SITE-SPECIFIC, KINETICALLY INERT CONJUGATION OF LABELS AND/OR CARRIERS TO TARGET MOLECULES SUCH AS HIS-TAGGED PROTEINS VIA METAL COMPLEX REAGENTS

Abstract

The present invention relates to means and methods for conjugating/attaching target molecules such as proteins to a label and/or carrier. Specifically, the present invention provides a complex comprising a metal cation coordinating (i) a metal cation ligand being a carbonate selected from CO.sub.3.sup.2− and HCO.sub.3— and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier. This complex can be used for attaching a label and/or a carrier to a target molecule, preferably a protein. The attachment of the label or carrier via the complex of the invention involves the replacement of the metal cation ligand with a coordinating group of the target molecule so that a product complex with the target molecule as primary ligand in the coordination sphere of the metal cation is formed. Accordingly, the present invention also provides for uses and methods involving the attachment of a label and/or carrier to a target molecule. Also provided are the products obtained by the labeling and or carrier-attaching methods of the invention and uses thereof. The invention further relates to methods for producing the complex of the invention and kits comprising the components for producing the complex of the invention.

Claims

1. A complex comprising: a) a metal cation; b) a metal cation ligand being CO.sub.3.sup.2− or HCO.sub.3.sup.−; and c) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier.

2. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain is a polydentate ligand that comprises one or more carboxylic acid groups and/or one or more amine groups and/or one or more aromatic amines and/or phosphates.

3. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain of c) is selected from: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), chelating peptides such as peptides with the consensus sequence (GHHPH).sub.nG; with n=1 to 3; see SEQ ID NOs: 1 to 3) or cadystin, triazacyclononane (TACN), diethylenetriamine-pentaacetate (DTPA), phytochelatin, carboxymethylaspartate (CMA), phosphonates, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionicdiacetic acid (NPDA), nitriloisopropionicdiacetic acid (NIPDA), N-(hydroxylethyl)ethylenediaminetriacetic acid (HEDTA), 1,4,7,10-tetraazacyclodo-decane-N,N′,N″,N′″-tetraacetic acid (DOTA), 1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazocyclodecane, 1,4,7-triazacyclonane-1,4,7-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4.7-diacetic acid (NODAGA), 1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid (TETA), ethylenedicysteine, ethylenediaminetetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DACT), bis(aminoethanethiol)carboxylic acid, ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetramine-hexaacetic acid (TTHA), 1,4,7-triazacyclononane phosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyridimidine and derivatives thereof.

4. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain of c) is selected from: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), chelating peptides with the consensus sequence (GHHPH).sub.nG, diethylenetriamine-pentaacetate (DTPA), nitrilopropionicdiacetic acid (NPDA), nitriloi sopropionicdiacetic acid (NIPDA), ethylenediamine-tetraacetic acid (EDTA), ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), carboxymethylaspartate (CMA) and derivatives thereof.

5. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain of c) comprises or is selected from NTA, IDA and derivatives thereof.

6. The complex of any one of claims 1 to 4, wherein the metal cation is a transition metal cation.

7. The complex of any one of claims 1 to 5, wherein the metal cation of the complex is a di-, tri- or tetravalent metal cation.

8. The complex of any one of claims 1 to 6, wherein the metal cation is a metal cation having a water ligand exchange rate of 10.sup.−1 s.sup.−1 or lower, preferably 10.sup.−2 s.sup.−1 or lower.

9. The complex of any one of claims 1 to 7, wherein the metal cation is selected from the group consisting of: Co.sup.3+, Cr.sup.3+, Rh.sup.3+, Ir.sup.3+, Pt.sup.2+, Pt.sup.4+, Ru.sup.2+, Ru.sup.3+, La.sup.3+, Eu.sup.3+, Os.sup.2+, Pd.sup.4+, Mo.sup.3+, Fe.sup.3+, Ru.sup.3+, Gd.sup.3+, Tc.sup.3+, Re.sup.3+, Sm.sup.3+, Tb.sup.3+, Ce.sup.3+, Pr.sup.3+, Nd.sup.3+, Pm.sup.3+, Dy.sup.3+, Ho.sup.3+, Er.sup.3+, Tm.sup.3+, Yb.sup.3++, V.sup.2+, Mn.sup.4+, Fe.sup.2+ and Lu.sup.3+.

10. The complex of any one of claims 1 to 7, wherein the metal cation is selected from the group consisting of: Co.sup.3+, Cr.sup.3+, Rh.sup.3+, Ir.sup.3+, Ir.sup.4+, Pt.sup.2+, Pt.sup.4+, Pd.sup.4+, Mo.sup.3+, Fe.sup.3+, Gd.sup.3+, Tb.sup.3+, Eu.sup.3+, Ru.sup.3+, La.sup.3+, Ru.sup.3+, Re.sup.3+, Re.sup.4+, V.sup.2+, Mn.sup.4+, Fe.sup.2+ and Os.sup.2+.

11. The complex of any one of claims 1 to 7, wherein the metal cation is Co.sup.3+.

12. The complex of any one of claims 1 to 11, wherein the metal cation ligand of b) is carbonate CO.sub.3.sup.2− or bicarbonate HCO.sub.3.sup.−.

13. The complex of any one of claims 1 to 12, wherein the complex comprises a [Co(III)(NTA)CO.sub.3].sup.2−, a [Co(III)(NTA)HCO.sub.3].sup.− complex or a hydrate thereof, wherein the label and/or carrier is attached to NTA.

14. The complex of any one of claims 1 to 13, wherein the label comprises a fluorophore, a diagnostic, a targeting moiety, a therapeutic agent, a PEG molecule, a lipid, biotin and/or its derivatives, proteins, peptides, a toxin and/or a reactive group selected from a thiol, azide, alkyne, nitrone, tetrazine and tetrazole.

15. The complex of any one of claims 1 to 13, wherein the label comprises or is a fluorophore.

16. The complex of any one of claims 1 to 13, wherein the label comprises or is biotin or derivatives thereof.

17. The complex of any one of claims 1 to 13, wherein the carrier is a polymer, a hydrogel, a microparticle, a nanoparticle, a sphere (including nano- and microsphere), a bead, a quantum dot, a prosthetic or a solid surface.

18. The complex of any one of claims 1 to 17, wherein the metal cation chelating domain comprises a linker between the chelating ligand and the label and/or carrier.

19. A composition comprising a complex as defined in any one of claims 1 to 18.

20. Use of a complex of any one of claims 1 to 18 or a composition of item 19 for the labeling of a target molecule, wherein the target molecule comprises a protein, peptide or nucleic acid, preferably a protein or DNA that can exchange the metal cation ligand in the complex and even more preferably wherein said target molecule comprising at least 4 histidine residues or histidine-like residues in a sequence [H.sub.nS.sub.m].sub.k, wherein H is a histidine residue or a histidine-like residue, wherein S is a spacer amino acid residue, wherein n is in each case independently 1 to 4, wherein m is in each case independently 0 to 6, and wherein k is 2 to 6.

21. Use of a complex of any one of claims 1 to 18 or a composition of claim 19 for the labeling of a target molecule, wherein said target molecule contains a histidine-rich region comprising at least two histidine residues, wherein said histidine-rich region is formed by a three-dimensional folding of the target molecule that brings said at least two histidine residues in spatial proximity, wherein the at least two histidine residues have a distance of 0 to 5 angstroms and are not consecutive in the amino acid sequence.

22. The use of claim 21, wherein the histidine-rich region is the Fc region of an antibody.

23. Use of the complex of any one of claims 1 to 18 or a composition of item 19 for attaching a label and/or carrier to an antibody, a domain thereof (e.g. the Fc region) or fragments thereof.

24. The use of claim 23, wherein the label is a toxin.

25. Use of a complex of any one of claims 1 to 18 or a composition of claim 19 for the labeling of a target molecule, said target molecule contains a region enriched in histidine-like residues, which develops during three-dimensional folding of the target molecule when histidine-like residues come in spatial proximity, wherein the at least two histidine-like residues have a distance of 0 to 5 angstroms and are not consecutive in the amino acid sequence.

26. The use of claim 20, 21 or 25, wherein the target molecule is a pharmaceutical, a diagnostic, a research agent, a cosmetic and/or a protein for environment treatments (e.g. water treatment).

27. The use of claim 20, 21 or 25, wherein the target molecule comprises or is a peptide or protein.

28. The use of claim 20, 21, 25, 26 or 27, wherein the target molecule comprises or is an enzyme, a targeting protein such as an antibody, a cytokine, a transport protein such as FABS for fatty acid transport, a storage protein such as ferritin, a mechanical support protein such as collagen, a growth factor, a hormone such as insulin or TSH, an interferon, a glycoprotein a synthetically engineered protein or a fragment thereof.

29. The use according to any one of claims 20 to 28, wherein the target molecule comprises the histidine residues or histidine like-residues at the N-terminus, the C-terminus or at an internal sequence region.

30. The use according to any one of claims 20 to 28, wherein the histidine residues or histidine-like residues are comprised in form of a His tag, preferably wherein the His tag consists of 2 to 10, preferably 4 to 8, and most preferably 6 to 8 consecutive residues.

31. Use of a complex of any one of claims 1 to 18 or a composition of claim 19 for producing a pharmaceutical, a diagnostic and/or a cosmetic.

32. A kit comprising: a) a metal cation, preferably a metal cation as defined in any one of claims 5 to 11; b) a metal cation ligand being CO.sub.3.sup.2− or HCO.sub.3.sup.−; and c) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier, preferably a metal cation chelating domain as defined in any one of claims 2 to 4 and 14 to 18.

33. A method for producing a complex according to any one of claims 1 to 18 comprising incubating in a solution: (i) a metal cation; (ii) a metal cation ligand as defined in claim 1 b); and (iii) a metal cation chelating domain as defined in claim 1 c).

34. The method of claim 33 further comprising collecting and/or purifying the complex according to any one of claims 1 to 18.

35. The method of claim 32 or 33, wherein the metal cation chelating domain is a metal cation chelating domain as defined in any one of claims 2 to 18.

36. The method of any one of claims 33 to 35, wherein the metal cation is a metal cation as defined in any one of claims 5 to 11.

37. The method of any one of claims 33 to 36, wherein the metal cation is Co.sup.3+, wherein the metal cation binding ligand is CO.sub.3.sup.2− or HCO.sub.3.sup.−, and wherein Co.sup.3+ and CO.sub.3.sup.2− or HCO.sub.3.sup.− are provided in form of a neutral complex with counter ions, such as in form of a salt, or in form of a charged complex comprising the Co.sup.2+ and CO.sub.3.sup.2− or HCO.sub.3.sup.−.

38. The method of claim 37, wherein the neutral complex is sodium tris-carbonatocobalte(III) trihydrate (Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O) or potassium tris-carbonatocobalte(III) trihydrate (K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O).

39. The method of any one of claims 33 to 38, wherein the incubation is performed in a buffer comprising HCO.sub.3.sup.− or CO.sub.3.sup.2−, preferably in a concentration of at least 1 mM, preferably at least 10 mM and most preferably 1 M.

40. A method for attaching a label and/or a carrier to a target molecule comprising the step of incubating the complex of any one of claims 1 to 18 or the composition of claim 19 with a target molecule, wherein the target molecule is a target molecule as defined in any one of claims 20 to 30.

41. The method of claim 40, wherein the method further comprises the step of recovering and/or purifying the target molecule with the label and/or carrier linked thereto.

42. The method of claim 40 or 41, wherein the solution has a pH of between 4.0 to 9.5, preferably 5.5 and 8.0.

43. The method of any one of claims 40 to 42, wherein the incubation is performed for at least 10 sec, preferably 1 min, most preferably 10 min.

44. The method of any one of claims 40 to 43, wherein the incubation is performed at a temperature between 0 and 95° C., preferably between 0 and 60° C. and most preferably between 0 to 42° C.

45. The method of any one of claims 40 to 44, wherein the method further comprises washing the complex of the invention in a solution comprising HCO.sub.3.sup.− or CO.sub.3.sup.2−, preferably at a concentration of at least 1 mM, preferably 10 mM and most preferably 1 M, before the incubation and/or wherein the incubation is performed in a solution comprising HCO.sub.3.sup.− or C03.sup.2-, preferably in a concentration of at least 1 mM, preferably 10 mM and most preferably 1 M.).

46. The method of any one of claims 40 to 45, wherein the incubation is performed in water or an aqueous solution.

47. The method of any one of claims 40 to 46, wherein the incubation is performed in a solution comprising one or more organic solvents selected from the group consisting of: DMSO, DMF, DMS, acetonitrile and isopropanol.

48. The method of any one of claims 40 to 47, wherein the incubation is performed in an aqueous solution containing one or more Good's buffer substances, Tris, phosphate and/or carbonate/bicarbonate.

