Fusion proteins useful for detecting analytes

• US 20070136825A1
• Filed: 09/13/2002
• Published: 06/14/2007
• Est. Priority Date: 09/18/2001
• Status: Abandoned Application

• Current Assignee: Carnegie Institution of Washington
• Original Assignee: Carnegie Institution of Washington
• Inventors: Lalonde, Sylvie, Fehr, Marcus, Frommer, Wolf-Bernd
• Application Number:
  US10/490,153
• Publication Number:
  US 20070136825A1
• Time in Patent Office: Days
• US Class Current: 800/3
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Disclosed are fusion proteins comprising two detection portions and a periplasmic binding protein (PBP) portion between this two detection portions which undergoes a conformational change upon binding of a compound. Also described are nucleic acid molecules comprising a nucleotide sequence encoding such a fusion protein as well as expression cassettes, vectors, transgenic plants and transgenic non-human animals comprising said nucleic acid molecules. In addition, described are methods for detecting an analyte in a sample or in a cell using said fusion protein, and optionally, a control sensor allowing a calibration of analyte detection made by said fusion protein. Also described are such control sensors and nucleic acid molecules encoding them, as well as diagnostic compositions, kits and uses of the fusion protein and the control sensor for in vitro or in vivo analyte detection and preparing a diagnostic composition.

Claims

1. A fusion protein comprising:

(a) two detection portions, wherein (i) the first detection portion is an energy-emitting protein portion and the second detection portion is a fluorescent protein portion;

or (ii) the two detection portions are portions of a split fluorescent protein; and

(b) a periplasmic binding protein (PBP) portion between said two detection portions, which undergoes a conformational change upon binding of a compound;

wherein said conformational change results in a change of the energy emitted by said two detection portions.

2. The fusion protein of claim 1, wherein the compound is selected from the group consisting of sugars, amino acids, peptides, organic acids, metals or ions, oxides, hydroxides or conjugates thereof, inorganic ions, (poly) amines and vitamins.

3. The fusion protein of claim 1 further comprising one or more linker peptides connecting the first and/or the second detection portion with the PBP portion.

4. The fusion protein of claim 1, wherein the PBP portion, the first and/or the second detection portion is truncated compared to the corresponding wild-type PBP portion, first and/or second detection portion.

5. The fusion protein of claim 1, wherein the first detection portion is a fluorescent protein portion.

6. The fusion protein of claim 1, wherein the change of the energy emitted is an increase or decrease of fluorescence resonance energy transfer (FRET).

7. The fusion protein of claim 6, wherein the first detection portion is enhanced cyan fluorescent protein (ECFP) and the second detection portion is enhanced yellow fluorescent protein (EYFP).

8. The fusion protein of claim 1, wherein the change of the energy emitted is an increase or decrease of bioluminescence resonance energy transfer (BRET).

9. The fusion protein of claim 1 further comprising a targeting signal sequence.

10. The fusion protein of claim 1, wherein said PBP portion is modified with respect to the corresponding wild-type PBP so that the binding affinity to the compound of said PBP portion is altered compared to the wild type PBP.

11. The fusion protein of claim 1, wherein said PBP has approximately the same binding affinity to the compound as the corresponding wild-type PBP.

12. The fusion protein of claim 1, wherein the PBP portion is a glucose/galactose binding protein (GGBP) in which the phenylalanine residue at a position corresponding to position 16 of a wild-type GGBP polypeptide as represented by SEQ ID NO:

4 is replaced by an alanine residue.

13. The fusion protein of claim 1, wherein the PBP portion is a GGBP in which at least one histidine residue is modified so that the side chain of said residue cannot be protonated.

14. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of claim 1.

15. An expression cassette comprising the nucleic acid molecule of claim 14 and operably linked thereto control sequences allowing expression in prokaryotic or eukaryotic cells.

16. A vector comprising the nucleic acid molecule of claim 14 or the expression cassette of claim 15.

17. The vector of claim 16, wherein the nucleic acid molecule is operably linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.

18. A method for producing cells capable of expressing the fusion protein of claim 1 comprising genetically engineering cells with the nucleic acid molecule of claim 14.

19. A host cell genetically engineered with the nucleic acid molecule of claim 14.

20. The host cell of claim 19 which is a bacterial, fungus, insect, plant or animal cell.

21. A method for producing the fusion protein of claim 1 comprising culturing the host cell of claim 19 under conditions allowing the expression of said fusion protein;

and recovering said fusion protein from the culture.

22. A fusion protein obtainable by the method of claim 21.

23. A transgenic plant or plant tissue comprising the plant cell of claim 20.

24. Harvestable parts of the transgenic plant of claim 23.

25. Propagation material of the transgenic plant of claim 23.

26. A transgenic non-human animal comprising the nucleic acid molecule of claim 14.

27. The transgenic non-human animal of claim 26 which is a mouse, a rat or a zebra fish.

28. A method for detecting an analyte comprising:

(a) contacting a sample with a fusion protein of claim 1, wherein the PBP portion of said fusion protein is capable of binding said analyte;

(b) supplying the fusion protein with energy suitable for exciting energy emission by said first and/or second detection portion; and

(c) measuring the energy emission.

