Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1997-08-18
2001-06-05
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S413000, C540S200000, C540S205000, C540S229000, C540S320000
Reexamination Certificate
active
06242581
ABSTRACT:
BACKGROUND OF THE INVENTION
Immobilized metal ion affinity chromatography (IMAC) was first introduced by Porath (Porath, J., J. Carlsson, I. Olsson, G. Belfrage [1975]
Nature
258:598-599.) under the term metal chelate chromatography and has been previously reviewed in several articles (Porath, J. [1992]
Protein Purification and Expression
3:263-281; and articles cited therein). The IMAC purification process is based on the employment of a chelating matrix loaded with soft metal ions such as Cu
2+
and Ni
2+
. Electron-donating groups on the surface of proteins, especially the imidazole side chain of histidine, can bind to the non-coordinated sites of the loaded metal. The interaction between the electron donor group with the metal can be made reversible by lowering the pH or by displacement with imidazole. Thus, a protein possessing electron-donating groups such as histidine can be purified by reversible metal complex/protein interactions.
Several different metal chelating ligands have been employed in IMAC to purify proteins. Iminodiacetic acid (IDA) ligand is a tridentate and thus anchors the metal with only three coordination sites (Porath, J., B. Olin [1983]
Biochemistry
22:1621-1630). Because of the weak anchoring of the metal, metal leakage has been known to occur. The tris(carboxymethyl)ethylenediamine (TED) ligand is pentadentate and forms a very strong metal-chelator complex. The disadvantage of this is that proteins are bound very weakly since only one valence is left for protein interaction. Nitrilo triacetic acid (NTA) is a tetradentate ligand which attempts to balance the metal anchoring strength with metal-ion protein interaction properties (Hochuli, E., H. Dobeli, A. Schacher [1987]
J. Chromatography
411:177-184). Other chelating ligands have been reported and are mentioned. See, e.g., Porath (1992), supra. However, these ligands also have certain disadvantages, including decreased bonding capacity, decreased specificity, and increased metal leakage.
In 1991, Ford et al. (Ford, C., I. Suominen, C. Glatz [1991]
Protein Expression and Purification
2:95-107) described protein purification using IMAC technology (Ni-NTA ligand) as applied to recombinant proteins having tails with histidine residues (polyhistidine recombinant proteins). This method takes advantage of the fact that two or more histidine residues can cooperate to form very strong metal ion complexes. The NTA chelating ligand immobilized on agarose and loaded with Ni
2+
has been useful in this method (Hochuli et al., supra; U.S. Pat. No. 5,047,513). It is available commercially through Qiagen, Inc. (Chatsworth, Calif.). However, this resin has the disadvantage that the interchanges between metal ions and poly-histidine recombinant proteins are not optimal. Metal leakage can occur, and background proteins can sometimes contaminate purification of recombinant proteins.
A metal chelating gel, i.e., carboxymethylated aspartate (CM-Asp) agarose complexed with calcium, has been used for purifying native calcium-binding proteins (Mantovaara, T., H. Pertoft, J. Porath [1989]
Biotechnology and Applied Biochemistry
11:564-570; Mantovaara, T., H. Pertoft, J. Porath [1991]
Biotechnology and Applied Biochemistry
13:315-322; Mantovaara, T., H. Pertoft, J. Porath [1991]
Biotechnology and Applied Biochemistry
13:120-126). However, the Ca
2+
-CM-Asp complex described by Mantovaara et al., has among its disadvantages that it does not bind strongly to histidine-tagged recombinant proteins. Another disadvantage, in addition to this inferior binding property, is its non-selectivity for histidine tags.
By contrast, the subject invention comprises the CM-Asp chelating ligand complexed to a transition metal in an octahedral geometry (coordination number of 6). In this unique configuration, the metal complex can be advantageously suited for purification of poly-histidine fused recombinant proteins. This is a novel use of the CM-Asp ligand and is part of the subject of the invention herein described.