49. The method of any one of claims 40 to 48, wherein the incubation is performed in the presence of Ca.sup.2+, preferably provided in form of CaCl.sub.2.

50. The method of any one of claims 40 to 49, wherein the incubation is performed in an aqueous solution containing one or more buffer substances, wherein the buffer substance(s) do not comprise an amine, carboxylic acid, aromatic amine and/or phosphate group.

51. The method of any one of claims 40 to 50, wherein the incubation is performed in an aqueous solution containing one or more buffer substances selected from the group consisting of: ACES, AMPSO, BES, BisTris, BisTris propane, borate, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES, carbonate/bicarbonate buffers, phosphate buffers (e.g. PBS) and Tris.

52. The method of any one of claims 40 to 51, wherein the incubation is performed in an aqueous solution containing one or more buffer substances selected from the group consisting of: BisTris, CAPS, CAPSO, HEPES, HEPBS, HEPPSO MES, MOPS, MOPSO, PIPES, TAPS, TES, phosphate buffers (e.g. PBS) and Tris.

53. The method of any one of claims 40 to 52, wherein the incubation is performed in an aqueous solution comprising a buffer substance selected from the group consisting of: Bis-Tris, MES, HEPES and PIPES.

54. The method of any one of claims 40 to 53, wherein the method does not involve an oxidation step such as a treatment with H.sub.2O.sub.2 in the presence of the label and/or carrier.

55. A labeled or carrier-attached target molecule obtainable by the method as defined in any one of claims 40 to 54.

56. A composition comprising the labeled or carrier-attached target molecule of claim 55.

57. Use of the labeled or carrier-attached target molecule of claim 55 or the composition of claim 56 as a research reagent.

58. The labeled or carrier-attached target molecule of claim 55 or the composition of claim 56 for use as a medicament.

Description

FIGURES

[0309] FIG. 1: Chemical reactivity of the [Co(III)(NTA)(His-protein)] complex.

[0310] NTA beads with immobilized His.sub.6-GFP (SEQ ID NO: 14) at Co.sup.2+ and Co.sup.3+ complex centers were incubated with different chelators and reducing agents in combination with 250 mM imidazole and the amount of eluted His.sub.6-GFP was measured. Due to the kinetic inertness of the Co.sup.3+ centers, His.sub.6-GFP is almost not eluted when immobilized on the beads in form of [Co(III)(NTA)(His.sub.6-GFP)]. Graph is adapted from Wegner and Spatz, 2013 (Wegner and Spatz, 2013).

[0311] FIG. 2: Schematic overview of methods for the labeling of a His-tagged protein via [Co(III)(NTA)] chemistry.

[0312] A Formation of the [Co(III)(NTA)(His-protein)] complex by oxidation of preformed [Co(II)(NTA)(His-protein)] complex as published in 2013 by Wegner & Spatz (Wegner and Spatz, 2013). NTA is preloaded with Co.sup.2+ ions (from Co(II)Cl.sub.2) and incubated with His-tagged protein. Finally, the Co.sup.2+ center is converted to Co.sup.2+ by oxidating the whole protein complex for 1 h with 20 mM H.sub.2O.sub.2. This method, however, has a major disadvantage since the oxidation step can affect function and stability of the conjugated protein. Furthermore, conjugates attached to the NTA moiety can be affected by the oxidation process.

[0313] B Formation of the [Co(III)(IDA)(His-protein)] complex by oxidation of preformed [Co(II)(IDA)(H.sub.2O).sub.3] complex as published in 2006 by Zatloukalová & Kucerová (Zatloukalova and Kucerová, 2006). IDA is preloaded with Co.sup.2+ ions (from Co(II)Cl.sub.2) and the Co.sup.2+ center is converted to Co.sup.3+ by oxidating the [Co(II)(IDA)(H.sub.2O).sub.3] complex for 1 h with 20 mM H.sub.2O.sub.2. Subsequently, the [Co(III)(IDA)(H.sub.2O).sub.3].sup.+ complex is incubated with His-tagged protein. This procedure is limited due to a very slow complex formation and a reduced binding efficacy. Furthermore, conjugates attached to the IDA moiety can be affected by the oxidation process.

[0314] C Formation of the [Co(III)(NTA)(His-protein)] complex by using cobalt(III) carbonate salts. NTA is preloaded with Co.sup.2+ ions from cobalt(III) carbonate salts (e.g. Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O) so as to form the complex of the invention. The complex is then incubated with His-tagged protein. This procedure is a very simple workflow for the conjugation of His-tagged proteins which can be performed under mild reaction conditions. The function of the proteins as well as the NTA conjugate, i.e. a label (e.g. a fluorophore) and/or carrier, can be fully preserved as the reaction can be performed continuously in physiological buffer conditions. Additionally, the carbonate ligand allows a faster and more efficient protein binding compared to the two water ligands in procedure B.

[0315] D Formation of the [Pt(IV)(NTA)(His-protein)] complex by using platinum(IV) nitrate salts. NTA is preloaded with Pt.sup.4+ ions from platinum(IV) nitrate solutions so as to form the complex of the invention. The complex is then incubated with His-tagged protein. This procedure is a very simple workflow for the conjugation of His-tagged proteins which can be performed under mild reaction conditions. Thereby the nitrate ligand allows a faster and more efficient protein binding compared to the two water ligands in procedure B.

[0316] FIG. 3: Oxidation of fluorophores with hydrogen peroxide. Different fluorophores were incubated for about 21 hours with 0.05% H.sub.2O.sub.2 following an incubation in 1% H.sub.2O.sub.2. The fluorescence of the fluorophores was measured every 15 (0.05% H.sub.2O.sub.2) to 30 (1% H.sub.2O.sub.2) minutes. All fluorophores show a decrease in fluorescence intensity during the H.sub.2O.sub.2 treatment illustrating that oxidation steps with H.sub.2O.sub.2 can interfere with the function and stability of labels.

[0317] FIG. 4: Protein degradation and His-tag cleavage during cobalt oxidation by H.sub.2O.sub.2. His-tagged protein (3.3 μM) was incubated for 1 hour with or without 66 μM CoCl.sub.2 and 20 mM H.sub.2O.sub.2. Protein stability and His-tag cleavage was analyzed by Western Blot using an α-His.sub.6-tag-antibody coupled to horse radish peroxidase (clone H-3)). Upon exposure to cobalt and H.sub.2O.sub.2 the protein spontaneously showed partial signs of degradation as well as cleavage of the His-tag. Gel lanes from presented image originate from the same gel/membrane.

[0318] FIG. 5: .sup.1H-NMR spectra of NTA and its cobalt complexes.

[0319] A .sup.1H-NMR measurements of NTA show a peak at ˜3.6 ppm, which correlates with the spectra created in silco (calculated peak at 3.57 ppm) using the software NMR Predict (https://www.nmrdb.org/new_predictor/index.shtml?v=v2.103.0; version April 2019), see Banfi and Patiny, 2008; Castillo et al. 2011; Aires-de-Sousa et al. 2002). B When NTA is complexed with Co.sup.2+ and D.sub.2O to [Co(II)(NTA)(D.sub.2O).sub.2].sup.− (synthesized with CoCl.sub.2*6H.sub.2O) the peak from ˜3.6 ppm is shifted to ˜3.8 ppm. C+D When the complexes are produced with Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O (C) or K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O (D) the peak shifts further to ˜1.9 ppm indicating the presence of the desired carbonato complex [Co(III)(NTA)(CO.sub.3)].sup.2−. All ppm shifts are normalized to the peak of remaining H.sub.2O at 4.7 ppm.

[0320] FIG. 6: Stability of the [Co(III)(NTA)(His.sub.6-PercevalHR)] complex obtained via [Co(III)(NTA)(CO.sub.3)].sup.2−. NTA beads with immobilized His.sub.6-PercevalHR (SEQ ID NO: 15) at Ni.sup.2+ and Co.sup.3+ complex centers (latter produced with Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O) were washed with PBS or imidazole (250 mM) and the amount of His.sub.6-PercevalHR remaining on the beads was determined by fluorescence measurements. [Co(III)(NTA)(His.sub.6-PercevalHR)] complexes produced via [Co(III)(CO.sub.3).sub.3] salts show thereby a similar chemical stability in imidazole as for complexes formed via the H.sub.2O.sub.2 oxidation procedure (compare to FIG. 1). Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant.

[0321] FIG. 7: Protein binding to [Co(III)(NTA)] complexes.

[0322] Agarose beads functionalized with [Ni(II)(NTA)(H.sub.2O).sub.2].sup.−, [Co(III)(NTA)(H.sub.2O).sub.2] or [Co(III)(NTA)(CO.sub.3)].sup.2− were incubated with His.sub.6-GFP (SEQ ID NO: 14).

[0323] A The remaining unbound protein was determined by fluorescence measurements of the supernatant at different time points. The [Co(III)(NTA)] complexes with an associated carbonate molecule bind the protein significantly faster than [Co(III)(NTA)] complexes with associated water molecules.

[0324] B After an incubation of 312 h the beads were washed with buffer or 250 mM imidazole and the amount His.sub.6-GFP remaining on the beads was determined by fluorescence measurements. All [Co(III)(NTA)(His-GFP)] complexes show thereby a similar chemical stability in imidazole. However, beads functionalized with [Co(III)(NTA)(CO.sub.3)].sup.2− complexes bind significantly more protein than beads preloaded with [Co(III)(NTA)(H.sub.2O).sub.2]. Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant

[0325] FIG. 8: Effect of buffer substance on protein binding.

[0326] A+B: Agarose beads functionalized with [Co(III)(NTA)(CO.sub.3)].sup.2− were incubated with His.sub.6-GFP (SEQ ID NO: 14) in different buffers. The remaining unbound protein was determined by fluorescence measurements of the supernatant at different time points. In MES and Bis-Tris based buffer solutions the complex formation was faster compared to Tris based buffers and more efficient compared to Tris and HEPES based buffers. Graph B is a zoom from graph A at the time points 0 and 3 hours after experiment start. Error bars: +/−SD.

[0327] FIG. 9: UV-Vis Spectra of [Co(III)(NTA)(CO.sub.3)].sup.2− and [Co(III)(NTA)(H.sub.2O).sub.2]

[0328] Visible absorption spectra of [Co(III)(NTA)(CO.sub.3)].sup.2− and [Co(III)(NTA)(H.sub.2O).sub.2] in aqueous solution at room temperature. [Co(III)(NTA)(CO.sub.3)].sup.2− is produced by incubating NTA with Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O and [Co(III)(NTA)(H.sub.2O).sub.2] is produced by oxidation with H.sub.2O.sub.2 of the [Co(II)(NTA)(H.sub.2O).sub.2] complex formed by incubating NTA with Co(II)Cl.sub.2*6H.sub.2O. A peak shift of each of the two maxima demonstrates the presence of the carbonate ligand at the [Co(III)(NTA)(CO.sub.3)].sup.2− complex.

[0329] FIG. 10: Co(III)(NTA)(His-GFP)] complex formation and stability in dependence of incubation time and temperature of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O with NTA NTA-functionalized agarose beads are incubated for different time periods at 4° C. (A), 25° C. (B) and 70° C. (C). Subsequently, resulting [CoIII)(NTA)(CO.sub.3)].sup.2− complexes are incubated for 48 h with His.sub.6-GFP. Stability of the final [Co(III)(NTA)(His-GFP)] complex is tested by a stringent wash with either a HEPES-based buffer or 250 mM imidazole in buffer. Protein amount immobilized on beads was determined by BCA-assay. With increasing Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation times the percentage of stable complexes as well as the immobilized His.sub.6-GFP on beads increases when protein is incubated subsequently for 48 h at 25° C. With elevating temperatures, saturation of immobilized protein is reached earlier. Error bars: +/−SD

[0330] FIG. 11: [Co(III)(NTA)(His-GFP)] complex formation and stability in dependence of [Co(III)(NTA)(CO.sub.3)].sup.2− with His.sub.6-GFP incubation time and temperature

[0331] [CoIII)(NTA)(CO.sub.3)].sup.2− functionalized magnetic agarose beads are incubated for different time periods at 4, 25 and 37° C. and stability of the final [Co(III)(NTA)(His-GFP)] complex is tested by a stringent wash with either protein binding buffer or 250 mM imidazole in buffer. Protein amount immobilized on beads was determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). A His-GFP immobilized on beads after imidazole treatment based in their relative fluorescence. With longer protein incubation times also more protein could be immobilized on the beads starting to reach a saturation plateau at 3.5 h. No effect of the protein incubation temperature on the yield of the final complex could be observed. B Percentage of His-GFP immobilized on beads after imidazole treatment in comparison to buffer wash. All produced complexes demonstrate a high stability towards treatment 250 mM imidazole. Error bars: +/−SD

[0332] FIG. 12: [Co(III)(NTA)(His-GFP)] complex formation and stability with K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O

[0333] NTA functionalized magnetic agarose beads are incubated for indicated times with K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O and followed by an incubation of His.sub.6-GFP with the produced [CoIII)(NTA)(CO.sub.3)].sup.2− complex. Stability of the final [Co(III)(NTA)(His-GFP)] complex is tested by a stringent wash with protein buffer (“buffer”) or 250 mM imidazole in buffer (“imidazole”). Protein amount immobilized on beads was determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). [CoIII)(NTA)(CO.sub.3)].sup.2− complexes produced via K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O can also coordinate with His-GFP to form stable [Co(III)(NTA)(His-GFP)] complexes.