29. The method of claim 28 additionally comprising:

(i) contacting a sample corresponding to the sample in step (a) with a control sensor, whereby said control sensor corresponds to the fusion protein used in step (a) with the exception that the PBP portion is modified and therefore incapable of binding said analyte ;

(ii) supplying the control sensor with the same energy as mentioned in (b);

(iii) measuring the energy emission; and

(iv) calibrating the energy emission measurement of step (c) with the measurement of step (iii).

30. A method for detecting an analyte in a cell comprising:

(a) supplying a cell which is genetically engineered with the nucleic acid molecule of claim 14, wherein the PBP portion of said fusion protein is capable of binding said analyte, with energy suitable for exciting energy emission by said first and/or second detection portion; and

(b) measuring the energy emission.

31. The method of claim 30 additionally comprising:

(i) supplying a cell corresponding to the cell of step (a), but being genetically engineered with a nucleic acid molecule encoding a control sensor and expressing said control sensor, with the same energy as mentioned in step (a);

whereby said control sensor corresponds to the fusion protein used in step (a) with the exception that the PBP portion is modified and therefore incapable of binding said analyte;

(ii) measuring the energy emission; and

(iii) calibrating the energy emission measurement of step (b) with the measurement of step (ii).

32. A method for detecting an analyte in a cell comprising:

(a) introducing into a cell by means of microinjection the fusion protein of claim 1, wherein the PBP portion of said fusion protein is capable of binding said analyte, or RNA which encodes and is capable of expressing said fusion protein in said cell;

(b) supplying the cell with energy suitable for exciting energy emission by the first and/or second detection portion of said fusion protein, and (c) measuring the energy emission.

33. The method of claim 32 additionally comprising:

(i) introducing into a cell corresponding to the cell used in step (a) by means of microinjection a control sensor or RNA which encodes and is capable of expressing said control sensor, whereby said control sensor corresponds to the fusion protein used in step (a) with the exception that the PBP portion is modified and therefore incapable of binding said analyte ;

(ii) supplying the cell of step (i) with the same energy as mentioned in step (a);

(iii) measuring the energy emission; and

(iv) calibrating the energy emission measurement of step (b) with the measurement of step (iii).

34. A method for the identification of a compound that affects the concentration and/or distribution of an analyte in a cell comprising:

(a) contacting a candidate compound with a cell that expresses a fusion protein of claim 1 which is suitable for detecting said analyte; and

(b) determining whether said contacting leads to a change in the energy emission of said fusion protein.

35. A method for the identification of a gene product involved in one or more enzymatic, transport or regulatory functions in a cell, wherein said fimctions affect the concentration and/or distribution of an analyte in the cell, said method comprising:

(a) providing a cell that expresses a fusion protein of claim 1 which is suitable for detecting said analyte, wherein the activity of a candidate gene product is altered in said cell compared to a corresponding wild-type cell ;

(b) culturing the cell of (a) under conditions that the fusion protein and, if appropriate, the candidate gene product is expressed and active; and

(c) determining whether, in the cell of (b), the energy emission of said fusion protein differs from that of the same fusion protein present in a cell corresponding to the cell of (b) in which the activity of said gene product is not altered.

40. A diagnostic composition comprising the fusion protein of claim 1.

41. A kit comprising the fusion protein of claim 1.

42. Use of the fusion protein of claim 1 for detecting analytes in vivo or in vitro.

43. Use of the fusion protein of claim 1 for preparing a diagnostic composition for diagnosing a condition which is correlated with a concentration of an analyte in cells, tissues or parts of a body.

44. Use of the fusion protein of claim 1 for the identification of a compound that affects the concentration and/or distribution of an analyte in a cell.

45. Use of the fusion protein of claim 1 for the identification of a gene product involved in one or more enzymatic, transport or regulatory functions, wherein said functions affect the concentration and/or distribution of an analyte in the cell.

36. A control sensor comprising:

(a) two detection portions, whereby (i) the first detection portion is an energy-emitting protein portion and the second detection portion is a fluorescent protein potion;

or (ii) the two detection portions are portions of a split fluorescent protein and (b) a PBP portion fused to said first and second detection portion which is modified with respect to the corresponding wild-type PBP portion and therefore is incapable of binding the compound which said wild-type PBP portion binds;

wherein said first and/or second detection portion emits energy.

37. The control sensor of claim 36, wherein the PBP portion is aglucose/galactose binding protein (GGBP) which shows the amino acid residue Ala at a position corresponding to the Asp residue at position 236 of a mature wild-type GGBP as shown in SEQ ID NO:

4.

38. The control sensor of claim 36, wherein the PBP portion is a maltose binding protein (MBP) which shows the amino acid residue Ala at a position corresponding to the Trp residue at position 340 of a mature wild-type MBP as shown in SEQ ID NO:

2.

39. A nucleic acid molecule comprising a nucleotide sequence encoding the control sensor of any one of claims 36 to 38.

46. Use of a control sensor of claim 36, the PBP portion of which is derived from a GGBP, for measuring the pH.