Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-06-20
2004-03-09
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S113000, C536S121000, C536S123100, C556S117000, C556S133000, C556S134000, C556S147000, C556S176000, C556S183000, C562S553000, C562S555000
Reexamination Certificate
active
06703498
ABSTRACT:
INTRODUCTION
1. Field of the Invention
This invention relates generally immobilized metal ion affinity chromatography.
2. Background of the Invention
Immobilized Metal Ion Affinity Chromatography (IMAC) is one of the most frequently used techniques for purification of fusion proteins containing affinity sites for metal ions. IMAC is a separation principle that utilizes the differential affinity of proteins for immobilized metal ions to effect their separation. This differential affinity derives from the coordination bonds formed between metal ions and certain amino acid side chains exposed on the surface of the protein molecules.
Since the interaction between the immobilized metal ions and the side chains of amino acids has a readily reversible character, it can be utilized for adsorption and then be disrupted using mild (i.e., non denaturing) conditions. Adsorbents that are currently commercially available include iminodiacetic acid (IDA), nitriloacetic acid (NTA), caboxymethylated aspartic acid (CM-Asp), and tris-carboxymethyl ethylene diamine (TED). These ligands offer a maximum of tri-(IDA), tetra-(NTA, CM-Asp), and penta-dentate (TED) complexes with the respective metal ion.
In most commercially available adsorbents, metal chelating ligands are provided at an average density of about 12 Å. Depending on the ligand, various metals can be chelated. Metal ions typically used in IMAC procedures have been classified into three categories—hard, intermediate, and soft—based on their preferential reactivity toward nucleophiles. The hard metal ions Fe
3+
, Ca
2+
, and Al
3+
show a preference for oxygen; the soft metal ions Cu
+
, Hg
2+
, Ag
+
, and the like show a preference for sulfur; and intermediate metal ions such as Cu
2
+, Ni
2+
, Zn
2+
, and Co
2+
coordinate nitrogen, oxygen, and sulfur. The number of cysteine residues on the surfaces of proteins is limited; therefore, histidine residues are the major targets for intermediate metal ions.
Because of its commercial success, there is continued interest in the development of new “IMAC” technologies and applications. The present invention satisfies this need.
Relevant Literature
See U.S. Pat. Nos. 4,569,794; 5,047,513; 5,284,933; 5,310,663; 5,962,641; 5,594,115; and 6,242,581 as well as Chaga et al., Biotechnol. Appl. Biochem. (1997)26: 7-14. Also of interest are: Ford et al., Protein Expression and Purification (1991) 2:95-107; Hochuli, et al., J. Chromatography (1987) 411:177-184; Mantovaara et al., Biotechnology and Applied Biochemistry (1989) 11:564-570; Mantovaara et al., Biotechnology and Applied Biochemistry (1991) 13:315-322; Mantovaara et al., Biotechnology and Applied Biochemistry (1991) 13:120-126); Porath et al., Nature (1975) 258:598-599; Porath & Olin, Biochemistry (1983) 22:1621-1630; and Porath, J., Protein Purification and Expression (1992) 3:263-281.
SUMMARY OF THE INVENTION
Water-soluble metal ion affinity compounds and methods for using the same are provided. The subject compounds include an aspartate based metal chelating ligand bonded to a water-soluble polymeric substrate, where the ligand is complexed with a metal ion. In certain embodiments, the subject compounds further include a member of a signal producing system, e.g., a directly or an indirectly detectable label moiety. Also provided are water-insoluble supports having the subject compounds present on, e.g., immobilized on, at least one surface thereof. The subject compounds find use in a variety of different applications, including analyte detection and analyte purification applications.
Definitions
The terms “affinity peptide,” “high affinity peptide,” and “metal ion affinity peptide” are used interchangeably herein to refer to a histidine-rich peptide that binds to a metal ion.
The terms “protein of interest” and “fusion partner polypeptide,” used interchangeably herein, refer to any protein to which the affinity peptide is fused for the purpose of purification or immobilization.
As used herein, the term “fusion protein” refers to the protein hybrid comprising a metal ion affinity peptide and a fusion partner polypeptide.
As used herein, the term “metal ion” refers to any metal ion for which the affinity peptide has affinity and that can be used for purification or immobilization of a fusion protein. Such metal ions include, but are not limited to, Ni
+2
, Co
+2
, Fe
+3
, Al
+3
, Zn
+2
and Cu
+2
. As used herein, the term “hard metal ion” refers to a metal ion that shows a binding preference for oxygen. Hard metal ions include Fe
3+
, Ca
2+
, and Al
3+
. As used herein, the term “soft metal ion” refers to a metal ion that shows a binding preference of sulfur. Soft metal ions include Cu
+
, Hg
2+
, and Ag
+
. As used herein, the term “intermediate metal ion” refers to a metal ion that coordinates nitrogen, oxygen, and sulfur. Intermediate metal ions include Cu
2+
, Ni
2+
, Zn
2+
, and Co
2+
.
As used herein, the terms “adsorbent” or “solid support” refer to a chromatography or immobilization medium used to immobilize a metal ion.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Water-soluble metal ion affinity compounds and methods for using the same are provided. The subject compounds include an aspartate based metal chelating ligand bonded to a water-soluble polymeric substrate, where the ligand is complexed with a metal ion. In certain embodiments, the subject compounds further include a member of a signal producing system, e.g., a directly or an indirectly detectable label moiety. Also provided are water-insoluble supports having the subject compounds present on, e.g., immobilized on, at least one surface thereof. The subject compounds find use in a variety of different applications, including analyte detection and analyte purification applications.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of such proteins and reference to “the labeling method” includes reference to one or more labeling methods and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of t
Bozicevic, Field & Francis
Clontech Laboratories, Inc.
Field Bret E.
Maier Leigh C.
Wilson James O.
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