[0334] Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant

[0335] FIG. 13: [Co(III)(NTA)(His-GFP)] complex formation in different buffer systems and its complex stability with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation

[0336] [CoIII)(NTA)(CO.sub.3)].sup.2−-functionalized magnetic agarose beads after 10 min of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time are incubated for different time periods with His.sub.6-GFP in different buffer systems. Stability of the final [Co(III)(NTA)(His-GFP)] complex is tested by a stringent wash with a buffer solution (“buffer”) followed by 250 mM imidazole in buffer (“imidazole”). Protein amount immobilized on beads was determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). A Protein amount immobilized on beads after imidazole treatment B His-GFP immobilized on beads after 15 min protein incubation before and after imidazole treatment. The formation of a stable [Co(III)(NTA)(His-GFP)] complex is possible with all protein binding buffer systems, but with varying efficiencies for bound protein and stability percentages of the final complex. Error bars: +/−SD

[0337] FIG. 14: [Co(III)(NTA)(His-GFP)] complex formation in different buffer systems and its complex stability with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation [CoIII)(NTA)(CO.sub.3)].sup.2−-functionalized magnetic agarose beads after 48 h of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time are incubated for different time periods with His.sub.6-GFP in different buffer systems. Stability of the final [Co(III)(NTA)(His-GFP)] complex is tested by a stringent wash with either analysis buffer (“buffer”) or 250 mM imidazole in analysis buffer (“imidazole”). Protein amount immobilized on beads was determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). A Protein amount immobilized on beads after imidazole treatment B−E His-GFP immobilized on beads after 1 min (B), 15 min (C), 1 h (D) or 24 h (E) protein incubation with or without imidazole treatment. The formation of a stable [Co(III)(NTA)(His-GFP)] complex is possible with all protein binding buffer systems, but with varying efficacies for bound protein. Error bars: +/−SD

[0338] FIG. 15: [Co(III)(NTA)(His-GFP)] complex formation at different pH values and its complex stability

[0339] [CoIII)(NTA)(CO.sub.3)].sup.2−-functionalized magnetic agarose beads after 48 h of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time are incubated for different time periods with His.sub.6-GFP in BisTris- or HEPES-based buffer systems at different pH values. Stability of the final [Co(III)(NTA)(His-GFP)] complex is tested by a stringent wash with either analysis buffer (“buffer”) or 250 mM imidazole in analysis buffer (“imidazole”). Protein amount immobilized on beads was determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). A Protein amount immobilized on beads after imidazole treatment B His-GFP immobilized on beads after 15 min protein incubation with or without imidazole treatment. The formation of a stable [Co(III)(NTA)(His-GFP)] complex is possible at all pH values, but with varying efficacies for bound protein. Thereby the efficiency increases with the protein incubation and with decreasing pH values during protein incubation. Error bars: +/−SD

[0340] FIG. 16: Stable immobilization of different proteins with His-tag or histidine-rich region via [Co(III)(NTA)(CO.sub.3).sup.2−

[0341] [Co(III)(NTA)(CO.sub.3).sup.2− funtionalized magnetic agarose beads are incubated with His-GFP, His-ProteinA, His-Sortase, His-Human Serum Albumin or anti-GFP mouse IgG1 and stability of the final [Co(III)(NTA)(protein)] complex is tested by a stringent wash with either protein binding buffer or 250 mM imidazole in buffer. A SDS-PAGE of protein supernatant after protein incubation with [Co(III)(NTA)(CO.sub.3)].sup.2−. Marker: 200, 150, 100, 75, 50, 37, 25 kDa. Lane 1, 4, 7, 10 and 13: protein remaining after [Co(III)(NTA)(CO.sub.3).sup.2− incubation. Lane 2, 5, 8, 11 and 14: protein remaining after [Ni(II)(NTA)(H.sub.2O).sub.2).sup.− incubation. Lane 3, 6, 9, 12 and 15: protein remaining after NTA incubation. Lane 1-3: His-GFP, lane 4-6: His-ProteinA, lane 7-9: His-Sortase, lane 10-12: His-HSA, lane 13-15: anti-GFP mouse IgG1. Except for His-ProteinA, where it is just a high percentage, all protein could be cleared from the supernatant after protein incubation. B Protein amount on beads determined by BCA-assay after imidazole treatment. Stable [Co(III)(NTA)(protein)] complex formation could be achieved for all proteins. C Activity of immobilized Sortase before and after imidazole treatment determined fluometrical with the SensoLyte® 520 Sortase A Activity Assay Kit. Sortase is still active after immobilization and the formed [Co(III)(NTA)(His-Sortase)] complex could resist a treatment with 250 mM imidazole. D+E GFP binding to anti-GFP mouse IgG1 immobilized on NTA (D) oder IDA (E) functionalized beads determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). Immobilized antibody was still functional demonstrated by GFP binding.

[0342] FIG. 17: IDA and TALON as metal binding domain. Formation and chemical stability of A [Co(III)(IDA)(His-GFP)] and B [Co(III)(TALON)(His-GFP)] complexes produced via [Co(III)(IDA)(CO.sub.3)].sup.− or [Co(III)(TALON)(CO.sub.3)] complexes, respectively. Incubation times of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O followed by His.sub.6-GFP protein are indicated in brackets (Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O/protein incubation time). Chemical stability was tested by a stringent wash with either a HEPES-based buffer or 250 mM imidazole in buffer. Protein amount immobilized on beads was measured based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). Significantly more His.sub.6-protein was chemically stable immobilized on the beads using [Co(III)(IDA/TALON)(CO.sub.3)].sup.2− complexes compared to beads without metal treatment. Thereby the chemically stability of complexes with the tridentate metal binding domain IDA was dramatically increased versus tetradentate TALON complexes.

[0343] Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant.

[0344] FIG. 18: Chemical reactivity of the [Co(III)(IDA)(His-protein)] complex.

[0345] Beads functionalized with [Co(III)(IDA)(His.sub.6-GFP)] complex were incubated with different conditions including chelators or reducing agents in combination with 250 mM imidazole and the amount of His-GFP remaining on the beads is measured by fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). Due to the kinetic inertness of the Co.sup.3+ centers, His.sub.6-GFP is almost not eluted, demonstrating the high stability of the produced [Co(III)(IDA)(His-protein)] complex. Error bars: +/−SD

[0346] FIG. 19: Immobilization efficiency of His.sub.6-GFP to [Co(III)(IDA)(CO.sub.3)].sup.− and [Co(III)(IDA)(H.sub.2O).sub.2].sup.+

[0347] Complex formation efficiency and chemical stability of [Co(III)(IDA)(His-GFP)] produced via Co(III)(IDA)(CO.sub.3)].sup.− or [Co(III)(IDA)(H.sub.2O).sub.2].sup.+complexes on magnetic beads after A 3 h and B 24 h of His.sub.6-GFP incubation. Co(III)(IDA)(CO.sub.3)].sup.− complexes were produced by incubating IDA-functionalized magnetic beads with Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O for indicated time (10 min or 48 h). Chemical stability was tested by a stringent wash with either a HEPES-based buffer or 250 mM imidazole in buffer. Protein amount immobilized on beads was measured based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). Significantly more His.sub.6-protein was chemically stable immobilized on the beads using [Co(III)(IDA)(CO.sub.3)].sup.− complexes prepared with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O incubation compared to beads functionalized with Co(III)(IDA)(H.sub.2O).sub.2].sup.+ complexes. [Co(III)(IDA)(CO.sub.3)].sup.− complexes prepared with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O incubation outcompeted Co(III)(IDA)(H.sub.2O).sub.2].sup.+ complexes after a protein incubation time of 24 h. All [Co(III)(IDA)(His-GFP)] complexes demonstrated a high chemical stability towards imidazole.

[0348] Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant

[0349] FIG. 20: [Co(III)(IDA/NTA)(His-GFP)] chemically stable complex formation via [Co(III)(IDA/NTA)(CO.sub.3)] versus oxygen treatment of [Co(H)(metal binding domain)(His-GFP)]. Chemical stability of A [Co(III)(IDA)(His-GFP)] or B [Co(III)(NTA)(His-GFP)] complexes produced via [Co(III)(metal binding domain)(CO.sub.3)].sup.2− complexes (Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O/protein incubation time) or oxidation of [Co(II)(metal binding domain)(His-GFP)] with oxygen (8 h) or hydrogen peroxide (20 mM, 1 h) treatment on magnetic beads was challenged with a treatment of a HEPES-based buffer or 250 mM imidazole in buffer. Protein amount immobilized on beads was measured based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). Significantly more His.sub.6-protein was chemically stable immobilized on the beads using [Co(III)(metal binding domain)(CO.sub.3)] complexes compared to a 8 hour oxygen treatment of [Co(II)(metal binding domain)(His-GFP)].

[0350] Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant

[0351] FIG. 21: Formation of Co(III)(HS-PEG-NTA)(His-GFP)] complexes on surfaces

[0352] His-GFP was immobilized via [Co(III)(HS-PEG-NTA(CO.sub.3)].sup.2− complexes to a gold nano-structured glass surface. Nanostructured gold dots are functionalized via thiol-PEG-NTA followed by an incubation with Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O to form [Co(III)(HS-PEG-NTA(CO.sub.3)].sup.2− complexes, to which subsequently His.sub.6-GFP is immobilized by formation of Co(III)(HS-PEG-NTA)(His-GFP)] complexes. All surfaces are passivated with a short PEG layer in-between the gold dots to avoid nonspecific protein interactions with the glass surface. The amount of immobilized His-GFP was determined based on the fluorescence on the surface (λ.sub.ex=490 nm, λ.sub.em=535 nm) confirming the GFP immobilization on the surface. “PEG only”: passivated surface; “PEG/GFP”: passivated surface incubated with His-GFP; “no metal”: passivated surfaces incubated with thiol-PEG-NTA and His-GFP; “[Co(III)(NTA)(His-GFP)]”: passivated surface incubated with thiol-PEG-NTA, Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O and His-GFP.

[0353] FIG. 22: Formation of [Co(III)(NTA-X-Biotin)(His-GFP)] complexes in solution His-GFP was coupled to a biotin moiety via [CoIII)(NTA-X-Biotin)(CO.sub.3)] complexes produced by incubating Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O with NTA-X-Biotin followed by His-GFP incubation with the resulting complex for different incubation times. The final complex was immobilized to beads functionalized with streptavidin via biotin-streptavidin interaction. Amount of immobilized protein as measure for biotinylated protein was determined based on the fluorescence of the beads slurry (λ.sub.ex=490 nm, λ.sub.em=535 nm). A [Co(III)(NTA-X-Biotin)(His-GFP)] complexes generated after 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O/NTA-X-Biotin and 30 min [CoIII)(NTA-X-Biotin)(CO.sub.3)]/His-GFP incubation immobilized on streptavidin beads. For all Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O to NTA ratio a significant amount of His-GFP could be biotinylated and immobilized on the beads. Thereby higher Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O ratios lead to increased labeling rates. B [Co(III)(NTA-X-Biotin)(His-GFP)] complexes generated after 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA-X-Biotin and 30 min or 48 h [CoIII)(NTA-X-Biotin)(CO.sub.3)]/His-GFP incubation immobilized on streptavidin beads after a stringent wash with 250 mM imidazole in buffer. With increasing protein incubation times the amount of immobilized and therefore chemical stable complex increases.

