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

Abstract

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 His-tagged recombinant polypeptides.

Claims

1. A method for immobilized metal ion chromatography (IMAC), comprising contacting a polypeptide sample with an immobilized chelator having 6 or more coordination groups, wherein the immobilized chelator is a solid phase having a polycarboxylic acid immobilized thereto, wherein said immobilized chelator has a structure of formula (la) or (Ib): ##STR00005## wherein SP is a solid phase; R.sup.1 is hydrogen or an organic residue, and PCA is the residue of a polycarboxylic acid, and wherein the immobilized chelator is complexed with a Ni.sup.2+ ion.

2. The method according to claim 1, wherein said coordination groups are selected from the group consisting of amino, carboxyl, carboxamide and hydroxamate groups.

3. The method according to claim 1, wherein said PCA is the residue of an amino polycarboxylic acid or a salt thereof.

4. The method according to claim 3, wherein the polycarboxylic acid is selected from the group consisting of ethylene diamino tetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethy lether)-N, N, N, N-tetraacetic acid (EGTA), diethylene triamino pentaacetic acid (DPTA), triethylene tetra mine-N, N, N, N, N, N-hexa-acetic acid (TTHA), and salts thereof.

5. The method according to claim 4, wherein said polycarboxylic acid is ethylene diamino tetraacetic acid (EDTA).

6. The method of claim 1, wherein the solid phase has a structure of formula (II): ##STR00006## wherein SP is the solid phase, and wherein one or more of the carboxylic acid groups may be deprotonated.

7. A method for immobilized metal ion chromatography (IMAC), comprising contacting a sample with an immobilized chelator comprising a solid phase having a polycarboxylic acid or salt thereof immobilized thereto, wherein the immobilized chelator has a structure of formula (la) or (Ib): ##STR00007## wherein SP is the solid phase; R.sup.1 is hydrogen or an organic residue, and PCA is the polycarboxylic acid or a salt thereof, wherein said polycarboxylic acid is complexed with a transition metal ion, wherein the immobilized polycarboxylic acid amide has at least 6 or more coordination groups, which are selected from amino, carboxyl, carboxamide and hydroxamate groups, and wherein 90% of the accessible primary amino groups on the surface of the solid phase are blocked.

8. The method according to claim 7, wherein the polycarboxylic acid is selected from the group consisting of ethylene diamino tetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), diethylene triamino pentaacetic acid (DPTA), triethylene tetramine-N, N, N, N, N, N-hexa-acetic acid (TTHA), and salts thereof.

9. The method according to claim 8, wherein said polycarboxylic acid is ethylene diamino tetraacetic acid (EDTA).

10. The method of claim 7, wherein the solid phase has a structure of formula (II): ##STR00008## wherein SP is the solid phase, and wherein one or more of the carboxylic acid groups may be deprotonated.

11. The method according to claim 7, wherein substantially all loosely bound metal ions have been removed.

12. The method according to claim 7, wherein said transition metal is a Ni.sup.2+ ion.

Description

FIGURE LEGENDS

(1) 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.

(2) FIG. 2: Comparison of the protein binding specificity of various Ni.sup.2+-containing chromatographic matrices in IMAC.

(3) 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).

(4) FIG. 3: Combination of various chelators and transition metal ions for IMAC.

(5) The protein binding properties of various matrices (inventive EDTA, EGTA and TTHA matrices and an NTA matrix) with different chelators in the presence of various transition metal ions are shown (cf. Example 6 for details).

(6) FIG. 4: Resistance of Ni-chelate matrices against extraction of Ni.sup.2+ by free chelators.

(7) The strength of Ni.sup.2+ binding to an inventive EDTA-amide matrix and comparative NTA matrices is shown (cf. Example 7 for details).

(8) FIG. 5: Resistance of various Ni-chelate matrices against thiol-containing reducing agents.

(9) The resistance of various Ni.sup.2+ containing chelator matrices (an inventive EDTA-amide matrix and comparative NTA matrices) against Ni.sup.2+ extraction and reduction in the presence of DTT is shown (cf. Example 8 for details).

(10) FIG. 6: Characterisation of spaced poly-histidine tags.

(11) 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.

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

EXAMPLES

Example 1

Preparation of Ni-EDTA-amide Sepharose 4B

(13) 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 NH.sub.4Cl (final concentration in water, final volume 2 liters). 4 mol NH.sub.3 are added from a 25% aqueous solution and the mixture is shaken o/n at room temperature.