BRIEF SUMMARY OF THE INVENTION
The present invention concerns a novel IMAC purification method which employs immobilized carboxymethylated aspartate (CM-Asp) ligands specifically designed for purification of recombinant proteins fused with poly-histidine tags. The new purification method is based upon the CM-Asp chelating matrix having the following structure:
A general description of the matrix used in the invention and illustrated above is:
When R
4
—R
5
—R
6
=H:
M=transition metal ion in a 2+ oxidation state with a coordination number of 6;
R
1
=a linking arm connecting the nitrogen atom of CM-Asp with R
2
;
R
2
=a functional linking group through which CM-Asp linking arm R
1
is connected to R
3
;
R
3
=a polymer matrix, e.g., those polymer matrices typically used in affinity or gel chromatography.
When R
1
—R
2
—R
3
=H:
R
4
=a linking arm connecting the methylene carbon atom of the carboxymethyl group of CM-Asp with R
5
;
R
5
=a functional linking group through which CM-Asp linking arm R4 is connected to R
6
;
R
6
=a polymer matrix, e.g., those polymer matrices typically used in affinity or gel chromatography.
In a preferred embodiment:
M=Fe
2+
, Co
2+
, Ni
2+
, Cu
2+
, or Zn
2+
;
R
1
=—CH
2
CH(OH)CH
2
—, —CH
2
(OH)CH
2
—O—CH
2
CH(OH)CH
2
—, —(CH
2
)
4
NHCH
2
CH(OH)CH
2
—, and —(CH
2
)
2
NHCH
2
CH(OH)CH
2
—;
R
2
=O, S, or NH; and
R
3
=agarose.
In a particularly preferred embodiment:
M=Co
2+
;
R
1
=CH
2
CH(OH)CH
2
;
R
2
=O; and
R
3
=agarose, cross-linked.
Prior to loading the 6×His recombinant protein to the resin, recombinant cells can be lysed and sonicated. The lysate can then be equilibrated with an aqueous buffer (pH 8) which itself does not form chelates with the metal. An example of an aqueous that can be used at this step in the described procedure is 50 mM sodium phosphate (pH 8.0)/10 mM Tris-HCl (pH 8.0)/100 mM NaCl, or the like. The equilibration buffer can contain denaturing agents or detergents, e.g., 10% “TRITON X-100,” 6 M guanidinium HCl, or the like. After binding the prepared 6×His recombinant protein on the metal CM-Asp chelating resin (the “CM-Asp resin complex”), the protein-bound resin is washed at pH 7.0 or 8.0. The elution of the protein can be carried out at a constant pH or with a descending pH gradient. In a preferred embodiment, protein elution can be achieved at a pH of about 6.0 to about 6.3. Alternatively, the 6×His recombinant protein bound to the CM-Asp chelating resin can be washed with low concentrations (less than 100 mM) of imidazole at pH 8.0 and then eluted by increasing the imidazole concentration to 40-100 mM.
Also included as an aspect of the subject invention is a scaled-up synthesis of the CM-Asp derivatized agarose chelating resin. It is an improved version of a previously reported small scale preparation (Mantovaara, T., H. Pertoft, J. Porath [1991]
Biotechnology and Applied Biochemistry
13:315-322). The improvement includes particular conditions for oxirane-agarose formation, temperature controlled conjugation of aspartic acid to the oxirane-agarose, and high ionic strength washing to remove extraneously bound metals. These conditions, temperatures, and ionic concentrations are described in detail herein.
An additional application of the subject invention includes screening for protein function on a microtiter plate or filter. The additional applications for the subject invention also include protein-protein interaction studies, as well as antibody and antigen purification. For example, by immobilization of the Co
2+
moiety onto 96-well plates by CM-Asp, such plates can be used for quantitation of 6×Histidine-tagged protein, protein-protein interaction studies, diagnostic screening for diseases, antibody screening, antagonist and agonist screening for drugs, and reporter gene
Kain Steven R.
Nelson Paul S.
Smith Thomas H.
Yang Te-Tuan
Borden Paula A.
Bozicevic, Field & Francis
Clontech Laboratories, Inc.
Field Bret E.
Gupta Anish
LandOfFree
Method for purification of recombinant proteins does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for purification of recombinant proteins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for purification of recombinant proteins will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2481986