Fusion protein of Fc-binding domain and calcium-binding photoprotein, gene encoding the same and use thereof
The invention provides a calcium-binding photoprotein which has a high ability to bind to IgG and is capable of more sensitively detecting IgG. A fusion protein of the present invention composed of a ZZ domain having the ability to bind to the Fc region of IgG and the calcium-binding photoprotein apoaequorin is used for the detection and assay of calcium ions and the detection of IgG.
1. A fusion protein comprising:
(1) a first region selected from the group of:
(a) a region having the amino acid sequence of SEQ ID NO: 1;
(b) a region having variants of the amino acid sequence of SEQ ID NO: 1, wherein said variants have from one to eight deleted, substituted, inserted and/or added amino acids, and which has the ability to bind to the Fc region of IgG; and
(c) a region which has an amino acid sequence that is at least approximately 94% identical to the amino acid sequence of SEQ ID NO: 1, and which has the ability to bind to the Fc region of IgG; and
(2) a second region selected from the group of:
(d) a region having the amino acid sequence of SEQ ID NO: 3;
(e) a region having variants of the amino acid sequence of SEQ ID NO: 3, wherein said variants have from one to eight deleted, substituted, inserted and/or added amino acids, and which has the ability to bind to a peroxide of coelenterazine or a peroxide of a coelenterazine derivative so as to form a holoprotein that emits light under the action of a calcium ion; and
(f) a region which has an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 3, and which has the ability to bind to a peroxide of coelenterazine or a peroxide of a coelenterazine derivative so as to form a holoprotein that emits light under the action of a calcium ion,
wherein the fusion protein has a minimum IgG-detection sensitivity of 1 ng/ml.
2. The fusion protein of claim 1 , wherein
(1) the first region is selected from the group of:
(a) a region having the amino acid sequence of SEQ ID NO: 1,
(b) a region having variants of the amino acid sequence of SEQ ID NO: 1, wherein said variants have from one to three deleted, substituted, inserted and/or added amino acids, and which has the ability to bind to the Fc region of IgG, and
(c) a region which has an amino acid sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO: 1, and which has the ability to bind to the Fc region of IgG; and
(2) the second region is selected from the group of:
(d) a region having the amino acid sequence of SEQ ID NO: 3,
(e) a region having variants of the amino acid sequence of SEQ ID NO: 3, wherein said variants have from one to four deleted, substituted, inserted and/or added amino acids, and which has the ability to bind to a peroxide of coelenterazine or a peroxide of a coelenterazine derivative so as to form a holoprotein that emits light under the action of a calcium ion, and
(f) a region which has an amino acid sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO: 3, and which has the ability to bind to a peroxide of coelenterazine or a peroxide of a coelenterazine derivative so as to form a holoprotein that emits light under the action of a calcium ion.
3. A fusion protein comprising:
(1) a region having a first amino acid sequence of SEQ ID NO: 1, and
(2) a region having a second amino acid sequence of SEQ ID NO: 3,
wherein the fusion protein has a minimum IgG-detection sensitivity of 1 ng/ml.
4. The fusion protein according to claim 1 , further comprising at least one amino acid sequence for enhancing at least one of translation and purification.
5. A fusion protein having the amino acid sequence of SEQ ID NO: 5.
6. A holoprotein comprising the fusion protein of claim 1 and a peroxide of coelenterazine or a peroxide of a coelenterazine derivative.
7. A kit comprising the fusion protein of claim 1 .
8. An immunoassay kit comprising the fusion protein of claim 1 .
9. A method for detecting or assaying calcium ions, comprising using the fusion protein of claim 1 .
10. An immunoassay method, comprising using the fusion protein of claim 1 .
11. A kit comprising the holoprotein of claim 6 .
12. An immunoassay kit comprising the holoprotein of claim 6 .
13. A method for detecting or assaying calcium ions, comprising using the holoprotein of claim 6 .
14. An immunoassay method, comprising using the holoprotein of claim 6 .
15. A holoprotein comprising the fusion protein of claim 2 and a peroxide of coelenterazine or a peroxide of a coelenterazine derivative.
16. A holoprotein comprising the fusion protein of claim 3 and a peroxide of coelenterazine or a peroxide of a coelenterazine derivative.
17. A holoprotein comprising the fusion protein of claim 4 and a peroxide of coelenterazine or a peroxide of a coelenterazine derivative.
18. A holoprotein comprising the fusion protein of claim 5 and a peroxide of coelenterazine or a peroxide of a coelenterazine derivative.