Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-07-13
2002-08-27
Russel, Jeffrey E. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C502S402000, C525S050000, C530S344000, C556S116000
Reexamination Certificate
active
06441146
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to new metal chelator resins and their manufacture process.
BACKGROUND OF THE INVENTION
Metal Chelate Affinity Chromatography (MCAC) (also denoted Immobilised Metal ion Affinity Chromatography (IMAC)) using affinity immobilised metal resins introduced by Porath et al. (Nature, 258, 589 (1975) and used for the purification of proteins which contain neighbouring histidine residues, has now become a powerful and versatile tool for the purification of natural and recombinant 6× His-tagged (or not) proteins and peptides.
The ligand used by these authors was the iminodiacetic acid (IDA). Electron paramagnetic resonance and absorption spectra studies have demonstrated that IDA is a tridentate ligand and the configuration of the complex IDA-M
2+
(1:1) with M
2+
=bivalent metal ions, is a square or tetrahedral one (R. Dallocchio et al.; J. Coord. Chem., 25, 265 (1992). This explains why immobilised IDA can form a stable complex with the ion Cu
2+
and Zn
2+
, but not with other heavy metal ions which need the octahedral configuration for a stable form.
It is also known that histidine is the only &agr;-aminoacid capable of forming octahedral complexes with different polyvalent metal ions as follows: His—M
2+
—His (B. Rao et al.; J. Inorg. Nucl. Chem.; 33, 809 (1971); M. M. Harding et al.; Acta Cryst., 16, 643 (1963)):
Each histidine gives 3 coordination bonds to the M
2+
i.e. the 3-N group of the imidazole ring and the NH
2
and COOH groups of the aminoacid; the 1-NH group of the imidazole ring does not participate in the formation of the complexes (C. C. Mc Donald et al.; JACS, 85, 3736 (1963)).
The complex formation is stereoselective (J. H. Rituma et al.; Recueil, 88, 411 (1969).
The complex chemistry of histamine and imidazole has been described (W. R. Walker et al.; J. Coord. Chem., 3, 77 (1973); Aust. J. Chem., 23, 1973 (1970)).
Furthermore, Single-crystal X-ray analysis (Simon H. Whitlow, Inorg. Chem., 12, 2286 (1973)) and Infrared Spectra studies (Y. Tomita et al.; JACS, 36, 1069 (1963) and J. Phys. Chem., 69, 404 (1965)) have demonstrated that Trisodium Nitrilotriacetate (Na
3
NTA) is a tetradentate ligand for different polyvalent metal ions M
2+
and the corresponding complexes NTA-M
2+
have an octahedral configuration:
At pH 5.5-10.0, NTA may be a mixture of HN
+
(CH
2
—COO
—
)
3
and N(CH
2
—COO
—
)
3
.
Only the carboxylate and uncharged N groups participate in the coordination bonding. The carboxylic and charged N groups do not participate in such linkages.
The NTA derivatives immobilised on Agarose introduced by E. Hochuli et al. (J. Chromatogr., 411, 177 (1987)) and U.S. Pat. No. 4,877,830 (1989) can be therefore, an interesting method for the purification of histidine containing proteins. Their ligands are H
2
N—(CH
2
)
n
—CH(COOH)—N (CH
2
—COOH)
2
(n=2,4) and the resulting resins are: Resin-NH—(CH
2
)
n
—CH(COOH)—N (CH
2
—COOH)
2
(n=2,4).
AIMS OF THE INVENTION
The present invention aims to provide new chelator resins having improved characteristics over the compounds of the state of the art and being suitable for metal chelate affinity chromatography.
The present invention is also related to the preparation process of such resins.
SUMMARY OF THE INVENTION
The present invention is related to an easy, rapid and inexpensive manufacture method of novel resins for IMAC and to said resins being hereafter called Pentadentate chelator (PDC) resins, which advantageously afford 5 coordination bonds to the M
2+
ions. Said coordination bonds may result in an improved stability of the obtained octahedral complexes and one coordination site is free for interaction and selective binding to accessible cysteine/histidine residues and chiefly histidine containing biomolecules that are preferably selected from the group consisting of proteins or peptides.
Furthermore, said PDC resins are able to chelate with different polyvalent metal ions including Cu
2+
, Ni
2+
, Zn
2+
and Co
2+
to give the corresponding metal chelate resins hereafter called Cu—PDC, Ni—PDC, Zn—PDC and Co—PDC respectively. These four resins will be thereafter used for the purification of histidine containing natural and recombinant proteins or peptides.