(14) The resulting NH.sub.2-Sepharose 4B is recovered by filtration, washed with water until free NH.sub.3 has be become undetectable, and resuspended in 0.5 M EDTA/Na.sup.+ pH 8.0 (final concentration, final volume 2 liters). Then, 50 mmol EDC are added and the mixture is shaken for 1 hour at room temperature. Thereafter, another 50 mmol EDC aliquot is added and the reaction is allowed to proceed o/n.

(15) The resulting EDTA-amide Sepharose is recovered by filtration and washed until the free EDTA-concentration has dropped below 1 mM. The Sepharose is then charged with 20 mM NiCl.sub.2 in Tris buffer pH 7.5, until free Ni.sup.2+ appears in the non-bound fraction. Free and loosely-bound Ni.sup.2+ ions are then removed by washing with water, 10 mM NTA pH 7.5, water, and is finally resuspended in 30% ethanol+10 mM imidazole/HCl+1 mM NTA pH 7.5 for long-term storage.

(16) The properties of the Ni.sup.2+ EDTA-amide Sepharose can be adjusted by varying the epoxy-activation step. Coupling at higher temperature (up to 40 C.) and using higher concentrations of epichlorhydrine and NaOH (up to 1.2 M epichlorhydrine and 1.0 M NaOH, respectively) will result in higher coupling-density, but also in higher background-binding. Lower temperature (down to 18 C.) using a lower concentration of epichlorhydrine and NaOH (down to 0.2 M epichlorhydrine and 0.1 M NaOH, respectively) will result in lower coupling density, and in even lower background binding, but also in lower specific binding capacity, in particular for proteins with short His-tags.

Example 2

Preparation of EDTA-Amide Magnetic Beads

(17) 5 ml of amine-terminated magnetic beads (Sigma #17643-5 ml) are washed in water and resuspended in a final volume of 5 ml in 0.5 M EDTA/Na.sup.+ pH 8.0. A 125 mol EDC aliquot is added and the reaction is shaken for 1 hour at room temperature. Thereafter, another 125 mol EDC aliquot is added and the reaction is continued o/n.

(18) The resulting EDTA-amid magnetic beads are recovered by magnetic separation, washed with water, and charged with Ni.sup.2+ or another metal ion analogously to Example 1.

Example 3

Preparation of High-Density EDTA-Amide Silica

(19) 100 g Davisil XWP1000 90-130 (Grace) are resuspended in 500 ml of 3% (v/v) aminopropyl triethoxy silane, 4% water, 93% ethanol and shaken gently for 2 hours at 40 C. and then o/n at room temperature. The amino-modified silica is recovered by filtration and free silane is removed by washing with pure water. Covalent coupling to EDTA and charging with Ni.sup.2+ are performed as described for Sepharose 4B. For long term-storage, the product can be dried out of water or isopropanol.

Example 4

Preparation of EDTA-Amide Silica with a Passivated Surface

(20) The Ni.sup.2+ EDTA-amide silica from Example 3 three still suffers from high background-binding when used in IMAC. This background may probably result from residual silanol- and amino groups (which for sterical reasons could not react with the activated EDTA) and/or from high surface concentrations of EDTA-amide. The background-problem can be solved by including a passivating silane during the modification of silica with aminopropyl-silane. The so far best passivating silane is the reaction product between glycidyl oxypropyl trimethoxy silane and 3-mercapto 1,2-propandiol.

(21) 3 ml glycidyl oxypropyl trimethoxy silane are mixed with 3 ml 2-mercapto 1,3-propandiol, 90 ml methanol, 4 ml water and 10 l 4-methyl morpholine, and the reaction is allowed to proceed for 30 minutes at 25 C. 150 l aminopropyl trimethoxy silane are added, and the resulting mixture is used to modify 30 g Davisil XWP1000 90-130 as described above. The ratio between the aminosilane and the passivating silane is in this example defined as 5%. It can, however, be varied between 1% and 50%. The resulting passivated amino-silica is then reacted with EDTA or another chelating group, charged with metal ions, and treated to remove loosely-bound metal ions as described above.

