TAG PEPTIDE HAVING A PROTEASE RECOGNITION SEQUENCE AND USE THEREOF

• US 20120052569A1
• Filed: 04/21/2010
• Published: 03/01/2012
• Est. Priority Date: 04/22/2009
• Status: Active Grant

• Current Assignee: Osaka University
• Original Assignee: Osaka University
• Inventors: Takagi, Junichi
• Granted Patent: US 8,481,310 B2
• Time in Patent Office: Days
• US Class Current: 435/331
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 16/10 from RNA viruses
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

Provided is a tag peptide in which a protease recognition sequence and an epitope of an antibody against the tag peptide are overlapped and thereby the protease recognition sequence per se is usable for detection or purification. Also provided is a purification method for recombinant proteins using the tag peptide and an antibody thereagainst. A preferable tag peptide comprises a Tobacco etch virus (TEV) protease recognition sequence as the protease recognition sequence, and its examples include a tag peptide comprising the amino acid sequence (1): RX₁X₂LYX₃QGKDG (wherein X₁, X₂ and X₃ may be the same or different and represent any amino acid residue).

Claims

1. A tag peptide having a protease recognition sequence, the protease recognition sequence overlapping with an epitope of an antibody against the tag peptide.

3. The tag peptide according to claim 1, comprising the following amino acid sequence (1):

4. The tag peptide according to claim 3, wherein the amino acid sequence (1) is the following amino acid sequence (2):

5. A tag peptide comprising the tag peptide according to claim 1 in combination with a second tag peptide.

6. The tag peptide according to claim 5, wherein the tag peptide allows an antibody against the tag peptide and an antibody against the second tag peptide to simultaneously bind thereto.

7. A polynucleotide encoding the tag peptide according to claim 1.

8. A recombinant vector containing the polynucleotide according to claim 7.

9. An antibody against the tag peptide according to claim 1.

10. The antibody according to claim 9 which is a monoclonal antibody produced by rat-mouse hybridoma 2H5 (FERM BP-11245).

12. A purification method for proteins, comprising the following steps (i) to (iii):

(i) making an antibody against the tag peptide according to claim 1 act on a sample containing a fusion protein of an objective protein and the tag peptide according to claim 1 for formation of a complex of the antibody with the fusion protein;

(ii) making an eluent act on the complex obtained in the step (i) for release of the fusion protein from the antibody; and

(iii) separating off the tag peptide by cleavage of the fusion protein obtained in the step (ii).

13. A kit for protein expression, purification, detection or quantification, comprising a recombinant vector containing a polynucleotide encoding the tag peptide according to claim 1 or an antibody against the tag peptide according to claim 1.

2. A tag peptide having a protease recognition sequence, the protease recognition sequence overlapping with an epitope of an antibody against the tag peptide and being a Tobacco etch virus (TEV) protease recognition sequence.

11. Rat-mouse hybridoma 2H5 (FERM BP-11245).