The present invention is also related to said PDC resins, for which the proteins cannot enter into the pores of the resin (molecular weights of proteins are greater than 5000 Daltons, by definition). Preferably, said resin is PDC-Sephadex® G-25 (obtained from Sephadex® G-25, Pharmacia, Uppsala, Sweden).
In addition, the binding of histidine containing proteins to the chelated metals depends on the complex Metal-PDC resins and the accessibility of histidine residues which in turn, depends on the configuration of the proteins of interest. Therefore, there is no universal rule that will predict the order of magnitude of binding of histidine containing biomolecules to Cu
2+
, Ni
2+
, Zn
2+
and Co
2+
.
The present invention is also related to a PDC KIT consisting of four separate columns Cu—PDC, Ni—PDC, Zn—PDC and Co—PDC, that determine the most appropriate metal chelate resin suitable for the purification of natural and recombinant biomolecules, preferably selected from the group consisting of proteins or peptides.
The Cu-PDC resins according to the invention are used as universal supports for immobilising covalently proteins using a water-soluble carbodiimide and also as concentration resins to reduce the volume of a protein solution.
A last aspect of the present invention concerns the use of the pentadentate chelator (PDC) resins, and especially the PDC-Sephadex® G-25, according to the invention to obtain water and buffers free of polyvalent metal ions. In particular, the PDC-Sephadex® G-25 according to the invention is useful for preparing “metallo-proteins” free of heavy metal ions or proteins free of heavy metal ions after the Immobilised Metal ion Affinity Chromatography steps.
REFERENCES:
patent: 3821126 (1974-06-01), Yamamoto et al.
patent: 4423158 (1983-12-01), Porath
patent: 4877830 (1989-10-01), Dobeli et al.
patent: 5047513 (1991-09-01), Dobeli et al.
patent: 63-55117 (1988-03-01), None
patent: 63-56503 (1988-03-01), None
Chemical Abstract 110: 39499j, Feb. 6, 1989.*
Brubaker et al, Flexidentate Chelation of . . . Inorganic Chemistry, vol. 5, No. 12, pp. 2110-2114, 1966.*
Translation of Japan Kokai 63-55117, Fractional Recovery Method of Gallium and Indium, Mar. 9, 1988.*
Translation of Japan Kokai 63-56503, Aminopolycarboxylic Acid-Type Chelote Resin And Its Manufacturing Method, Mar. 11, 1988.*
Nature vol. 258, Porath, et al., Metal Chelate Affinity Chromatography, A Anew Approach to Protein Fractionation, pp. 598-599, Dec. 18, 1975.
J. Coord. Chem., 1992 vol. 25. Coordination of Copper (II) to Polyaminopolycarbozylic Acids in Aquesous Solution, pp. 265-270, Micera, et al.
J. inorg. Nucl. Chem, 1971, vol. 33, pp. 809-816, Thermodynamics of the Interaction of Transition Metal Ions With Histamine, Rao, et al.
Acta Cryst. 1963 16, pp. 643-650, The Crystal Structure of Di (Histidino) Zincpentahydrate, Harding, et al.
J. Amer. Chem. Soc. 85, pp. 3736-3742, 1963, A Nuclear Magnetic Resonance Study of Structures of Cobalt (II)-Histidine Complexes, McDonald, et al.
88 (1969) Recueil, 411, Steroselectivity in the Complex Formationof Histodine With Cobalt (II) and Nickel (II), pp. 411-416, Ritsma, et al.
J. Coord. Chem., 1973, vol. 3, pp. 77-84, Histidine and Histamine Complexes of Copper and Zinc, Walker, et al.
Inorganic Chemistry, vol. 12, No. 10, 1973, Structure of Sodium Nitrilotriacetatocopper (II), Monohydrate, Simon H. Whitlow, pp. 2286-2289.
Aust. J. Chem., 1970, 23, 1973, pp. 1973-1979, Imidazolato-Bridged Complexes of Copper (II), Bridson et al.
The Journal of Physical Chemistry, vol. 69, No. 2, Feb.1965, Infrared Spectra of Nitrilotriacetate Chelates in Aqueous Solution, Tomita, et al.
Journal of Chromatography, 411, (1987), 177-184, New Metal Chelate Adsorbent Selective For Pro
Knobbe Martens Olson & Bear LLP
Russel Jeffrey E.
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