التفاصيل البيبلوغرافية
| العنوان: |
AFFINITY PEPTIDES AND METHOD FOR PURIFICATION OF RECOMBINANT PROTEINS |
| Document Number: |
20120157659 |
| تاريخ النشر: |
June 21, 2012 |
| Appl. No: |
12/842764 |
| Application Filed: |
July 23, 2010 |
| مستخلص: |
This invention describes a process for separating a fusion protein or polypeptide in the form of its precursor from a mixture containing said fusion protein and impurities, which comprises contacting said fusion protein with a resin containing immobilized metal ions, said fusion protein covalently operably linked directly or indirectly to an immobilized metal ion-affinity peptide, binding said fusion protein to said resin, and selectively eluting said fusion protein from said resin. |
| Inventors: |
Hernan, Ronald A. (Ballwin, MO, US); Mehigh, Richard J. (St. Louis, MO, US); Brockie, Ian R. (St. Louis, MO, US); Jenkins, Elizabeth (Sherman, IL, US) |
| Assignees: |
SIGMA-ALDRICH CO. (St. Louis, MO, US) |
| Claim: |
1. A polypeptide, protein or protein fragment represented by the formula R1-Sp1-(His-Z1-His-Arg-His-Z2-His)-Sp2-R2, wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal ion-affinity peptide, R1 is hydrogen, a polypeptide, protein or protein fragment, Sp1 is a covalent bond or a spacer comprising at least one amino acid residue, R2 is hydrogen, a polypeptide, protein or protein fragment, Sp2 is a covalent bond or a spacer comprising at least one amino acid residue, Z1 is an amino acid residue selected from the group consisting of Ala, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z2 is an amino acid residue selected from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val. |
| Claim: |
2. The polypeptide, protein or protein fragment of claim 1, wherein Z1 is selected from the group consisting of Ala, Asn, Ile, Lys, Phe, Ser, Thr, and Val, and Z2 is selected from the group consisting of Ala, Asn, Gly, Lys, Ser, Thr and Tyr. |
| Claim: |
3. The polypeptide, protein or protein fragment of claim 1, wherein Z1 and Z2 are selected from the group consisting of: (a) Z1 is Asn and Z2 is Gly; (b) Z1 is Asn and Z2 is Lys (c) Z1 is Lys and Z2 is Gly. (d) Z1 is Lys and Z2 is Lys. (e) Z1 is Ile and Z2 is Asn; (f) Z1 is Thr and Z2 is Ser; (g) Z1 is Ser and Z2 is Tyr; (h) Z1 is Val and Z2 is Ala; and (i) Z1 is Ala and Z2 is Lys. |
| Claim: |
4. The polypeptide, protein or protein fragment of claim 1, wherein R1 or R2 is hydrogen. |
| Claim: |
5. The polypeptide, protein or protein fragment of claim 1, wherein R1 or R2 is an amino acid residue. |
| Claim: |
6. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising a proteolytic cleavage site, a fusion protein, a secretion sequence, a leader sequence for cellular targeting an antibody epitope or an internal ribosomal sequences. |
| Claim: |
7. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising a proteolytic cleavage site. |
| Claim: |
8. The polypeptide, protein or protein fragment of claim 7, wherein the proteolytic cleavage site is cleaved with enterokinase. |
| Claim: |
9. The polypeptide, protein or protein fragment of claim 1, wherein any one of Sp1, Sp2, R1 and R2 comprises at least one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 1-17. |
| Claim: |
10. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising the enzyme glutathione-S-transferase of the parasite helminth Schistosoma japonicum. |
| Claim: |
11. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 15). |
| Claim: |
12. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising the amino acid sequence DLYDDDDK (SEQ ID NO: 16). |
| Claim: |
13. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising the amino acid sequence Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 17). |
| Claim: |
14. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence [table included] where: D, Y and K are their representative amino acids; X20 and X21 are independently a hydrogen or a covalent bond; each X1 and X4 is independently a covalent bond or at least one amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; each X2, X3, X7 and X8 is independently an amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; X5 is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid or a combination of multiple or alternating histidine residues, said combination comprising His-Gly-His, or -(His-X)m—, wherein m is 1 to 6 and X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X9 is a covalent bond or an aspartate residue; and n is 0, 1 or 2. |
