Claims
- 1. An immobilized metal ion affity chromatography purification method for purification of a recombinant proteins, said method comprising:
(a) providing carboxymethylated aspartate ligand complexed with a transiton metal ion in a 2+ oxidation state, having a coordination number of 6; (b) loading a mixture of cell lysate comprising a recombinant protein having a polyhistidine tail to bind with said ligand; and (c) eluting said recombinant protein with a suitable elutant to obtain a purified recombinant protein.
- 2. The method, according to claim 1, wherein said transition metal-complexed carboxymethylated aspartate ligand forms a carboxymethylated aspartate chelating matrix which comprises said transition metal and a polymer matrix.
- 3. The method, according to claim 2, wherein said transition metal is connected to said polymer matrix by a linking arm and a finctional inking group.
- 4. The method, according to claim 3, wherein said linking arm is selected from the group consisting of-CH2CH(OH)CH2-, -CH2(OH)CH2-0-CH2CH(OH)CH2-, CH2)4NHCH2CH(OH)CH2-, and CH2)2NHCH2CH(OH)CH2-.
- 5. The method, according to claim 3, wherein said finctional linking group is selected from the group consisting of 0, S, and NH.
- 6. The method, according to claim 2, wherein said polymer matrix is agarose.
- 7. The method, according to claim 2, wherein said carboxymethylated aspartate chelating matrix has the structure
- 8. The method, according to claim 2, wherein said carboxymethylated aspartate chelating matrix has the structure
- 9. An immobilized metal ion affinity chromatography complex comprising a carboxymethylated aspartate ligand and a tnansition metal complexed thereto, wherein said transition metal ion has a 2+ oxidation state and a coordination number of 6.
- 10. The complex, according to claim 9, wherein said complex has the structure:
- 11. The method, according to claim 10, wherein said polymer matrix comprises a polymer matrix suitable for use in afinty or gel chromatography.
- 12. The complex, according to claim 10, wherein
M=Fe2+ , Col+, Ni2+ , Cue, or Zne; R=CH2CH(OH)CH2-, H2(OH)CH2 H2CH(OH)CH2-, or CH2)2NHCH2CH(OH)CH,- R2=O, S, or NH; and R3=agarose or polystyrene.
- 13. The complex, according to claim 12, wherein
M=Co2+ ; R1=CH2CH(OH)CH2; R2=O; and R3=agarose, cross-linked or polystyrene
- 14. A method for synthesizing carboxymethylated aspartate agarose chelating resin, said method comprising
(a) forming oxirane-agarose; (b) conjugating aspartic acid to oxirane-agarose; and (c) washing said aspartic acid-oxirane-agarose conjugate to remove extraneously bound metals using a high ionic strength solution.
- 15. The method, according to claim 14, wherein said conditions for oxirane-agarose formation comprise carrying out the formation at about room temperature, overnight, adjusting to about pH 7.0.
- 16. The method, according to claim 14, wherein said temperature control conditions for conjugating aspartic acid to said oxirane-agarose comprise mixing at less than about 25° C., reacting at about 80° C. for 4 hours, then cooling to room temperature overnight.
- 17. The method, according to claim 14, wherein said washing step (c) comprises use of a solution of at least 7.5% sodium hydroxide.
- 18. The complex according to claim 9, wherein said complex has the structure:
- 19. The method, according to claim 18, wherein said polymer matrix comprises a polymer matrix suitable for use in affinity or gel chromatography.
- 20. The complex according to claim 18, wherein
M=Fe2+ , Co2+ , Ni2+ , Cu2+ , or Zn2+ ; R4=dCH2)4NHCH2CH(OH)CH2- or dCH2)4NH-; R5=O, S, NH, or CO; and R6=agarose or polystyrene.
- 21. The complex, according to claim 20, wherein
M=Co2+ ; R={CH2)4NHCH2CH(OH)CH2- or {CH2)4NH-; R5=O or CO; and R6=agarose, cross linked, or polystyrene.
- 22. A method for synthesizing carboxymethylated aspartate chelating matrices, said method comprising the steps:
(a) Michael addition of the a-amino function of monoprotected a,u-diamino acids to maleic acid; (b) deprotecting the w-amino fimctionality; and (c) attaching the chelator primary amine molecule to a solid matrix.
- 23. A method for screening for protein finction on a microtiter plate or filter, said method comprising the steps:
(a) immobilizing a complex of claim 1 to the plate or filter; (b) binding said immobilized complex to the protein for which the function is being screened; and (c) performing an assay for protein finction on the bound protein. HAtSHAPPS ci5cl.doc/DNB/clr
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of co-pending application Ser. No. 08/698,747, filed Aug. 16, 1996.
Continuations (1)
|
Number |
Date |
Country |
Parent |
08912406 |
Aug 1997 |
US |
Child |
09839696 |
Apr 2001 |
US |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
08698747 |
Aug 1996 |
US |
Child |
08912406 |
Aug 1997 |
US |