Modified biotin-binding protein

摘要

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; and6) 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.

主权项

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; 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 16th aspartic acid residue of SEQ ID NO: 2 with an amino acid that does not form a hydrogen bond, wherein the protein satisfies 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; and iv) showing high expression in a soluble fraction of Escherichia coli, and wherein the protein satisfies 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; l) 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, and wherein at least one of the amino acid residues specified in 1) to 6) are replaced as in specified in 1) to 6).