Example 5

Comparison of Ni2+-Containing Chromatographic Supports in IMAC

(22) The following chromatographic supports were tested:

(23) TABLE-US-00001 NTA-Qiagen: Ni.sup.2+-NTA Agarose purchased from Qiagen EDTA-amide silica I: Ni.sup.2+-charged EDTA-amide silica with 20% coupling density (Example 4) EDTA-amide silica II: Ni.sup.2+-charged EDTA-amide silica with 5% coupling density (Example 4) EDTA-amide Sepharose: Ni.sup.2+-charged EDTA-amide Sepharose 4B (Example 1)

(24) E. coli cells were resuspended in buffer (50 mM Tris/HCl pH 7.5, 2 mM magnesium acetate, 50 mM NaCl, 5 mM mercaptoethanol). A lysate was prepared and cleared by ultracentrifugation. Then, either 1 M of a fusion protein (NES-YFP-H.sub.10) comprising a nuclear export signal (NES), the yellow fluorescent protein (YFP) and a C-terminal deca His-tag, or 1 M of a fusion between the maltose binding protein (MBP) with a C-terminal hexa His-tag (MBP-H.sub.6), or 0.5 M of a fusion protein containing a deca His-tag, a zz-tag and the mouse exportin CRM1 (H.sub.10-zz-CRM1) were added and used as starting materials for the binding experiments. For purification of the deca-His-tagged proteins, starting materials were supplemented with 1 mM imidazole.

(25) 400 l starting material each were rotated o/n at 4 C. with 10 l chromatographic support. After washing with 5 ml buffer, bound proteins were eluted with 0.4 M imidazole pH 7.5. Analysis was by SDS-PAGE on 12% acrylamide gel, followed by Coomassie-staining. The load corresponds to 0.5 l matrix.

(26) FIG. 2 shows the results for NES-YFP-H.sub.10 (panel A), MBP-H.sub.6 (panel B) and H.sub.10-ZZ-CRM1 (panel C). All tested matrices exhibit efficient binding of His-tagged proteins. The matrices of the present invention however show significantly lower backgrounds compared to the NTA-Qiagen matrix.

Example 6

Testing of Various Chelators and Transition Metal Ions for IMAC

(27) Amino-silica with 5% coupling density (as described in Example 4) was conjugated with either EDTA, EGTA, NTA, or TTHA. Each of the resulting matrices was then charged with either Ni.sup.2+, Co.sup.2+, Zn.sup.2+, or Cu.sup.2+. The binding assays for NES-YFP-H.sub.10 were performed as described in Example 5, however, 100 mM NaCl were included in the wash buffer.

(28) FIG. 3 shows that the tested combinations of matrices and metal ions show efficient binding of NES-YFP-H.sub.10.

Example 7

Resistance of Ni-Chelate Matrices Against Extraction of Ni2+ by Free Chelators

(29) Each 1 ml of Ni.sup.2+-EDTA-amide silica (50% coupling density; prepared according to Example 4), Ni.sup.2+-NTA-amide silica (50% coupling density), or Ni.sup.2+-NTA-agarose (Qiagen) were washed over a time of 45 min with either 30 ml Tris-buffer, or 10 mM EDTA pH 7.6, or 10 mM EGTA pH 7.6, or 40 mM NTA pH 7.6, followed by equilibration in binding buffer (50 mM Tris/HCl pH 7.5, 500 mM NaCl, 5 mM MgCl.sub.2). 10 l of each pre-treated matrix was then used to bind a His.sub.10-MBP-GFP fusion (10 M concentration) out of 600 l E. coli lysate. The bound fraction was eluted with 75 l 1 M imidazol/HCl pH 7.5. 1 l of each eluate was then analysed by SDS-PAGE followed by Coomassie-staining. The results are shown in FIG. 4A. The inventive Ni.sup.2+-EDTA-amide silica matrix was completely resistant against all tested chelator-solutions. The comparative matrices were significantly less resistant. Ni.sup.2+-NTA-agarose was discharged by any of the chelator-treatments. Ni.sup.2+-NTA-amide silica was completely discharged by 10 mM EDTA and 40 mM NTA, but retained a small activity after treatment with EGTA.