| Claim: |
15. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence [table included] where: D, Y, K are their representative amino acids; X20 and X21 are independently a hydrogen or a covalent bond; each X2, X3, X7 and X8 is independently an amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; X5 is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid or a combination of multiple or alternating histidine residues, said combination comprising His-Gly-His, or -(His-X)m—, wherein m is 1 to 6 and X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X9 is a covalent bond or an aspartate residue; and n is at least 2. |
| Claim: |
16. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence [table included] where: D, Y, and K are their representative amino acids; X20 and X21 are independently a hydrogen or a covalent bond; X10 is a covalent bond or an amino acid; each X2, X3, X7 and X8 is independently an amino acid residue selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; X5 is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid or a combination of multiple or alternating histidine residues, said combination comprising His-Gly-His, or -(His-X)m—, wherein m is 1 to 6 and X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; X9 is a covalent bond or an aspartate residue; and n is at least 2. |
| Claim: |
17. The polypeptide, protein or protein fragment of claim 1, wherein Sp1 or Sp2 is a spacer comprising at least one amino acid residue, said spacer comprising an antigenic domain, wherein the antigenic domain comprises the sequence [table included] where: D, Y and K are their representative amino acids; X20 and X21 are independently a hydrogen or a covalent bond; each X11 is a covalent bond or an amino acid; each X12 is an amino acid selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; each X13 is a covalent bond or at least one amino acid selected from the group consisting of aromatic amino acid residues and hydrophilic amino acid residues; X14 is a covalent bond or a spacer domain, the spacer domain comprising at least one amino acid or alternating histidine residues, said combination comprising His-Gly-His, or -(His-X)m—, wherein m is 1 to 6 and X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X15 is a covalent bond or an aspartate residue; and n is 0 or at least 1. |
| Claim: |
18. A polypeptide, protein or protein fragment represented by the formula R1-Sp1-(His-Z1-His-Arg-His-Z2-His)t-Sp2-R2, wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal ion-affinity peptide, t is at least 2, R1 is hydrogen, a polypeptide, protein or protein fragment, Sp1 is a covalent bond or a spacer comprising at least one amino acid residue, R2 is hydrogen, a polypeptide, protein or protein fragment, Sp2 is a covalent bond or a spacer comprising at least one amino acid residue, Z1 is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z2 is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val. |
| Claim: |
19. The peptide of claim 18, wherein Z1 and Z2 are selected from the group consisting of: (a) Z1 is Asn and Z2 is Lys; and (b) Z1 is Lys and Z2 is Gly. |
| Claim: |
20. A polypeptide, protein or protein fragment represented by the formula R1-Sp1-[(His-Z1-His-Arg-His-Z2-His)-Sp2]t-R2, wherein (His-Z1-His-Arg-His-Z2-His) (SEQ ID NO: 24) is a metal ion-affinity peptide, t is at least 2, R1 is hydrogen, a polypeptide, protein or protein fragment, Sp1 is a covalent bond or a spacer comprising at least one amino acid residue, R2 is hydrogen, a polypeptide, protein or protein fragment, Sp2 is a covalent bond or a spacer comprising at least one amino acid residue, Z1 is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z2 is an amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val; and each Sp2 of the recombinant polypeptides, proteins or protein fragments may be the same or different. |
| Claim: |
21. The peptide of claim 20, wherein Z1 and Z2 are selected from the group consisting of: (a) Z1 is Asn and Z2 is Lys; and (b) Z1 is Lys and Z2 is Gly. |
| Current U.S. Class: |
530/324 |
| Current International Class: |
07; 07; 07 |
| رقم الانضمام: |
edspap.20120157659 |
| قاعدة البيانات: |
USPTO Patent Applications |