[0354] Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant

[0355] FIG. 23: His.sub.6-GFP immobilization on [Pt(IV)(NTA)(NO.sub.3)] functionalized beads NTA-functionalized agarose beads were incubated for 10 min with platinum(IV) nitrate followed by an incubation for 30 min with His.sub.6-GFP. Chemical stability of the formed [Pt(IV)(NTA)(His.sub.6-GFP)] complex was challenged with 250 mM imidazole and immobilized protein on beads was determined by BCA assay. The stable immobilization of a significant amount of protein via [Pt(IV)(NTA)(NO.sub.3)] complexes could be demonstrated. Additionally, the usage of the metal binding ligand nitrate enabled a facile and fast protein binding to the complex. Error bars: +/−SD; p-values: <0.001: ***; <0.01: **; <0.05: *; >0.05: not significant

EXAMPLES

Example 1: Comparison of the Chemical Stability of the [Co(II)(NTA)(His.SUB.6.-GFP)] and the [Co(III)(NTA)(His.SUB.6.-GFP)] Complexes

[0356] Aliquots of beads with immobilized [Co(II)(NTA)(His.sub.6-GFP)] and [Co(III)(NTA)(His.sub.6-GFP)] were incubated with either strong chelators or with widely used reducing agents in combination with 250 mM imidazole to demonstrate that Co.sup.3+ based complexes are superior to commonly used Ni.sup.2+ or Co.sup.2+ based complexes regarding chemical stability.

[0357] His.sub.6-GFP (SEQ ID NO: 14) was expressed in E. coli BL21(DE3) using the plasmid pET His.sub.6 GFP TEV LIC (Addgene #29663) (Pedelacq et al. 2006) and purified via Ni.sup.2+-NTA-beads as described by Wegner and Spatz (Wegner and Spatz, 2013).

[0358] Ni.sup.2+-NTA agarose resin (Novagen) was washed 1) with 9 bead volumes ddH.sub.2O, 2) with 3 bead volumes 0.1 M EDTA pH 7.5, 3) thrice with 9 bead volumes buffer A (50 mM Tris-HCl pH 7.4, 300 mM NaCl), 4) with 1.5 bead volumes 0.1 M CoCl.sub.2*6H.sub.2O 5) with 9 bead volumes buffer B (bufferA with 250 mM imidazole) and 6) thrice with 9 bead volumes buffer A. Finally, His.sub.6-GFP was loaded on the beads by incubating them in one bead volume of 10 μM His.sub.6-GFP in buffer A. Between each step the bead slurry was centrifuged for 1 min at 300 g and the supernatant was decanted. To obtain [Co(III)(NTA)(His.sub.6-GFP)] complexes the beads with immobilized His.sub.6-GFP on Co.sup.2+-NTA were incubated for 1 h at room temperature in buffer A with 20 mM H.sub.2O.sub.2 (the beads later used as control containing Co.sup.2+ were incubated in buffer A without H.sub.2O.sub.2). Subsequently, after washing the beads several times with buffer A, beads were resuspended in 2 bead volumes buffer A and distributed in 150 μl aliquots for the stability experiment. Finally, 50 μl of each test reagent (final concentrations: chelators: 250 mM imidazole, 25 mM NTA, 25 mM EDTA; reducing agents (cysteamine, DTT, TCEP, ascorbate): 1 mM supplemented with 250 mM imidazole) was added to an aliquot. Following 1 h incubation at room temperature, 100 μl supernatant was analyzed for GFP fluorescence (λ.sub.ex=480 nm, λ.sub.em=510 nm) using a plate reader (TECAN, infinite 2000). All experiments were performed in duplicates.

[0359] As depicted in FIG. 1 only very small amounts of eluted protein were observed upon incubation with the tested chelators or reducing agents when His.sub.6-GFP was bound to Co.sup.3+ centers. In contrast, His.sub.6-GFP bound to Co.sup.2+ beads was completely eluted under the same conditions. Thus, the [Co(III)(NTA)(His.sub.6-GFP)] complex is both inert towards the disruption by strong chelators and the reduction to Co.sup.2+.

Example 2: Oxidation of Fluorophores by Hydrogen Peroxide

[0360] The utilization of H.sub.2O.sub.2 to oxidize Co.sup.2+ to Co.sup.3+ as employed in the method described in Example 1 and as previously described in the prior art (see Wegner and Spatz, 2013) does not only harm the attached protein but can also negatively affect the functionality of NTA-conjugates such as labels or carriers. For example, the fluorescence of several fluorophores can decrease upon oxidation by H.sub.2O.sub.2 as shown in the following.

[0361] Fluorophore conjugates were diluted in phosphate buffered saline (PBS) (Thermo; 18912014) (final concentrations: 5 μg/ml fluorescein (Riedel de Haen; 28802); 185 μg/ml Alexa488 coupled antibody (Invitrogen; A11039); 9 μg/ml FITC coupled antibody (Thermo; MA1-81891); 5 μM atto488 coupled Ni.sup.2+-NTA (Sigma; 39625)) and 100 μl of each fluorophore solution was incubated for about 21 h with 0.05% H.sub.2O.sub.2 followed by another 22 h in 1% H.sub.2O.sub.2 in a black 96 well plate. Fluorescence intensity (λ.sub.ex=490 nm, λ.sub.em=535 nm) was measured every 15 min (0.05% H.sub.2O.sub.2) or 30 min (1% H.sub.2O.sub.2) on a plate reader (TECAN; Spark).

[0362] The fluorescence measurements depicted in FIG. 3 illustrate that all analysed fluorophores showed a pronounced decrease in fluorescence intensity upon incubation with H.sub.2O.sub.2.

Example 3: Protein Degradation and His-Tag Cleavage during Cobalt Oxidation by H.SUB.2.O.SUB.2

[0363] The utilization of H.sub.2O.sub.2 to oxidize Co.sup.2+ to Co.sup.3+ as employed in the method described in Example 1 and as previously described by Wegner and Spatz (Wegner and Spatz 2013) can spontaneously provoke a Fenton-like reaction (Hanna, Kadiiska et al. 1992), which can lead to protein degradation and cleavage of the histidine residue (Davies 1987, Stadtman 1990) as demonstrated in an anti-His-tag Western Blot.

[0364] The fluorescent protein PercevalHR (SEQ ID NO: 15) was expressed in E. coli DH5α using the plasmid pRsetB-PercevalHR (Addgene #49081) (Tantama et al. 2013)) and purified via a Ni.sup.2+-NTA column as described in (Tantama et al. 2013).

[0365] 3.3 μM Hiss-tagged PercevalHR protein was mixed with 33 μM CoCl.sub.2*6H.sub.2O in protein buffer (50 mM Tris pH7.4, 150 mM NaCl) and incubated for 2 min at room temperature. Subsequently, 20 mM H.sub.2O.sub.2 was added and the mixture was incubated for 1.5 h at 21° C. For control samples without cobalt and/or H.sub.2O.sub.2 an equivalent volume of protein buffer was used. Finally the reaction was quenched with 33 mM EDTA pH 8.0.

[0366] For Western Blot analysis the protein samples were mixed with SDS sample buffer (25 mM Tris-HCl pH 6.8, 192 mM Glycin, 0.1% (w/v) SDS, 0.002% (w/v) bromophenol blue, 100 mM DTT (final concentrations)), denatured at 70° C. for 10 min and 84 pmol of protein was loaded on a SDS-PAGE gel (7% (w/v) acrylamide-bisacrylamid (37.5:1), 375 mM Tris-HC1 pH 8.8, 0.1% (w/v) SDS, 0.1 (w/v) ammoniumpersulfate , 0.1% (v/v) TEMED; run conditions: 120 V constant, Laemmli Running buffer (25 mM Tris-HCl pH 8.8, 192 mM Glycin, 0.1% (w/v) SDS)). Following protein separation, the proteins were blotted on a nitrocellulose membrane (Whatman, 10401196) and the membrane was washed for 5 min at room temperature with TBS-T (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween20) and subsequently blocked for 1 h at room temperature with 5% (w/v) bovine serum albumin in TBS-T. Finally, the membrane was incubated with 200 ng/ml horse radish peroxidase labelled anti-His-tag antibody (clone H-3) (Santa Cruz, sc-8036 HRP), washed tricethrice for 10 min with TBS-T and incubated for 5 min at room temperature in luminol-based enhanced chemiluminescence horseradish peroxidase (HRP) substrate solution (Thermo, 34076). The chemoluminest signals from the His-tagged proteins were detected using a LAS3000 system (FUJIFILM).

[0367] The Western Blot in FIG. 4 demonstrates the possible protein degradation as well as spontaneous His-tag cleavage upon incubation of the protein with cobalt in combination with H.sub.2O.sub.2 by smeary and less intense protein bands.

Example 4: Synthesis of the [Co(III)(NTA)(CO.SUB.3.)].SUP.2− Complex

[0368] In order to fully circumvent the use of H.sub.2O.sub.2 a new method was developed which employs Co(III) carbonate salts (e.g. Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O and K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O) for the formation of the [Co(III)(NTA)(His-protein)] complex. The complex formation process is schematically depicted in FIG. 2C. In a first step the Co(III) salt is incubated with NTA to form [Co(III)(NTA)CO.sub.3].sup.2−. Proton magnetic resonance spectroscopy (.sup.1H-NMR) was applied to demonstrate the successful formation of the [Co(III)(NTA)CO.sub.3].sup.2− complex.

[0369] Synthesis of Sodium tris-carbonatocobalte(III) trihydrate.

[0370] Sodium tris-carbonatocobalte(III) trihydrate (Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O) was synthesized as described by Bauer and Drinkard (Bauer and Drinkard 1960). Briefly, a mixture of 0.1 mole (29.1 g) of Co(II)(NO.sub.3)*6H.sub.2O (Sigma; 1.02554) in 50 ml ddH.sub.2O and 10 ml of 30% hydrogen peroxide (Riedel-de Haen; 18312) was added dropwise with stirring to an ice-cold slurry of 0.5 mole(=42.0 g) sodium bicarbonate (Merck; 1.06329) in 50 ml ddH.sub.2O. The mixture was incubated on ice with continuous stirring for 1 h. Subsequently, the olive product was filtered and washed thrice with each of cold water, absolute ethanol and dry ether. Finally, the product was dried overnight under vacuum and stored at −20° C. in nitrogen atmosphere.

[0371] Synthesis of Potassium tris-carbonatocobalte(III) trihydrate.

[0372] Potassium tris-carbonatocobalte(III) trihydrate (K.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O) was synthesized in solution as described by Shibata (Shibata 1983; adaptation of Mori et al. 1956). Briefly, a mixture of 0.1 mole (24 g) of Co(II)Cl.sub.2*6H.sub.2O (Honeywell; 255599) in 24 ml ddH.sub.2O and 40 ml of 30% hydrogen peroxide was added dropwise with stirring to an ice-cold slurry of 0.7 mole (70 g) potassium bicarbonate (Honeywell; 237205) in 70 ml ddH.sub.2O. Subsequently, the resulting green solution is filtered by suction and directly used for following experiments.

[0373] Preparation of the [Co(III)(NTA)(CO.sub.3)].sup.2− Complex

[0374] In order to produce the [Co(III)(NTA)(CO.sub.3)].sup.2− complex from the sodium salt, 580 μmole (210 mg) Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O was added to 2 ml of 1 M sodium bicarbonate and 2 M nitrilotriacetic acid trisodium salt (Sigma; N0253) in ddH.sub.2O and the slurry was sonicated for 30 min. Following an incubation of 72 h at 70° C., 3 ml of 1 M sodium bicarbonate were added to the now pinkish slurry and the mixture was sonicated for 2 h at 70° C. Subsequently, the violet supernatant was submitted to NMR analysis. For complexes produced from potassium salt, K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O was synthesized in solution from 0.1 mole CoCl.sub.2*6H.sub.2O (see above) and subsequently 0.1 mole nitrilotriacetic acid trisodium salt (25.7 g) was added with 60 ml ddH.sub.2O as described in (Shibata 1983). After 3 h of incubation under continuous stirring at 60° C., the resulting violet solution was filtered and the pH was adjusted with aqueous acetic acid to pH 7.3. Finally, the solution was incubated over night at 4° C., cleared from the white precipitate and submitted to NMR analysis. To obtain the complex [Co(II)(NTA)(D.sub.2O)].sup.− a mixture of 5 mM nitrilotriacetic acid (Sigma; 72559) and 5 mM CoCl.sub.2*6H.sub.2O was produced from stock solutions in D.sub.2O and incubated for 15 min at room temperature prior to NMR measurement. To dissolve the nitrilotriacetic acid in D.sub.2O (Carl Roth; HN81.3) a small volume of 10 M NaOH was added to the corresponding stock solution. For the measurement of pure NTA a 5 mM solution in D.sub.2O was prepared from a stock solution prepared as described above. The .sup.1H-NMR spectra were measured at room temperature on a Jeol ECZ400S spectrometer at a resonance frequency of 400 MHz. To improve the signal-to-noise ratio, up to 32 signals were added prior to the Fourier transformation. Intensities of resulting spectra were normalized at 7 ppm and all ppm values were adjusted to a water peak at 4.70 ppm.

