Mutant apoprotein of photoprotein with low calcium sensitivity

Patent metadata

Field	Value
Publication number	US-9238681-B2
Application number	US-201414307075-A
Country	US
Kind code	B2
Filing date	Jun 17, 2014
Priority date	Apr 5, 2011
Publication date	Jan 19, 2016
Grant date	Jan 19, 2016

Abstract

Calcium-binding photoproteins showing the luminescence pattern with a slow decay of are desired. The invention provides a mutant apoprotein comprising an amino acid sequence wherein the 23rd to 34th amino acids in the amino acid sequence of SEQ ID NO: 2 are substituted with an amino acid represented by formula I below: Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa 34; having a function to bind to the peroxide of coelenterazine or the peroxide of a coelenterazine analog to form a photoprotein capable of emitting light under the action of calcium ions; and, having a half decay time of the luminescence emitted by binding of the photoprotein to calcium ions being not less than 2 seconds.

First claim

The invention claimed is:

1. An isolated polynucleotide comprising a polynucleotide encoding an isolated mutant apoprotein comprising an amino acid sequence wherein the 23rd to 34th amino acids in the amino acid sequence of SEQ ID NO: 2 are substituted with an amino acid represented by formula I below: Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34, wherein:
Xaa23 is Asp, Glu, Gin, Ser, Thr, or Asn,
Xaa24 is Lys, Arg, His, Leu, or Thr,
Xaa25 is Asp, Glu, Gin, Ser, Thr, or Asn,
Xaa26 is an optional amino acid,
Xaa27 is Asp, Glu, Gin, Ser, Thr, or Asn,
Xaa28 is Gly,
Xaa29 is an optional amino acid,
Xaa30 is Ile, Leu, or Val,
Xaa31 is Asp, Glu, Gln, Ser, Thr, or Asn,
Xaa32 is an optional amino acid,
Xaa33 is an optional amino acid, and
Xaa34 is Asp, Glu, Gin, Ser, Thr, or Asn;
having a function to bind to the peroxide of coelenterazine or the peroxide of a coelenterazine analogue to form a photoprotein capable of emitting light under the action of calcium ions; and
having a half decay time of the luminescence emitted by binding of the photoprotein to calcium ions being not less than 2 seconds.

2. A recombinant vector comprising the polynucleotide encoding the isolated mutant apoprotein according to claim 1 .

3. A transformant transformed with the recombinant vector according to claim 2 .

4. A method for producing the isolated mutant apoprotein according to claim 1 , which comprises culturing a transformant comprising a recombinant vector comprising a polynucleotide encoding the isolated mutant apoprotein to produce the isolated mutant apoprotein.

5. A kit comprising the polynucleotide encoding the isolated mutant apoprotein according to claim 1 , a recombinant vector comprising the polynucleotide, or a transformant transformed with the vector.

6. A method for measuring the activity of a sequence involved in promoter regulation, which comprises
detecting a luminescence from a photoprotein consisting of the isolated mutant apoprotein according to claim 1 and a peroxide of coelenterazine or a peroxide of coelenterazine analogue in the host cell transfected with a vector comprising a polynucleotide encoding the isolated mutant apoprotein.

7. A method for measuring changes in intracellular calcium levels, which comprises expressing the polynucleotide according to claim 1 to form a photoprotein.

Assignees

• Jnc Corp

Inventors

• Inouye Satoshi
• Miura Yuiko

Classifications

• C07K2319/21

  containing a His-tag · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• C07K2319/036

  targeting to the medium outside of the cell, e.g. type III secretion · CPC title

• C07K14/435 Primary

  from animals; from humans · CPC title

• C07K14/43595 Primary

  from coelenteratae, e.g. medusae · CPC title