Epitope tag and method for detection and/or purification of tagged polypeptides
Provided herein is a novel epitope that can be used as a tag in methods for rapid and effective characterization, purification, and subcellular localization of polypeptides of interest, which comprise the tag. The tag is specifically recognized by an epitope specific antibody, which can be used to detect, capture, quantify, and/or purify polypeptides of interest that are tagged with the epitope. Also provided is novel epitope specific antibody.
1. A kit for purification of a polypeptide comprising an epitope peptide, wherein the kit comprises:
a) an antibody that specifically binds to the epitope peptide, wherein the epitope peptide comprises from about 8 to about 25 amino acids, wherein the amino acid sequence comprises a sequence as defined in SEQ ID NO: 1 (RX 4 X 5 AX 7 SX 9 W),
wherein X 4 can be K or a substitution;
wherein X 5 can be A or R or a conservative substitution of A or R;
wherein X 7 can be V or a conservative substitution of V,
wherein X 9 can be H or a conservative substitution of H, wherein the antibody comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 6; and
b) a secondary binding partner, wherein the secondary binding partner is specific for the antibody, specific for the polypeptide, or specific for a complex comprising both, and wherein the secondary binding partner comprises a detectable moiety and/or an immobilized or immobilizable moiety.
2. The kit of claim 1 , wherein the epitope peptide comprises from about 12 to about 25 amino acids, wherein the amino acid sequence comprises a sequence as defined by SEQ ID NO: 32 (X 1 X 2 RX 4 X 5 AX 7 SX 9 WX 11 X 12 ),
wherein X 1 can be P or A;
wherein X 2 can be D or a conservative substitution of D;
wherein X 4 can be K or a substitution;
wherein X 5 can be A or R or a conservative substitution of A or R;
wherein X 7 can be V or a conservative substitution of V, and
wherein X 9 can be H or a conservative substitution of H; and
wherein X 11 and X 12 can be Q or a conservative substitution of Q.
3. The kit of claim 1 , wherein the epitope peptide has an amino acid sequence as defined by SEQ ID NO: 3 (RKAAVSHW); or an amino acid sequence as defined by SEQ ID NO: 4 (PDRKAAVSHWQQ); or an amino acid sequence as defined by SEQ ID NO: 5 (PVRSAALSQWSS), or an amino acid sequence as defined by SEQ ID NO:33 (PDRVRAVSHWSS), or an amino acid sequence as defined by SEQ ID NO: 34 (ADRVRAVSHWSS).
4. The kit of claim 1 , wherein the antibody is a nanobody, wherein the nanobody comprises an amino acid sequence comprising framework region 1, CDR1, framework region 2, CDR2, framework region 3, CDR3, and framework region 4, wherein CDR3 comprises SEQ ID NO:12, and wherein a cysteine is present in the framework region 2, which is capable of forming a disulfide bridge with the cysteine present in CDR3.
5. The kit of claim 1 , wherein the antibody is an antibody having the amino acid sequence as defined in SEQ ID NO: 6.
6. The kit of claim 1 , wherein the antibody is attached to a solid support.
7. The kit of claim 6 , wherein the solid support is selected from the group comprising polystyrene, polypropylene, polyvinylchloride, polyacrylamide, celluloses, dextrans, synthetic polymers and co-polymers, latex, silica, agarose, metal, glass, and carbon.
8. A method of purifying a polypeptide comprising the steps of:
a) contacting a sample comprising a polypeptide that comprises an epitope peptide with the antibody of the kit of claim 1 , wherein prior to or after binding, the antibody is attached to a solid support;
b) washing the solid support of step a) to remove unbound and unspecifically bound constituents; and
c) eluting the polypeptide.
9. A method of purifying a polypeptide comprising the steps of:
a) contacting a sample comprising a polypeptide that comprises an epitope peptide with the antibody of the kit of claim 1 ;
b) contacting the sample obtained after step a) with a secondary binding partner specific for the antibody, specific for the polypeptide, or specific for a complex comprising both, wherein the secondary binding partner is attached to a solid support prior to or after binding;
c) washing the solid support of step b) to remove unbound and unspecifically bound constituents; and
d) eluting the polypeptide.
10. The method of claim 8 , wherein the antibody is an antibody specific for the epitope peptide, wherein the antibody is a nanobody, wherein the nanobody comprises an amino acid sequence comprising framework region 1, CDR1, framework region 2, CDR2, framework region 3, CDR3, and framework region 4, wherein CDR3 comprises SEQ ID NO: 12, and wherein a cysteine is present in the framework region 2, which is capable of forming a disulfide bridge with the cysteine present in CDR3.
11. The method of claim 9 , wherein the antibody is an antibody specific for the epitope peptide, wherein the antibody is a nanobody, wherein the nanobody comprises an amino acid sequence comprising framework region 1, CDR1, framework region 2, CDR2, framework region 3, CDR3, and framework region 4, wherein CDR3 comprises SEQ ID NO: 12, and wherein a cysteine is present in the framework region 2, which is capable of forming a disulfide bridge with the cysteine present in CDR3.
12. The method of claim 8 , wherein the antibody comprises SEQ ID NO: 6.
13. The method of claim 9 , wherein the antibody comprises SEQ ID NO: 6.