[0375] By measuring the H.sup.1-NMR spectra of pure NTA as well as its complexes with cobalt and water or carbonate ligands the formation of the [Co(III)(NTA)CO.sub.3].sup.2− complex was confirmed (FIG. 5). The spectrum of the hydrogens of pure NTA shows—beside the ubiquitous water peak at 4.70 ppm—a peak at ˜3.6 ppm, which is in agreement with a simulation (3.57 ppm calculated) performed by the software NMR Predict (https://www.nmrdb.org/new_predictor/index.shtml?v=v2.103.0) (Banfi and Patiny, 2008; Castillo et al. 2011; Aires-de-Sousa et al. 2002). When NTA forms a complex with cobalt and water (here due to H.sup.1-NMR measurements heavy water (D.sub.2O) is used) ([Co(II)(NTA)(D.sub.2O).sub.2].sup.−; FIG. 5B) or carbonate ([Co(III)(NTA)(CO.sub.3)].sup.2−; Figure C+D) the original peak at ˜3.6 ppm shifted to ˜3.8 ppm for D.sub.2O as ligand and to ˜1.9 ppm with carbonate, respectively. A similar peak shift was observed regardless of whether sodium or potassium cobalt(III) carbonate salt was used for production of the complex. The shifts of the NTA peak demonstrate that the magnetic environment of the hydrogen atoms in NTA changes as additional atoms come in close proximity to the hydrogen atoms during the complexation process. The observation of different peak shifts for complexes produced with cobalt(III) carbonate salt therefore strongly indicates the presence of a carbonate ligand instead of a water ligand in the final cobalt-NTA complex.

Example 5: Chemical Stability of the [Co(III)(NTA)(His.SUB.6.-PercevalHR)] Complex formed Via [Co(III)(NTA)CO.SUB.3.]

[0376] The [Co(III)(NTA)(CO.sub.3)].sup.2− complex with NTA linked to agarose beads was incubated with the His-tagged protein PercevalHR to form [Co(III)(NTA)(His.sub.6-PercevalHR)] immobilized on agarose beads. Subsequently, the chemical stability of the [Co(III)(NTA)(His.sub.6-PercevalHR)] complex was evaluated. As control, a conventional Ni.sup.2+-NTA based matrix was employed.

[0377] The fluorescent protein PercevalHR (SEQ ID NO: 15) was expressed in E. coli DH5α using the plasmid pRsetB-PercevalHR (Addgene #49081) (Tantama, Martinez-Francois et al. 2013) and purified via a Ni.sup.2+-NTA column as described in (Tantama, Martinez-Francois et al. 2013).

[0378] NTA agarose resin (Qiagen, 1022963) was washed 1) with 10 bead volumes ddH.sub.2O, 2) with 3 bead volumes 100 mM EDTA pH7.5, 3) thrice with 10 bead volumes ddH.sub.2O and subsequently 10 bead volumes 1 mM Na.sub.3[CO(III)(CO.sub.3).sub.3]*3H.sub.2O or 1 mM Ni(II)SO.sub.4 in 1 M NaHCO.sub.3 were added. After an incubation for 48 h in a thermoshaker at 23° C. shaking at 1100 rpm, the beads were wash twice with 10 bead volumes ddH.sub.2O and once with 10 bead volumes protein buffer (50 mM Tris pH7.4, 150 mM NaCl). Finally, one bead volume of 10 μM His.sub.6-PercevalHR (SEQ ID NO: 15) in protein buffer was added and incubated for 48 hat 4° C. shaking at 1100 rpm on a thermoshaker to bind the protein to the matrix. Following two washes with 10 bead volumes protein buffer, 3 bead volumes protein buffer were added and 10 μl bead slurry was analyzed for PercevalHR fluorescence (λ.sub.ex=500 nm, λ.sub.em=545 nm) using a plate reader (TECAN, infinite 2000). To test the stability of the complex 10 bead volumes of either 250 mM imidazole in protein buffer or protein buffer alone were added and subsequently removed by washing the beads with 10 bead volumes protein buffer. Finally, beads were resuspended in 3 bead volumes protein buffer and 10 μl of the bead slurry were analyzed for remaining fluorescence. All experiments were performed in triplicates.

[0379] As depicted in FIG. 6 the [Ni(II)(NTA)(His.sub.6-PercevalHR)] complexes used as control showed a low stability towards the chelator imidazole treatment. In sharp contrast, the [Co(III)(NTA)(His.sub.6-PercevalHR)] complexes formed via cobalt(III)carbonate salt and the [Co(III)(NTA)(CO.sub.3)] pre-complex show strong stability towards imidazole. The measured stability of the complexes is similar to the [Co(III)(NTA)(His.sub.6-PercevalHR)] complexes produced with the indirect oxidation method (as employed in example 1). Accordingly, this data confirms that surprisingly [Co(III)(NTA)(His.sub.6-PercevalHR)] can be formed without an oxidation step at high efficiency by using a [Co(III)(NTA)(CO.sub.3)].sup.2− pre-complex.

Example 6: Binding Kinetics of His.SUB.6.-GFP to [Co(M)(NTA)CO.SUB.3.].SUP.2−

[0380] Example 4 documents that a [Co(III)(NTA)CO.sub.3].sup.2− complex can be formed. Further, Example 5 indicates that the [Co(III)(NTA)CO.sub.3].sup.2− complex linked to beads via NTA can be synthesized and surprisingly used to form a [Co(III)(NTA)(His-protein)] complexes on beads. We speculated that the carbonate as ligand at the cobalt(III) center may facilitate the formation of the [Co.sup.III(NTA)(His-protein)] complex. To confirm this finding, the binding efficacy of His.sub.6-GFP to [Co(III)(NTA)(H.sub.2O).sub.2] and to [Co(III)(NTA)(CO.sub.3)].sup.2− was directly compared.

[0381] Functionalization of NTA Agarose Beads

[0382] NTA agarose resin (Qiagen, 1022963) was washed 1) with 10 bead volumes ddH.sub.2O, 2) with 10 bead volumes 100 mM EDTA pH7.5, 3) twice with 10 bead volumes ddH.sub.2O and once with 6.7 bead volumes ddH.sub.2O for [Co(II)(NTA)(H.sub.2O).sub.2].sup.− and [Co(III)(NTA)(H.sub.2O).sub.2] complexes or twice with 10 bead volumes ddH.sub.2O and once with 6.7 bead volumes 1 M NaHCO.sub.3 for [Co(III)(NTA)(CO.sub.3)].sup.2− complexes, respectively. Subsequently 8.7 bead volumes 1 mM Co(II)Cl.sub.2*6H.sub.2O in ddH.sub.2O (for [Co(II)(NTA)(H.sub.2O).sub.2] and [Co(III)(NTA)(H.sub.2O).sub.2] complexes) or 1 mM Na.sub.3[CO(III)(CO.sub.3).sub.3]3H.sub.2O in 1 M NaHCO.sub.3 (for [Co(III)(NTA)(CO.sub.3)].sup.2− complexes) were added. After incubating for 18 h in a thermoshaker at 25° C. at 1100 rpm the beads were washed with 6.7 bead volumes of protein buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl) (for [Co(II)(NTA)(H.sub.2O)2].sup.−, [Co(III)(NTA)(H.sub.2O).sub.2] complexes and one sample of [Co(III)(NTA)(CO.sub.3)].sup.2− complexes) or 1 M NaHCO.sub.3 (for one sample of [Co(III)(NTA)(CO.sub.3)].sup.2− complexes referred to as “[Co(III)(NTA)(CO.sub.3)] in 1M NaHCO.sub.3” in FIG. 7), respectively. For samples with final [Co(III)(NTA)(H.sub.2O).sub.2] complexes the second wash was performed with 6.7 bead volumes of 20 mM H.sub.2O.sub.2 in ddH.sub.2O and incubated for 1 h at 25° C. on a thermoshaker (1100 rpm).

[0383] Binding kinetics of His.sub.6-GFP to the Functionalized NTA Agarose Beads

[0384] The produced beads were incubated with 3.3 bead volumes of 20 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl) and incubated at 4° C. shaking at 1100 rpm on a thermoshaker. For one sample of the [Co(III)(NTA)(CO.sub.3)].sup.2− (referred to as “[Co(III)(NTA)(CO.sub.3)] in 1M NaHCO.sub.3” in FIG. 7), 1 M NaHCO.sub.3 was used instead of protein buffer. Fraction of unbound protein was analyzed by measuring the fluorescence intensity (λ.sub.ex=490 nm, λ.sub.em=535 nm) of 100 μl supernatant in a plate reader (TECAN, Spark) at various time points.

[0385] Chemical Stability of the [Co(III)(NTA)(His.sub.6-GFP)] Complex

[0386] After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), as described above, was terminated, the amount of protein bound to the beads was analyzed. To this end, beads were washed thrice with 6.7 bead volumes protein buffer, resuspended in 6.7 bead volumes protein buffer and finally 100 μl bead slurry were analyzed for GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). To test the stability of the complex 1.7 bead volumes of 1.25 M imidazole in protein buffer (final concentration 250 mM) were added and incubated for 10 min at 25° C. at 1100 rpm on a thermoshaker following three bead washes with 6.7 bead volumes of protein buffer. Finally, beads were resuspended in 6.7 bead volumes protein buffer and 100 μl bead slurry was analyzed for remaining fluorescence. All experiments were performed in triplicates.

[0387] The results of these experiments clearly demonstrate that the Co.sup.3+ complexes with an associated carbonate molecule bind the protein significantly faster than complexes in which water molecules are bound to Co.sup.3+ (FIG. 7A). Consistently, beads functionalized with [Co(III)(NTA)(CO.sub.3)].sup.2− complexes bind significantly more protein than beads preloaded with [Co(III)(NTA)(H.sub.2O).sub.2] (FIG. 7B). In addition, FIG. 7B again confirms that [Co(III)(NTA)(His.sub.6-GFP)] complexes show a strong stability towards chelators (here imidazole).

[0388] The experiments further indicate that washing the bead-attached [Co(III)(NTA)(CO.sub.3)].sup.2− with 1M NaHCO.sub.3 prior to protein binding further facilitates His.sub.6-GFP binding. Without being bound by theory, it is believed that the presence of HCO.sub.3.sup.− and/or CO.sup.2− in the buffer prevents conversion of [Co(III)(NTA)(CO.sub.3)].sup.2− complexes into the slower enhancing [Co(III)(NTA)(H.sub.2O).sub.2] complexes. While in the present experiment His-protein binding was performed also in presence of 1M NaHCO.sub.3 for the “[Co(III)(NTA)(CO.sub.3)] in 1M NaHCO.sub.3” sample (see above), it is envisaged that the presence of 1M NaHCO.sub.3 during the protein binding, if at all, only contributes to a very small degree. This is because during protein binding it is desired that the carbonate ligand is released from the Co.sup.2+ complex which may be rather hindered than facilitated by the presence of 1M NaHCO.sub.3.

Example 7: Binding Kinetics of a His-Tagged Protein to [Co(M)(NTA)(CO.SUB.3.)].SUP.2− in Different Buffer Systems

[0389] It was determined whether the formation of the [Co(III)(NTA)(His-Protein)] complex can be improved by changing the composition of the reaction buffer.

[0390] The beads functionalized with [Co(III)(NTA)(CO.sub.3)].sup.2− were prepared as described in Example 6 with small modifications: Bead washing steps 1) to 3) were performed with 5 bead volumes of the corresponding solutions. In 3) the second and third washing step were done with 1 M NaHCO.sub.3. The metal was loaded in 6.5 bead volumes. After metal binding the beads were washed with 5 bead volumes 1 M NaHCO.sub.3.

[0391] The incubation with His-tagged GFP (SEQ ID NO: 14) was also performed as described in Example 6 except that 5 bead volumes of 10 μM protein solution were used and that the protein buffer was adapted by replacing 50 mM Tris pH7.4 with either 50 mM Bis-Tris pH6.0, 50 mM HEPES pH7.0, 50 mM MES pH6.0 or 50 mM Tris-HCl pH7.5. Additionally, the first 24 hours of protein incubation were performed at room temperature instead of 4° C.

[0392] The experiments demonstrated that the reaction speed and efficacy could be significantly improved when Tris based buffers are replaced by HEPES or especially non-coordinating buffers such as MES or BisTris based buffers. After 24 hours of incubation 95% of all protein was immobilized on the beads when using MES and BisTris based buffers compared to 75% with Tris based buffer (FIG. 8A). Further, comparing the results of FIGS. 7A and 8A, indicates that the increase of the temperature for the first 24 h of protein incubation and/or washing with 1M NaHCO.sub.3 facilitates His-protein coordination. After already three hours of incubation about 80% of the protein could be bound to beads when using MES or BisTris based buffers. By contrast in Tris based buffer less than 50% of the protein got immobilized on the beads. It was demonstrated that it is possible to coordinate His-tagged proteins with the [Co(III)(NTA)(CO.sub.3)].sup.2− complex with very high efficacy in reasonable time scales.

