Fragment antibody and method for crystallizing protein using fragment antibody

Patent metadata

Field	Value
Publication number	US-10513562-B2
Application number	US-201716345639-A
Country	US
Kind code	B2
Filing date	Nov 7, 2017
Priority date	Nov 9, 2016
Publication date	Dec 24, 2019
Grant date	Dec 24, 2019

Abstract

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.

First claim

The invention claimed is:

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.

Assignees

• Fujifilm Wako Pure Chemical Corp
• Univ Osaka

Inventors

• Takagi Junichi

Classifications

• C07K2319/73

  containing coiled-coiled motif (leucine zippers) · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title

• C07K16/44 Primary

  against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids} · CPC title

• C07K16/286

  against neuromediator receptors, e.g. serotonin receptor, dopamine receptor · CPC title