Affinity tag nucleic acid and protein compositions, and processes for using same
The present invention concerns compositions and processes that use affinity tags for isolating, and detecting or quantifying analytes, including nucleic acids, proteins and polypeptides. Compositions include nucleic acid compositions and protein compositions with affinity binding pairs, including metal binding peptides and immobilized metals, or peptide affinity groups.
1. A process for isolating an analyte of interest, said process comprising the steps of:
providing
(i) a sample containing or suspected of containing said analyte of interest;
(ii) a first antibody having an affinity for said analyte;
(iii) a fusion antibody comprising:
(a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody; or
(b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody; and
(iv) a matrix comprising a metal or a second member of said affinity peptide group;
contacting said sample with said first antibody, thereby forming a first complex between said first antibody and any analyte present in said sample;
contacting said first complex with said fusion antibody, thereby forming a second complex between said first complex and said fusion antibody;
contacting said second complex with said matrix to bind said second complex to said matrix;
removing unbound material from said matrix, and
releasing said analyte of interest from said second complex, thereby isolating said analyte of interest.
2. The process of claim 1 , wherein said peptide affinity group comprises: S-protein and S-peptide; Glutathione S-Transferase (GST) tag and Glutathione (GSH); Protein Kinase A catalytic subunit (PKA) recognition peptide and PKA; Hemagglutinin (HA) epitope tag peptide and HA; Ketosteroid Isomerase (KSI) tag and oligo Phe; Ketosteroid Isomerase (KSI) tag and oligo Leu; oligo Arg and oligo Glu; or oligo Arg and oligo Asp.
3. The process of claim 1 , wherein said metal binding peptide comprises oligohistidine or an oligopeptide comprising the sequence HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG (SEQ ID NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID NO:5), SPHHGGHPHHG (SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO:10), or HGLGHGHEQQHGLGHGH (SEQ ID NO:9).
4. A process for detecting or quantifying an analyte of interest, said process comprising the steps of:
providing
(i) a labeled sample containing or suspected of containing labeled analyte of interest;
(ii) a first antibody having an affinity for said analyte;
(iii) a fusion antibody comprising:
(a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody; or
(b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody; and
(iv) a matrix comprising a metal or a second member of said affinity peptide group;
contacting said sample with said first antibody, thereby forming a first complex between said first antibody and any analyte present in said sample;
contacting said first complex with said fusion antibody, thereby forming a second complex between said first complex and said fusion antibody;
contacting said second complex with said matrix to bind said second complex to said matrix;
removing unbound material from said matrix; and
detecting or quantifying said labeled analytes bound to said matrix by means of detecting or quantifying a signal from said labels.
5. The process of claim 4 , wherein said peptide affinity group comprises: S-protein and S-peptide; Glutathione S-Transferase (GST) tag and Glutathione (GSH); Protein Kinase A catalytic subunit (PKA) recognition peptide and PKA; Hemagglutinin (HA) epitope tag peptide and HA; Ketosteroid Isomerase (KSI) tag and oligo Phe; Ketosteroid Isomerase (KSI) tag and oligo Leu; oligo Arg and oligo Glu; or oligo Arg and oligo Asp.
6. The process of claim 4 , wherein said metal binding peptide comprises oligohistidine or an oligopeptide comprising the sequence HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG (SEQ ID NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID NO:5), SPHHGGHPHHG (SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO:10), or HGLGHGHEQQHGLGHGH (SEQ ID NO:9).
7. The process of claim 4 , wherein said analyte of interest comprises a protein or polypeptide.
8. The process of claim 4 , further comprising the step of releasing said analyte of interest from said second complex prior to said detecting or quantifying step.
9. The process of claim 4 , wherein said labeled analytes are detected fluorescently, chemiluminescently, colorimetrically or enzymatically.
10. A process for detecting or quantifying an analyte of interest, said process comprising the steps of:
providing
(i) a labeled sample containing or suspected of containing labeled analytes of interest;
(ii) a first antibody having an affinity for said analyte;
(iii) a fusion antibody comprising:
(a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody; or
(b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody; and
(iv) a matrix comprising a metal or a second member of said affinity peptide group;
forming a first complex comprising said matrix, said fusion antibody and said first antibody;
contacting said first complex with said labeled sample, thereby forming a second complex between said first complex and any labeled analytes that may be present in said sample;
removing unbound material from said matrix; and
detecting or quantifying said labeled analytes bound to said matrix by means of detecting or quantifying a signal from said labels.
11. The process of claim 10 , wherein said matrix comprises an array of different first antibodies, thereby detecting or quantifying multiple analytes of interest.
12. The process of claim 10 , wherein said labeled analytes are detected fluorescently, chemiluminescently, colorimetrically or enzymatically.
13. The process of claim 1 , wherein the fusion antibody comprises (iii)(a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody.
14. The process of claim 1 , wherein the fusion antibody comprises (iii)(b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody.
15. The process of claim 4 , wherein the fusion antibody comprises (iii)(a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody.
16. The process of claim 4 , wherein the fusion antibody comprises (iii)(b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody.
17. The process of claim 10 , wherein the fusion antibody comprises (iii)(a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody.
18. The process of claim 10 , wherein the fusion antibody comprises (iii)(b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for the first antibody.