 , wherein the fourth promoter is an inducible promoter, and wherein said method further comprises introducing into each cell of said plurality of cells an induction molecule for said inducible promoter under conditions sufficient to contact said inducible promoter with said induction molecule.
17. The method of claim 1 , wherein said plurality of cells comprises microbial cells.
18. The method of claim 17 , wherein said microbial cells comprise Escherichia coli ( E. coli ) cells.
19. The method of claim 1 , further comprising isolating said inhibitor of said protein phosphatase from said cell.
20. The method of claim 1 , wherein said isoprenoid pathway is a mevalonate pathway.