[0393] It is of note that the pH values indicated for the different protein buffers indicate the pH before adding the solution to the beads. Due to residual NaHCO.sub.3 from the second washing step remaining on the beads, the pH in all samples was 8.5 to 9 (as verified by pH measurement) during incubation with the protein. Accordingly, due to the very similar pH values in all buffer solutions the experiments clearly demonstrate that the buffer substance per se has an influence on the His-protein binding. The observed better performance of the good buffers MES and BisTris relative to Tris buffer suggests that using Good buffers which cannot form complexes with Co.sup.3+ is advantageous vis-à-vis the use of buffers which can form such complex, such as Tris buffer.

Example 8: UV-Vis Analysis of [Co(III)(NTA)(CO.SUB.3.)].SUP.2−

[0394] Example 4 documents that a [Co(III)(NTA)(CO.sub.3)].sup.2− complex can be formed by NMR. In the following examples the formation of the [Co(III)(NTA)CO.sub.3].sup.2− complex is proven by another technique, namely the absorbance measurement by UV-Vis.

[0395] Preparation of Co(III)(NTA)(CO.sub.3)].sup.2− Complex:

[0396] In order to produce the [Co(III)(NTA)(CO.sub.3)].sup.2− complex a solution of 1 mM Na.sub.3[CO(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 by dissolving the salt in the solution with 1 h of sonication followed by a filtration step through a 0.22 μm filter is prepared. Subsequently, a mixture of 0.95 mM of the Na.sub.3[CO(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 solution and 0.95 mM NTA trisodium salt (Sigma; N0253) dissolved in ddH.sub.2O is prepared in 1 M NaHCO.sub.3. Following an incubation of 1 h at 25° C., the visible absorbance of the light violet solution was measured in 1 cm cuvettes (Brand; 759150) on a UV-Vis-NIR Spectrophotometer (Cary5000).

[0397] Preparation of Co(III)(NTA)(H.sub.2O).sub.2] Complex:

[0398] In order to produce the [Co(III)(NTA)(H.sub.2O).sub.2] complex a mixture of 0.95 mM Co(II)Cl.sub.2*6H.sub.2O, 0.95 mM NTA trisodium salt (Sigma; N0253) and 20 mM H.sub.2O.sub.2 is prepared in ddH.sub.2O. Following an incubation of 24 h at 25° C., the visible absorbance of the light violet solution was measured in 1 cm cuvettes (Brand; 759150) on a UV-Vis-NIR Spectrophotometer (Cary5000) against a blank with ddH.sub.2O.

[0399] The results in FIG. 9 clearly demonstrate the coordination of carbonate as metal binding ligand in the [Co(III)(NTA)(CO.sub.3)].sup.2− complex by a shift of the two peaks from 402 nm for the [Co(III)(NTA)(H.sub.2O).sub.2] complex to 390 nm for the [Co(III)(NTA)(CO.sub.3)].sup.2− complex or from 567 nm to 573 nm, respectively.

Example 9: His.SUB.6.-GFP Coordination to [Co(III)(NTA)(CO.SUB.3.)].SUP.2− and Stability of the Formed [Co(III)(NTA)(His-GFP)] Complex After Different Incubations Times and Temperatures of Na.SUB.3.[Co(M)(CO.SUB.3.).SUB.3.]*3H.SUB.2.O with NTA

[0400] Example 4 documents that a [Co(III)(NTA)CO.sub.3].sup.2− complex can be formed by the incubation of NTA with Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O. In the following example the effect of different incubation times and temperatures of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O with NTA on His.sub.6-GFP coordination to the resulting [Co(III)(NTA)(CO.sub.3)].sup.2− complexes as well as the chemical stability of the final Co(III)(NTA)(His-GFP)] complexes towards imidazole is investigated.

[0401] Functionalization of NTA Agarose Beads

[0402] NTA functionalized agarose beads (Qiagen; 1022963) were washed 1) with 27 bead volumes ddH.sub.2O, 2) with 27 bead volumes 100 mM EDTA pH8.0, 3) once with 27 bead volumes ddH.sub.2O and twice with 27 bead volumes 1 M NaHCO.sub.3. Subsequently 16 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]3H.sub.2O in 1 M NaHCO.sub.3 was added and beads were incubated at 4° C., 25° C. or 70° C. for 1 min, 10 min, 30 min, 1 h, 24 h or 48 h as indicated in a thermo shaker at 1400 rpm. After the incubation the beads were washed twice with 16 bead volumes of 1 M NaHCO.sub.3.

[0403] Immobilization of His.sub.6-GFP to Functionalized [CIII)(NTA)(CO.sub.3)].sup.2− Agarose Beads

[0404] The produced beads were incubated with 12 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 48 h. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 16 bead volumes protein buffer and resuspended in 16 bead volumes protein buffer.

[0405] Chemical Stability of the [Co(III)(NTA)(His.sub.6-GFP)] Complex

[0406] After the indicated [Co(III)(NTA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions (each 7.2 bead volumes) and washed once either with 17.8 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively.

[0407] After a final wash with 17.8 bead volumes protein buffer, beads were resuspended in 17.8 bead volumes protein buffer. Immobilized protein amount on 25 μl beads slurry was determined by BCA assay (Thermo, 23227) in microplate based on the manufacturer's guidelines. The experiment were performed in triplicates.

[0408] The results of these experiments shown in FIG. 10 clearly demonstrate that there is a positive correlation of the incubation times of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O and NTA with the amount of immobilized His-GFP on the agarose beads after buffer as well as imidazole treatment when the protein was incubated for such a long period as 48 h. For incubations at 70° C. the reaction seems to reach its saturation point already after 1 min as Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O coordination to NTA is unsurprisingly accelerated at higher temperatures. Additionally, for incubations performed at 4° C. and 25° C. as depicted in FIGS. 10A and B the incubation times correlate positively with the complex stability. For incubations at 25° C. high complex stability is already reached at 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation, while for 4° C. incubations this stability is reached only after 24 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation. It is envisaged that stable complex formation is accelerated with higher temperatures.

Example 10: Kinetics of His-Protein Binding to Beads Functionalized with [Co(III)(NTA)(CO.SUB.3.)].SUP.2− at Different Temperatures

[0409] In the following example protein binding to [Co(III)(NTA)(CO.sub.3)].sup.2− functionalized magnetic agarose beads after different His-protein incubation times and at three different temperatures as well as the stability towards imidazole of the resulting [Co(III)(NTA)(His-GFP)] complexes is examined.

[0410] Functionalization of NTA Magnetic Agarose Beads

[0411] NTA functionalized agarose beads (Thermo; 78605) were washed 1) with 26 bead volumes ddH.sub.2O, 2) with 26 bead volumes 100 mM EDTA pH8.0, 3) twice with 26 bead volumes ddH.sub.2O and once with 26 bead volumes 1 M NaHCO.sub.3. Subsequently 160 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or 1 M NaHCO.sub.3 for samples without metal were added and beads were incubated at 25° C. for 48 h on a thermo shaker at 1400 rpm. After the incubation the beads were washed thrice with 160 bead volumes of 1 M NaHCO.sub.3.

[0412] Immobilization of His.sub.6-GFP to Functionalized [CoIII) (NTA)(CO.sub.3)].sup.2− Magnetic Agarose Beads

[0413] The produced beads were incubated with 120 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in 50 mM HEPES pH7.2, 150 mM NaCl and incubated at 4, 25 or 37° C. as indicated shaking at 1400 rpm on a thermo shaker for 1 min, 10 min, 30 min, 1 h, 2 h, 3.5 h or 24 h as indicated. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 160 bead volumes protein buffer and finally resuspended in 160 bead volumes of the corresponding washing buffer.

[0414] Chemical Stability of the [Co(III)(NTA)(His.sub.6-GFP)] Complex

[0415] After the indicated [Co(III)(NTA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions and washed once either with bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein bufferFinally, 10 μl bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). The experiments were performed in triplicates.

[0416] As depicted in FIG. 11A the amount of immobilized protein on imidazole treated beads increases with the incubation time of His-GFP with the [Co(III)(NTA)(CO.sub.3)].sup.2− functionalized beads. Thereby the temperature during incubation does not effect the final yield of formed [Co(III)(NTA)(His.sub.6-GFP)] complex. For all protein incubation times and temperatures a high stability of the resulting complex towards imidazole could be observed (FIG. 11B).

Example 11: [Co(III)(NTA)(His-GFP)] Complex Formation and Stability using K.SUB.3.[Co(III)(CO.SUB.3.).SUB.3.]*3H.SUB.2.O to form [Co(III)(NTA)(CO.SUB.3.)].SUP.2− Complexes

[0417] Example 4 demonstrates the formation of the [Co(III)(NTA)(CO.sub.3)].sup.2− complex by incubating K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O with NTA. In the following example the coordination of His-GFP to Co(III)(NTA)(CO.sub.3)].sup.2− complex produced with K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O as well as the chemical stability towards imidazole of the resulting [Co(III)(NTA)(His-GFP)] complex is examined.

[0418] Functionalization of NTA Magnetic Agarose Beads

[0419] NTA functionalized magnetic agarose beads (Thermo; 78605) were washed 1) with 600 bead volumes ddH.sub.2O, 2) with 600 bead volumes 100 mM EDTA pH8.0, 3) once with 600 bead volumes ddH.sub.2O and twice with 600 bead volumes 1 M NaHCO.sub.3. Subsequently 160 bead volumes of 1 mM K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or only 1 M NaHCO.sub.3 of samples without metal were added. K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O was produced as described in example 4. The concentration calculation is based in the assumption of a 100% reaction efficiency of the synthesis process. After incubating the samples as indicated for 10 min or 48 h as indicated in a thermo shaker at 25° C. at 1400 rpm the beads were washed thrice with 160 bead volumes of 1 M NaHCO.sub.3.

[0420] Immobilization of His.sub.6-GFP to the Functionalized NTA Magnetic Beads

[0421] The produced beads were incubated with 120 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 1 h or 48 h as indicated. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 160 bead volumes protein buffer and resuspended in 160 bead volumes protein buffer.

[0422] Chemical Stability of the [Co(III)(NTA)(His.sub.6-GFP)] Complex

[0423] After the indicated [Co(III)(NTA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions (each 72 bead volumes) and washed once either with 178 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer and 10 82 l bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). Experiment was performed in triplicates.

[0424] The results depicted in FIG. 12 clearly demonstrate, that His-GFP can coordinate to Co(III)(NTA)(CO.sub.3)].sup.2− complexes formed by the incubation of K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O with NTA. The resulting [Co(III)(NTA)(His-GFP)] complexes show high chemical stability towards imidazole treatment. The use of K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O offers the possibility to perform incubations with metal binding domains at higher concentration than with Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O as K.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O is soluble in higher concentrations.

Example 12: His.SUB.6.-GFP Coordination to [Co(III)(NTA)(CO.SUB.3.)].SUP.2− and Stability of the Formed [Co(III)(NTA)(His-GFP)] Complex in Different Protein Binding Buffers

[0425] Example 7 demonstrates the effect of different buffer substances on the binding kinetics of a His-tagged protein to [Co(III)(NTA)(CO.sub.3)].sup.2−. In the following example protein binding to [Co(III)(NTA)(CO.sub.3)].sup.2− functionalized magnetic agarose beads of two different Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation times in various different buffers systems as well as the stability towards imidazole of the resulting [Co(III)(NTA)(His-GFP)] complex is examined.

[0426] Functionalization of NTA Magnetic Agarose Beads

[0427] NTA functionalized agarose beads (Thermo; 78605) were washed 1) with 182 bead volumes ddH.sub.2O, 2) with 182 bead volumes 100 mM EDTA pH8.0, 3) once with 182 bead volumes ddH.sub.2O and twice with 182 bead volumes 1 M NaHCO.sub.3 for samples with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time or twice with 182 bead volumes ddH.sub.2O and once with 182 bead volumes 1 M NaHCO.sub.3 for samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time. Subsequently 160 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or 1 M NaHCO.sub.3 for samples without metal were added and beads were incubated at 25° C. for 10 min or 48 h as indicated in a thermo shaker at 1400 rpm. After the incubation the beads were washed twice for samples with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time or thrice for samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time with 160 bead volumes of 1 M NaHCO.sub.3.

[0428] Immobilization of His.sub.6-GFP to Functionalized [CoIII)(NTA)(CO.sub.3)].sup.2− Magnetic Agarose Beads

[0429] The produced beads were incubated with 120 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in either Tris-, HEPES-, MES-, MOPS-, BisTris-, ACES-, PIPES-, BES-, CAPS-, TAPS-based protein buffer (50 mM buffer pH 7.2, 150 mM NaCl) or PBS and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 1 min, 15 min, 1 h or 24 h as indicated. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 160 bead volumes protein buffer for samples with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time analysis or analysis buffer (50 mM HEPES pH 7.2, 150 mM NaCl) for samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time and finally resuspended in 160 bead volumes of the corresponding washing buffer.

