Modified biotin-binding protein

Patent metadata

Field	Value
Publication number	US-9611303-B2
Application number	US-201514661672-A
Country	US
Kind code	B2
Filing date	Mar 18, 2015
Priority date	Jun 24, 2009
Publication date	Apr 4, 2017
Grant date	Apr 4, 2017

Abstract

The present invention provides a modified biotin-binding protein comprising an amino acid sequence represented by SEQ ID NO: 2 or its modified sequence and having a biotin-binding activity and replacement selected from the group consisting of:
1) replacement of the 36th serine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 2) replacement of the 80th tryptophan residue of SEQ ID NO: 2 with a hydrophilic amino acid residue; 3) replacement of the 116th aspartic acid residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 4) replacement of the 46th proline residue of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue and replacement of the 78th threonine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond; 5) replacement of the 46th proline residue of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue and replacement of the 116th aspartic acid residue of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond; and 6) replacement of the 46th proline residue of SEQ ID NO: 2 with a threonine, serine, or tyrosine residue, replacement of the 78th threonine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond, and replacement of the 116th aspartic acid residue of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond.

First claim

The invention claimed is:

1. A protein comprising an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having an identity of not less than 95% to the amino acid sequence of SEQ ID NO: 2, the protein having replacement selected from the group consisting of:
1) replacement of the 36th serine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond and replacement of the 116th aspartic acid residue of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond; and
2) replacement of the 36th serine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond, replacement of the 78th threonine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond, and replacement of the 116th aspartic acid residue of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond.

2. The protein according to claim 1 , selected from the group consisting of:
1-a) a modified biotin-binding protein (TM2 S36A-D116A) in which the 36th serine residue of SEQ ID NO: 2 is replaced with alanine and the 116th aspartic acid residue of SEQ ID NO: 2 is replaced with alanine; and
2-a) a modified biotin-binding protein (TM2 S36A-T78A-D116A) in which the 36th serine residue of SEQ ID NO: 2 is replaced with alanine, the 78th threonine residue of SEQ ID NO: 2 is replaced with alanine, and the 116th aspartic acid residue of SEQ ID NO: 2 is replaced with alanine.

3. The protein according to claim 1 or 2 , satisfying at least one of the following properties:
i) allowing purification using biotin;
ii) having protease resistance; and
iii) binding to biotin under weak acidic conditions and not binding to biotin under neutral conditions.

4. A protein comprising an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence having an identity of not less than 95% to the amino acid sequence of SEQ ID NO: 2, the protein comprising:
3) replacement of the 78th threonine residue of SEQ ID NO: 2 with an amino acid residue that does not form a hydrogen bond and replacement of the 116th aspartic acid residue of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond.

5. The protein according to claim 4 , wherein
3-a) the 78th threonine residue of SEQ ID NO: 2 is replaced with alanine, and the 116th aspartic acid residue of SEQ ID NO: 2 is replaced with alanine (TM2 T78A-D116A).

6. The protein according to claim 4 or 5 , satisfying at least one of the following properties:
i) allowing purification using biotin;
ii) maintaining a tetramer structure of a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
iii) having protease resistance;
iv) showing high expression in a soluble fraction of E. coli , and
v) not allowing purification using iminobiotin.

7. The protein according to claim 1 or 4 , showing a biotin-binding affinity lower than that of a protein comprising the amino acid sequence represented by SEQ ID NO: 2.

8. The protein according to claim 1 or 4 , satisfying at least one of the following provisions a) to p):
a) the 14th asparagine residue of SEQ ID NO: 2 is not modified or is replaced with glutamine or aspartic acid;
b) the 18th serine residue of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;
c) the 34th tyrosine residue of SEQ ID NO: 2 is not modified or is replaced with serine or threonine;
d) the 36th serine residue of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;
e) the 40th aspartic acid residue of SEQ ID NO: 2 is not modified or is replaced with a residue other than asparagine;
f) the 69th tryptophan residue of SEQ ID NO: 2 is not modified;
g) the 76th serine residue of SEQ ID NO: 2 is not modified or is replaced with threonine or tyrosine;
h) the 78th threonine residue of SEQ ID NO: 2 is not modified or is replaced with serine or tyrosine;
i) the 80th tryptophan residue of SEQ ID NO: 2 is not modified;
j) the 96th tryptophan residue of SEQ ID NO: 2 is not modified;
k) the 108th tryptophan residue of SEQ ID NO: 2 is not modified;
1) the 116th aspartic acid residue of SEQ ID NO: 2 is not modified or is replaced with glutamic acid or asparagine;
m) the 46th proline residue of SEQ ID NO: 2 is not modified;
n) the 66th alanine residue of SEQ ID NO: 2 is not modified;
o) the 97th leucine residue of SEQ ID NO: 2 is not modified or is modified to isoleucine; and
p) the 113th valine residue of SEQ ID NO: 2 is not modified, wherein the amino acid residues specified in 1) to 3) are replaced as in specified in 1) to 3).

9. The protein according to claim 1 or 4 , comprising an amino acid sequence having an identity of not less than 98% to the amino acid sequence represented by SEQ ID NO: 2.

10. A carrier to which the protein according to claim 1 or 4 is immobilized.

11. A nucleic acid encoding the protein according to claim 1 or 4 .

12. A vector containing the nucleic acid according to claim 11 .

Assignees

• Japan Tobacco Inc

Inventors

• Takakura Yoshimitsu
• Tsunashima Masako
• Sofuku Kozue

Classifications

• C07K14/375 Primary

  from Basidiomycetes · CPC title

• C07K2319/22

  containing a Strep-tag · CPC title

• G01N2333/36

  from Actinomyces; from Streptomyces (G) · CPC title