Fragment antibody and method for crystallizing protein using fragment antibody
View Patent ↗An object of the present invention is to provide a fragment antibody which can be conveniently produced as one having antigen-binding activity, and which has a greater ability to crystallize itself alone or as a complex with an antigen molecule than that of Fv-clasp (v1) even in a case where the fragment antibody is obtained in an E. coli expression system. The present invention relates to a fragment antibody including a complex of a peptide (VH(112C)-SARAH) in which an N-terminus of a SARAH domain is linked to a C-terminus of a heavy chain domain (VH region) of an antibody, and an amino acid residue of antibody residue 112 according to Chothia numbering scheme in the VH region is mutated to cysteine; and a peptide (VL-SARAH(37C)) in which an N-terminus of a SARAH domain is linked to a C-terminus of a light chain domain (VL region) of an antibody, and an amino acid residue at position 13 from the C-terminus in the SARAH domain is mutated to cysteine.
1. A fragment antibody comprising a complex of:
(a) a peptide (VH(112C)-SARAH) in which an N-terminus of a SARAH domain is linked to a C-terminus of a heavy chain domain (VH region) of an antibody, and an amino acid residue of antibody residue 112 according to Chothia numbering scheme in the VH region is mutated to cysteine; and
(b) a peptide (VL-SARAH(37C)) in which an N-terminus of a SARAH domain is linked to a C-terminus of a light chain domain (VL region) of an antibody, and an amino acid residue at position 13 from the C-terminus in the SARAH domain is mutated to cysteine,
wherein (c) the VH(112C)-SARAH and the VL-SARAH(37C) are linked by a disulfide bond between the two cysteines.
2. The fragment antibody according to claim 1 , wherein the SARAH domain in the VH(112C)-SARAH is represented by any one selected from SEQ ID NOs: 1 to 8, and the SARAH domain in the VL-SARAH(37C) is represented by any one selected from SEQ ID NOs: 9 to 16.
3. The fragment antibody according to claim 1 , wherein the SARAH domain in the VH(112C)-SARAH is represented by SEQ ID NOs: 1 or 2, and the SARAH domain in the VL-SARAH(37C) is represented by SEQ ID NOs: 9 or 10.
4. A fragment antibody for promoting protein crystallization, the fragment antibody comprising a complex of:
(a) a peptide (VH(112C)-SARAH) in which an N-terminus of a SARAH domain is linked to a C-terminus of a heavy chain domain (VH region) of an antibody, and an amino acid residue of antibody residue 112 according to Chothia numbering scheme in the VH region is mutated to cysteine; and
(b) a peptide (VL-SARAH(37C)) in which an N-terminus of a SARAH domain is linked to a C-terminus of a light chain domain (VL region) of an antibody, and an amino acid residue at position 13 from the C-terminus in the SARAH domain is mutated to cysteine,
wherein (c) the VH(112C)-SARAH and the VL-SARAH(37C) are linked by a disulfide bond between the two cysteines.
5. The fragment antibody for promoting protein crystallization according to claim 4 , wherein the SARAH domain in the VH(112C)-SARAH is represented by any one selected from SEQ ID NOs: 1 to 8, and the SARAH domain in the VL-SARAH(37C) is represented by any one selected from SEQ ID NOs: 9 to 16.
6. The fragment antibody for promoting protein crystallization according to claim 4 , wherein the SARAH domain in the VH(112C)-SARAH is represented by SEQ ID NOs: 1 or 2, and the SARAH domain in the VL-SARAH(37C) is represented by SEQ ID NOs: 9 or 10.
7. A method for crystallizing a protein, comprising contacting the fragment antibody according to claim 1 with said protein.
8. A method for crystallizing a protein, comprising contacting the fragment antibody according to claim 2 with said protein.
9. A method for crystallizing a protein, comprising contacting the fragment antibody according to claim 3 with said protein.