[0430] Chemical Stability of the [Co(III)(NTA)(His.sub.6-GFP)] Complex

[0431] After the indicated [Co(III)(NTA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads for samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time were splitted in two portions (each 72 bead volumes) and washed once either with 178 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer. Immobilized protein amount on 10 μl beads slurry was analyzed via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). For samples with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time beads were analysed before and after a washing treatment with first 160 bead volumes of 250 mM imidazole in protein binding buffer followed by 160 bead volumes protein binding buffer. The experiments were performed in triplicates.

[0432] The results clearly demonstrated that in with all buffer systems his-tagged protein could be immobilized to the beads in a chemically stable manner. Thereby the proportion of stable complex is higher in samples with 48 h of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time (FIG. 14) compared to samples with 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time (FIG. 13). For all buffer systems and Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation times the amount of immobilized protein after imidazole treatment increases with the protein incubation time. Thereby the different buffer systems have a greater influence on reaction kinetics in samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time compared to 10 min Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time. It is envisaged that the usage of HEPES, MES, MOPS and PIPES buffers on samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation times results in an increased amount of stably immobilized protein on the beads at short protein incubation times (FIG. 14B-D). For longer incubation times it is envisaged (FIG. 14E) HEPES, MES, MOPS or ACES are good buffers of choice.

Example 13: Effect of pH on His.SUB.6.-GFP Coordination to [Co(III)(NTA)(CO.SUB.3.)].SUP.2− and the Stability of the Formed [Co(III)(NTA)(His-GFP)] Complex

[0433] Example 7 indicates the effect of the pH of the protein binding buffer on the binding kinetics of a His-tagged protein to [Co(III)(NTA)(CO.sub.3)].sup.2−. In the following example protein binding to [Co(III)(NTA)(CO.sub.3)].sup.2− functionalized magnetic agarose beads in two protein binding buffer systems with each 5 different pH values as well as the stability towards imidazole of the resulting [Co(III)(NTA)(His-GFP)] complex is examined.

[0434] Functionalization of NTA Magnetic Agarose Beads

[0435] NTA functionalized agarose beads (Thermo; 78605) were washed 1) with 26 bead volumes ddH.sub.2O, 2) with 26 bead volumes 100 mM EDTA pH8.0, 3) twice with 26 bead volumes ddH.sub.2O and once with 26 bead volumes 1 M NaHCO.sub.3. Subsequently 160 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or 1 M NaHCO.sub.3 for samples without metal were added and beads were incubated at 25° C. 48 h in a thermo shaker at 1400 rpm. After the incubation the beads were washed thrice for samples with 48 h Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA incubation time with 160 bead volumes of 1 M NaHCO.sub.3.

[0436] Immobilization of His-GFP to Functionalized [CoIII)(NTA)(CO.sub.3)].sup.2− Magnetic Agarose Beads

[0437] The produced beads were mixed with 120 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in either BisTris- or HEPES-based protein buffer (50 mM buffer, 150 mM NaCl) with pH 5.5, 6.0, 6.5, 7.0 or 7.5 for the BisTris-based system and pH 7.5, 8.0, 8.5, 9.0 or 9.5 for the HEPES-based systems and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 1 min, 15 min, 1 h or 24 h as indicated. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 160 bead volumes analysis buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and finally resuspended in 160 bead volumes analysis buffer.

[0438] Chemical Stability of the [Co(III)(NTA)(His.sub.6-GFP)] Complex

[0439] After the indicated [Co(III)(NTA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions (each 72 bead volumes) and washed once either with 178 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer. 10 μl bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). The experiments were performed in triplicates.

[0440] The results clearly demonstrated that in at all pH values his-tagged protein could be immobilized to the beads in a chemically stable manner. Thereby a high percentage of His-GFP could be immobilized in a chemically stable manner and the amount of immobilized protein before and after imidazole treatment increases with the protein incubation time (FIG. 15A). The complex formation efficiency of the final [Co(III)(NTA)(His-GFP)] complex are dramatically increased with protein binding buffers at lower pH values (FIG. 15B). Thereby not only efficiency differences based on the pH, but also originating from the buffer system could be observed (pH7.5 BisTris versus HEPES).

Example 14: Immobilization of Different Proteins via their His-Tags or Histidine-Rich Regions to [Co(III)(NTA)(Co).SUB.3.].SUP.2− or [Co(III)(IDA)(Co).SUB.3.].SUP.− Functionalized Beads

[0441] Several examples demonstrate the immobilization of His.sub.6-GFP via [Co(III)(NTA)(Co).sub.3].sup.2− or [Co(III)(IDA)(Co).sub.3].sup.− to beads. In the following example the immobilization of different his-tagged proteins as well as an antibody coordinated with its histidine-rich region of his Fc part to beads is tested. Additionally, the functionality of an immobilized enzyme (sortase) and the antibody (GFP binding to immobilized anti-GFP IgG1) is investigated.

[0442] NTA functionalized magnetic agarose beads (Thermo; 78605) or IDA functionalized magnetic beads (Cube Biotech; 30805) were washed 1) with 33 bead volumes ddH.sub.2O, 2) with 33 bead volumes 100 mM EDTA pH8.0, 3) once with 33 bead volumes ddH.sub.2O and twice with 33 bead volumes 1 M NaHCO.sub.3 for samples without metal and with cobalt center or thrice with 33 bead volumes ddH.sub.2O for samples with nickel center. Subsequently 160 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or 1 M NaHCO.sub.3 for samples without metal or 1 mM NiSO.sub.4 in ddH.sub.2O for samples with nickel center were added and beads were incubated at 25° C. 10 min in a thermo shaker at 1400 rpm. After the incubation the beads were washed thrice with 160 bead volumes of 1 M NaHCO.sub.3 or ddH.sub.2O for samples with nickel center.

[0443] Immobilization of Protein to Functionalized [CoIII)(NTA/IDA)(Co.sub.3)].sup.2− Magnetic Agarose Beads

[0444] The produced beads were mixed with 120 bead volumes of protein (10 μM His-GFP (SEQ ID NO: 14); 10 μM His-Protein A (Abcam; ab52953); 10 μM His-sortase A (SEQ ID NO: 16); 1 μM His-Human serum albumin (antikoerperonline; ABIN2181228); 0.2 μM anti-GFP mouse IgG1 (Biolegend; 902605)) in protein binding buffer (50 mM HEPES pH7.2, 150 mM NaCl) and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 48 h or 30 min for IDA samples, respectively. After the incubation of functionalized beads with protein a sample of the protein supernatant was saved for later analysis by SDS-PAGE and beads were washed once with 160 bead volumes protein buffer and finally resuspended in 160 bead volumes analysis buffer or for antibody sample continued with GFP incubation as described in a separate part.

[0445] Chemical Stability of the [Co(III)(NTA/IDA)(Protein)] Complex

[0446] After the indicated [Co(III)(NTA/IDA)(protein)] complex formation processes were performed, the amount of protein bound to the beads after chemical stress was analyzed. To this end, beads were washed once with 178 bead volumes of 250 mM imidazole in protein binding buffer. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer. Protein amount on 25 μl beads slurry was determined by BCA assay (Thermo, 23227) in microplate based on the manufacturer's guidelines.

[0447] Determination of Functionality of Sortase A Immobilized on Beads

[0448] Activity of immobilized sortase A on 20 μl beads slurry was determined with the SensoLyte® 520 Sortase A Activity Assay Kit from Anaspec (#72228) as descripted by the manufacturer.

[0449] GFP Binding to Immobilized α-GFP Antibody

[0450] To assess the functionality of the immobilized antibody, beads functionalized with [Co(III)(NTA/IDA)(IgG1)] were incubated for 1 h at 25° C. with 120 bead volumes of 0.54 μM GFP (without His-tag) (Abcam; ab84191) in protein binding buffer. After the incubation beads were washed once with 160 bead volumes protein buffer and finally resuspended in 160 bead volumes analysis buffer and subjected in case of samples with IDA to chemical stability determination as described above. Finally, GFP bound to the immobilized antibody was determined based on GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) of 10 μl beads slurry using a plate reader (TECAN, Spark).

[0451] SDS-PAGE of Protein Supernatants

[0452] SDS-PAGE of protein supernatant after protein incubation was performed as described in example 3 except that a 12% (w/v) acrylamide-bisacrylamid (37.5:1) gel was used and that per well 6 μl of protein supernatant was loaded. Visualization of bands was performed with an Instant Blue Coomassie stain (Expedion; ISB1L).

[0453] Purification of His-Sortase

[0454] The enzyme sortase A (SEQ ID NO: 16) was expressed in E. coli BL21(DE3) using the plasmid pET29_eSrtA (Addgene #75144) (Chen, Don et al. 2011) and purified via a Ni.sup.2+-NTA column as described in (Chen, Dorr et al. 2011).

[0455] The results presented in FIG. 16 clearly demonstrate that beside His.sub.6-GFP also other His-tagged proteins or even antibodies via their histidine-rich region can be cleared out of the supernatant during protein binding (FIG. 16A) and finally immobilized stably on [Co(III)(NTA)(Co).sub.3].sup.2− or [Co(III)(IDA)(Co).sub.3].sup.− functionalized beads (FIG. 16B). Additionally it could be demonstrated that immobilized protein is still functional as e.g. the enzyme sortase A is still active (FIG. 16C) or anti-GFP antibody immobilized on NTA or IDA beads can still bind its antigen GFP.

Example 15: Complex Formation and Stability using Other Metal Binding Domains than NTA

[0456] Example 4 documents that a [Co(III)(NTA)CO.sub.3].sup.2− complex can be formed. Further, Example 5 indicates that the [Co(III)(NTA)CO.sub.3].sup.2− complex linked to beads via NTA can be synthesized and surprisingly used to form a [Co(III)(NTA)(His-protein)] complexes on beads. We speculated that also other metal binding domains than NTA can be used to form [Co(III)(metal binding domain)(His-protein)] complexes. In order to test the versatility of the method complex formation with iminodiacetic acid (IDA), a tridentate metal binding domain, and TALON, a commercial tetradentate metal binding domain was examined.

[0457] Functionalization of IDA/TALON Magnetic Beads

[0458] IDA functionalized magnetic beads (Cube Biotech; 30805) or TALON functionalized magnetic agarose resin (Takara, 635636) were washed 1) with 20 bead volumes ddH.sub.2O, 2) with 20 bead volumes 100 mM EDTA pH8.0, 3) twice with 20 bead volumes ddH.sub.2O and once with 20 bead volumes 1 M NaHCO.sub.3. Subsequently 160 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or only 1 M NaHCO.sub.3 for samples without metal were added. After incubating the samples as indicated for 10 min or 48 h in a thermo shaker at 25° C. at 1400 rpm the beads were washed thrice with 160 bead volumes of 1 M NaHCO.sub.3.

[0459] Immobilization of His.sub.6-GFP to the Functionalized IDA/TALON Magnetic Beads

[0460] The produced beads were incubated with 160 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 30 min, 1 h or 48 h as indicated. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 160 bead volumes protein buffer and resuspended in 160 bead volumes protein buffer.

[0461] Chemical Stability of the [Co(III)(IDA/TALON)(His.sub.6-GFP)] Complex

[0462] After the indicated [Co(III)(IDA)(His-GFP)] or [Co(II)(TALON)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions (each 72 bead volumes) and washed once either with 178 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer and 10 μl bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). Both experiments were performed in triplicates.

[0463] The experiments clearly demonstrate the protein immobilization with IDA (FIG. 17A) and TALON (FIG. 17B) as metal binding domain. Therefore the cobalt(III)-mediated protein immobilization via cobalt(III) carbonate complexes is not restricted to the use of NTA as metal binding domain. Additionally, the tridentate metal binding domain IDA shows even improved characteristics such as an improved stability for short incubation times compared to (see FIG. 20B). The tetradentate metal binding domain TALON also demonstrates protein immobilization.

Example 16: Investigation of the Chemical Stability of the [Co(III)(IDA)(His-GFP)] Complex

[0464] Example 1 demonstrates the chemical stability of the [Co(III)(NTA)(His-GFP)] complex. In Example 15 the formation of [Co(III)(IDA)(His-GFP)] and its stability towards imidazole is proven. In following example [Co(III)(IDA)(His-GFP)] functionalized beads were incubated with either strong chelators or with widely used reducing agents in combination with 250 mM imidazole to demonstrate that complexes of IDA with a Co.sup.3+ metal center form chemical stabile complexes such as complex composed of NTA and Co.sup.3+ can do as demonstrated in Example 1.