(30) The chelator-washed matrices from the panel shown in FIG. 4A were equilibrated in 100 mM Tris/HCl pH 7.5, mixed with an equal volume of 1% dimethylglyoxime (dissolved in Ethanol) and incubated for 15 min at 60 and subsequently o/n at room temperature, before photographs were taken. The results are shown in FIG. 4B. A pink dimethylglyoxime-Ni.sup.2+ precipitate, representing loosely-bound Ni.sup.2+, occurred with untreated Ni.sup.2+-NTA-amide silica and with untreated Ni.sup.2+-NTA agarose, but not with the inventive Ni.sup.2+-EDTA-amide silica. Pre-washing of Ni.sup.2+-NTA-amide silica or of Ni.sup.2+-NTA agarose with the above-mentioned chelators removed loosely-bound Ni.sup.2+ to the same extent as it reduced the capacity to bind the histidine-tagged protein.

(31) Thus, the binding of Ni.sup.2+ ions to the inventive EDTA-amide matrix is significantly stronger than to comparative NTA matrices.

Example 8

Resistance of Ni-Chelator Matrices Against Thiol-Containing Reducing Agents

(32) The resistance of various Ni.sup.2+ containing chelator matrices against dithiothreitol (DTT), a thiol-containing reducing agent, was tested.

(33) NTA-agarose (Qiagen), NTA-amide silica and EDTA-amide silica were either left untreated or were incubated o/n at room temperature with 1 M DTT, buffered with 1 M Tris/HCl pH 7.5. The results are shown in FIG. 5A. The DTT treatment converted Ni.sup.2+ from NTA-agarose (Qiagen) and NTA-amide silica into brownish reduction products. In contrast, Ni.sup.2+ EDTA-amide silica remained fully unaffected.

(34) The above matrices (50 l) were resuspended in either 1 ml 1 M Tris/HCl pH 7.5 or 1 M Tris/HCl pH 7.5+1 M DTT. 5 min later, a His.sub.10-tagged red fluorescent protein was added and the binding reactions were rotated o/n at room temperature. The matrices were allowed to sediment by gravity and photographs were taken. The results are shown in FIG. 5B (upper panel). Red colour on the beads indicates binding of the his-tagged protein. In the absence of DTT, all matrices bound the his-tagged protein very well. The DTT-treatment fully abolished binding to Ni.sup.2+ NTA agarose (Qiagen) and Ni.sup.2+ NTA-amide silica. The non-bound fractions contained brownish reduction products of Ni.sup.2+ that were released from the beads. In contrast, binding of the His-tagged red fluorescent protein to the inventive Ni.sup.2+ EDTA amide silica remained unaffected by the DTT treatment.

(35) The bound fractions were eluted with 1 M imidazol pH 7.3, and analysed by SDS-PAGE followed by Coomassie staining. The results are shown in FIG. 5B (lower panel). The analysis confirmed that 1 M DTT completely abolished binding of the his-tagged protein to Ni.sup.2+ NTA-agarose or Ni.sup.2+ NTA-amide silica, while binding to the inventive Ni.sup.2+ EDTA-amide silica was not affected at all.

Example 9

Characterisation of Spaced Poly-Histidine Tags

(36) A mixture of DHFR derivatives tagged with various histidine tags was bound to Ni.sup.2+ EDTA amide silica and eluted slowly with a gradient of increasing imidazole concentration. The results are shown in FIG. 6A. (Actual concentrations are given above the lanes). Panel shows analysis of eluted fractions by SDS-PAGE/Coomassie-staining. Tags with greater number of histidines confer tighter binding to the matrix, elution at higher imidazole concentration, and thus better separation from contaminants that are not poly-histidine tagged. The following tags have been used (amino acids in single letter code):

(37) TABLE-US-00002 (SEQIDNO:1) MRGS6= MRGSHHHHHH (SEQIDNO:2) H10= MHHHHHHHHHH (SEQIDNO:3) spacedH14= MSKHHHHSGHHHTGHHHHSGSHHH (SEQIDNO:4) spacedH21= MSKHHHHSGHHHTGHHHHSGSHHHTGHHHHSGSHHH (SEQIDNO:5) spacedH28= MSKHHHHSGHHHTGHHHHSGSHHHTGHHHHSGSHHH TGHHHHSGSHHH

(38) Although His-tags with more histidines confer a more specific binding to Ni-chelate matrices, they pose the problem that continuous stretches of too many histidines compromises expression levels and solubility in E. coli. Interrupting continuous polyhistidine stretches with Gly, Ser, and/or Thre-containing spacers rectifies these problems. In the representative example shown, tagging of DHFR with a spaced His.sub.14 tag doubled the yield of soluble protein during recombinant expression in E. coli as compared to a conventional, continuous His.sub.10 tag (FIG. 6B).