Immobilisation of chelating groups for immobilised metal ion chromatography (IMAC)

#The present invention refers to a method for binding a polycarboxylic acid to a solid phase. Further, the invention refers to a solid phase having a polycarboxylic acid immobilized thereto and methods of using the solid phase, e.g. for purifying recombinant polypeptides.

#A very powerful strategy for purifying recombinant proteins is the use of a “His-tag”, which comprises typically 6-10 consecutive histidines. His-tags bind tightly to free coordination sites of Ni2+-ions. They can be released by a high concentration of imidazole, which competes for coordination sites on Ni2+. Such cycle of specific absorption and desorption can be used for one-step purification of a desired protein, resulting in enrichment factors of 100 or even more.

#The most widely used variant of this technique uses NαNα-bis(carboxymethyl)-lysine coupled via the ε-amino group to agarose beads. The active group is NTA (nitrilo triacetic acid) charged with Ni2+, while the spacer is an amino butyl group. Such Ni2+-NTA-matrix has, however, several serious disadvantages, such as the high costs for NαNα-bis(carboxymethyl)-lysine.

#FIG. 1: Scheme for the coupling of EDTA to an amine-containing support. The reaction is carried out under conditions where a selective reaction occurs between a single carboxyl group of EDTA with the amino group on the support.

#FIG. 2: Comparison of the protein binding specificity of various Ni2+-containing chromatographic matrices in IMAC.

#The protein binding characteristics of various His-tagged proteins, namely a comparative matrix (NTA-Qiagen) and three inventive matrices (EDTA-amide silica I, EDTA-amide silica II and EDTA-amide sepharose) are shown (cf. Example 5 for details).

#A) The binding characteristics of inventive spaced poly-His tags (spaced H14, spaced H21 and spaced H28) and comparative His tags (MRGS6 and H10) are shown.

#B) The expression of proteins containing an inventive spaced His-tag and a comparative His-tag (H10) are shown (cf. Example 9 for details).

#1 liter Sepharose 4B is prewashed on a glass-funnel with 1 liter 0.1 M NaOH (in water), followed by 5 times one liter of pure water, transferred to a 5 liter flask, and filled up with water to a volume of 2 liters. 0.8 mol of NaOH are added, the temperature adjusted to 25° C., followed by addition of 1.0 mol epibromhydrine. The mixture is then shaken for 2 hours at 25° C., and then chilled on ice. The resulting epoxy-activated Sepharose is subsequently recovered by filtration through a glass funnel, washed with water, and resuspended in 2 M NH4Cl (final concentration in water, final volume 2 liters). 4 mol NH3 are added from a 25% aqueous solution and the mixture is shaken o/n at room temperature.