[0465] Functionalization of IDA Magnetic Beads

[0466] IDA functionalized magnetic beads (Cube Biotech; 30805) were washed 1) with 20 bead volumes ddH.sub.2O, 2) with 20 bead volumes 100 mM EDTA pH8.0, 3) twice with 20 bead volumes ddH.sub.2O and once with 20 bead volumes 1 M NaHCO.sub.3. Subsequently 160 bead volumes 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or 1 M NaHCO.sub.3 for samples without metal were added and beads were incubated at 25° C. for 10 min in a thermo shaker at 1400 rpm. After the incubation the beads were washed thrice with 20 bead volumes of 1 M NaHCO.sub.3. The produced beads were incubated with 160 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in 50 mM HEPES pH 7.2, 150 mM NaCl and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 30 min. After the incubation with His.sub.6-GFP (SEQ ID NO: 14), beads were washed thrice with 160 bead volumes protein buffer.

[0467] Chemical Sstability of the [Co(III)(IDA)(His.sub.6-GFP)] Complex

[0468] Subsequently, 160 bead volumes of each test reagent (final concentrations: 250 mM imidazole, 25 mM NTA or 25 mM EDTA in protein buffer or 1 mM DTT, TCEP or ascorbate supplemented with 250 mM imidazole in protein buffer or 50 mM Glycin pH10.0 as indicated was added to the coresponding sample. Following 1 h incubation at 25° C. and 1400 rpm shaking, the supernatant was removed and beads were washed thrice with 160 bead volumes protein buffer and dissolved in 160 bead volumes protein buffer. Finally, 10 μl bead slurry was analyzed for its remaining immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). The experiment were performed in triplicates.

[0469] The experiments clearly demonstrates the high chemical stability of [Co(III)(IDA)(His.sub.6-GFP)] complexes towards different chemicals including chelators and reducing agents. As depicted in FIG. 18 in comparison to beads treated with buffer only a very small reduction of immobilized was observed upon incubation with the tested chelators or reducing agents when His.sub.6-GFP was bound to [Co(III)(IDA)(CO.sub.3)] complexes. Thus, the [Co(III)(IDA)(His.sub.6-GFP)] complex is both inert towards the disruption by strong chelators and the reduction to Co.sup.2+.

Example 17: Comparison of [Co(III)(IDA)(His-GFP)] Complex Formation via [Co(III)(IDA)(CO.SUB.3.)].SUP.− Versus [Co(III)(IDA)(H.SUB.2.O).SUB.2.].SUP.+

[0470] Example 4 documents that a [Co(III)(NTA)CO.sub.3].sup.2− complex can be formed. Further, Example 5 indicates that the [Co(III)(NTA)CO.sub.3].sup.2− complex linked to beads via NTA can be synthesized and surprisingly used to form a [Co(III)(NTA)(His-protein)] complexes on beads. In example 6 it is shown that the binding kinetics of His-GFP to [Co(III)(NTA)(CO.sub.3)].sup.2− is improved compared to the kinetics for complex formation using [Co(III)(NTA)(H.sub.2O).sub.2]. In example 15 it is demonstrated IDA is as possible metal binding domain for the formation [Co(III)(IDA)(His-GFP)] complex via the [Co(III)(IDA)CO.sub.3].sup.−. We speculated that the carbonate as ligand at the cobalt(III) center may facilitate also the formation of the [Co(III)(IDA)(His-protein)] complex. To confirm this finding, the complex formation efficiency of His.sub.6-GFP to [Co(III)(IDA)(H.sub.2O).sub.2] and to [Co(III)(IDA)(CO.sub.3)].sup.− as well as the chemical stability of the final [Co(III)(IDA)(His-GFP)] complex was directly compared.

[0471] Functionalization of IDA Magnetic Beads

[0472] IDA functionalized magnetic beads (Cube Biotech; 30805) were washed 1) with 80 bead volumes ddH.sub.2O, 2) with 80 bead volumes 100 mM EDTA pH8.0, 3) twice with 80 bead volumes ddH.sub.2O and once with 80 bead volumes 1 M NaHCO.sub.3 for [Co(III)(IDA)(CO.sub.3)].sup.2− complexes or ddH.sub.2O for [Co(II)(IDA)(H.sub.2O).sub.2].sup.− complexes or no metal samples, respectively.

[0473] Subsequently 160 bead volumes 1 mM Co(II)Cl.sub.2*6H.sub.2O in degassed ddH.sub.2O (for [Co(II)(IDA)(H.sub.2O).sub.2] and [Co(III)(IDA)(H.sub.2O).sub.2].sup.+ samples) or 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 (for [Co(III)(IDA)(CO.sub.3)].sup.− complexes) were added. To samples without metal 160 bead volumes ddH.sub.2O was added. After incubating for 10 min (or 48 h for indicated samples with [Co(III)(IDA)(CO.sub.3)].sup.− complex) in a thermo shaker at 25° C. at 1400 rpm the beads were washed thrice with 160 bead volumes of 1 M NaHCO.sub.3 for samples with [Co(III)(IDA)(CO.sub.3)].sup.2− complexes or ddH.sub.2O for [Co(II)(IDA)(H.sub.2O).sub.2] samples or no metal samples, respectively. [Co(III)(IDA)(H.sub.2O).sub.2].sup.+ were washed once with 160 bead volumes, incubation in 160 bead volumes of 20 mM H.sub.2O.sub.2 for 1 h at 25° C. on a thermo shaker with 1400 rpm and finally washed once with 160 bead volumes of ddH.sub.2O.

[0474] Immobilization of His.sub.6-GFP to the Functionalized IDA Magnetic Beads

[0475] The produced beads were incubated with 160 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) or PBS as indicated and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 3 h or 24 h as indicated. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 160 bead volumes protein buffer and resuspended in 160 bead volumes protein buffer.

[0476] Chemical Stability of the [Co(III)(IDA)(His.sub.6-GFP)] Complex

[0477] After the indicated Co(III)(IDA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions (each 72 bead volumes) and washed once either with 178 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer and 10 μl bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). Experiment was performed in triplicates.

[0478] The results of these experiments clearly demonstrate that beads functionalized with [Co(III)(IDA)(CO.sub.3)].sup.− complexes obtained by incubating NTA and Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O for 10 min bind significantly more protein than beads preloaded with [Co(III)(IDA)(H.sub.2O).sub.2].sup.+ (FIG. 19). For beads functionalized with [Co(III)(IDA)(CO.sub.3)].sup.− complexes obtained after 48 h of Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O incubation, a significant advantage over Co(III)(IDA)(H.sub.2O).sub.2].sup.+ complexes is only visible after 24 h protein incubation. It is emphasized that preferred incubation times for IDA are 10 min for Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O/NTA and 30 min to 3 h for the subsequent protein incubation. With these incubation time combinations a significant improved immobilization efficiency to [Co(III)(IDA)(CO.sub.3)].sup.− complexes compared to [Co(III)(IDA)(H.sub.2).sub.2].sup.+ is achieved. In addition, FIG. 19 again confirms that [Co(III)(IDA)(His.sub.6-GFP)] complexes show a strong stability towards chelators (here imidazole).

Example 18: Comparison of [Co(III)(Metal Binding Domain)(His-GFP)] Chemically Stable Complex Formation via [Co(III)(Metal Binding Domain)(CO.SUB.3.)].SUP.2− Versus Oxygen Treatment of [Co(II)(Metal Binding Domain)(His-GFP)]

[0479] Example 5 documents that a high percentage of [Co(III)(NTA)(His-protein)] complexes formed via [Co(III)(NTA)(CO.sub.3)].sup.2−are chemically stabile. We speculated that the [Co(III)(metal binding domain)(His-protein)] complex formation starting with [Co(III)(metal binding domain)CO.sub.3].sup.2− may result in an increased amount of chemically stable [Co(III)(metal binding domain)(His-protein)] complexes than the treatment of [Co(II)(metal binding domain)(His-protein)] with oxygen. To confirm this finding, the amount of formed chemically stable [Co(III)(metal binding domain)(His-protein)] complex via [Co(III)(metal binding domain)(CO.sub.3)] or by 8 hours of oxygen treatment of [Co(II)(metal binding domain)(His-protein)] was directly compared using NTA or IDA as metal binding domain, respectively.

[0480] Functionalization of IDA/NTA Magnetic Agarose Beads

[0481] IDA functionalized magnetic beads (Cube Biotech; 30805) or NTA functionalized magnetic agarose resin (Thermo, 78605) were washed 1) with 80 bead volumes ddH.sub.2O, 2) with 80 bead volumes 100 mM EDTA pH8.0, 3) twice with 80 bead volumes ddH.sub.2O and once with 160 bead volumes ddH.sub.2O for [Co(II)(IDA/NTA)(H.sub.2O).sub.2] complexes or 1 M NaHCO.sub.3 for [Co(III)(IDA/NTA)(CO.sub.3) complexes or no metal samples, respectively. For samples involving with [Co(II)(IDA/NTA)(H.sub.2O).sub.2] complexes, all washes were performed with degassed, 20 min nitrogen aerated solutions as well as in tubes overlayed with nitrogen.

[0482] Subsequently 160 bead volumes 1 mM Co(II)Cl.sub.2*6H.sub.2O in degassed, 20 min nitrogen aerated ddH.sub.2O (for [Co(II)(IDA/NTA)(H.sub.2O).sub.2]) or 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 (for [Co(III)(IDA/NTA)(CO.sub.3)] complexes) were added. To samples without metal 1 M NaHCO.sub.3 was added. After incubating for 10min (or 48 h for indicated samples with [Co(III)(NTA)(CO.sub.3)].sup.2− complex) in a thermo shaker at 25° C. at 1400 rpm the beads were washed thrice with 160 bead volumes of ddH.sub.2O for [Co(II)(IDA/NTA)(H.sub.2O).sub.2] samples or 1 M NaHCO.sub.3 for samples with [Co(III)(IDA/NTA)(CO.sub.3)] complexes or no metal samples, respectively.

[0483] Immobilization of His.sub.6-GFP to the Functionalized IDA/NTA Magnetic Beads

[0484] The produced beads were incubated with 120 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 30 min or 48 h for no metal samples or as indicated in FIG. 9. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed twice (sample “[Co(III)(IDA/NTA)(His-GFP)] via H.sub.2O.sub.2” only once) with 160 bead volumes protein buffer and finally 160 bead volumes protein buffer was added. One sample of [Co(II)(IDA/NTA)(His-GFP)] (referred to as “[Co(III)(IDA/NTA)(His-GFP)] via O.sub.2” in FIG. 20) was aerated with O.sub.2 for 8 hours, while in another sample of [Co(II)(IDA/NTA)(His-GFP)] (referred to as “[Co(III)(IDA/NTA)(His-GFP)] via H.sub.2O.sub.2” in FIG. 9) 6.4 bead volumes of 500 mM H.sub.2O.sub.2 (final 20 mM) were added and incubated for 1 h at 25° C. with 1400 rpm on a thermo shaker followed by a wash with 160 bead volumes protein buffer.

[0485] Chemical Stability of the [Co(III)(IDA/NTA)(His.sub.6-GFP)] Complex

[0486] After the indicated Co(III)(IDA)(His-GFP)] or Co(II)(NTA)(His-GFP)] complex formation processes were performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were washed once with 160 bead volumes protein buffer, resuspended in 160 bead volumes protein buffer, splitted in two portions (each 72 bead volumes) and washed once either with 178 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 178 bead volumes protein buffer, beads were resuspended in 178 bead volumes protein buffer and 10 μl bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). IDA experiments were performed in triplicates; NTA experiments in singlets in three independent experiments.

[0487] The results of these experiments as documented in FIG. 20 clearly demonstrate that significantly more His.sub.6-GFP protein can be immobilized chemically stable on beads using [Co(III)(IDA)(CO.sub.3)].sup.2− complexes (FIG. 20A) and [Co(III)(NTA)(CO.sub.3)].sup.2− complexes (FIG. 20B) than when aerating beads functionalized with Co(II)(metal binding domain)(His-protein)] complexes for 8 hours with oxygen. Beside a higher percentage of stable complex, using [Co(III)(metal binding domain)(CO.sub.3)].sup.2− is also faster than the oxidation method.

Example 19: Protein Immobilization via [Co(III)(HS-PEG-NTA)(CO.SUB.3.)].SUP.2− Complexes on Surfaces

[0488] Several examples of this invention demonstrate the formation of [Co(III)(NTA)(His-protein)] complexes via [Co(III)(NTA)(CO.sub.3)].sup.2−. In this example the protein immobilization with this principle is tested on glass surfaces with nano-structured gold dots.

[0489] Production and Passivation of Nano-Structured Glass Surfaces

[0490] Nanostructured surfaces were produced by diblock-copolymer micelle nanolithography as previously described (Spatz, Mossmer et al. 2000, Roman, Martin et al. 2003, