Metal chelating compositions for protein purification, detection or binding

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The present invention is generally directed to metal chelating compositions and to methods for making and using the same for protein purification, detection or binding and, in particular, to nitrilotriacetic acid derivatives that have improved binding specificity and stability and to methods for making and using these nitrilotriacetic acid derivatives for protein purification, protein detection or protein binding.

Metal chelate affinity chromatography has been used as a technique for the purification of proteins for many years. Early resins used in this process were simple chelators such as iminodiacetic acid (IDA) coupled to agarose supports (Porath et al. Nature, 258:598–599, 1975) and charged with various metals such as Cu2+, Zn2+ and Ni2+. These resins were found to selectively capture proteins and peptides from natural sources (Porath and Olin, Biochemistry, 22:1621, 1983; Lonnerdal and Keen, J. Appl. Biochem., 4:203, 1983; Sulkowski, Protein Purification: Micro to Macro, pages 149–162, Edited by R. Burgess, Published by Liss New York, N.Y., 1987). With the advent of molecular biological techniques, metal chelate chromatography assumed a more important role in the purification of proteins with the use of a 6-histidine tag. See, for example, Dobeli et al., U.S. Pat. No. 5,284,933. The poly histidine tag bound very strongly to the immobilized nickel and could be used for the identification and purification of these recombinant molecules. The tridentate chelator IDA was quite selective for these tagged proteins but the nickel was found to leach slowly from the resin reducing the capacity and causing interference with some downstream uses of the proteins.

More recently, a tetradentate chelator known as nitrilotriacetic acid resin was developed for use with metals having six coordination sites. This resin has become the preferred resin for the purification of poly histidine containing proteins since it has very little metal leaching and good selectivity. However, considerable amount of effort is required to obtain this selectivity. For example, the addition of various amounts of imidazole is necessary to determine whether protein must be optimized to achieve the desired results.

In U.S. Pat. No. 4,877,830, Dobeli et al. describe nitrilotriacetic acid resins suitable for protein purification represented by the general formula:

[carrier matrix]-spacer-NH—(CH2)x—CH(COOH)—N(CH2COO—)2Ni2+

wherein x is 2, 3 or 4, the carrier matrix is one used in affinity or gel chromatography such as cross-linked dextrans, agarose or polyacrylamides, and the spacer is preferably —O—CH2—CH(OH)—CH2— or —O—CO—. Dobeli et al., U.S. Pat. No. 4,877,830 at col. 2, lines 23–37. These resins are prepared by reacting an N-terminal protected compound of the formula:

R—HN—(CH2)x—CH(NH2)—COOH

wherein R is an amino protecting group and x is 2,3 or 4, with bromoacetic acid in an alkaline medium and subsequently cleaving off the protecting group and reacting this product with an activated resin. See, e.g., Hochuli et al., Journal of Chromatography, 411(1987) 177–184.

In U.S. Pat. No. 5,625,075, Srinivasan et al. describe a metal radionuclide chelating compound having multiple sulfur and nitrogen atoms. These chelating compounds incorporate two nitrogen atoms and three sulfur atoms, two nitrogen atoms and four sulfur atoms, or three nitrogen atoms and three sulfur atoms.

While these compounds provide improved specificity relative to some resins containing nitrilotriacetic acid derivatives, a need remains for chelating compounds having greater binding specificity for polyhistidine containing proteins.

Advantages are gained by the use of neutral ether, thioether, selenoether or amide linkage(s) in the linking moiety instead of positively charged amine linkages. Oxygen, sulfur, and selenium atoms and amides have some affinity for metals which may provide enhanced chelation properties, binding the metal more firmly than traditional tetradentate chelators having positive amine linkages. In addition, the use of a non-charged atoms between the nitrilo nitrogen and the carrier appears to reduce non-specific binding of proteins. Use of S, O, Se or amide in the linking moiety, L, therefore, in place of amine or other charged moieties tends to increase the stability and specificity of the composition for protein purification.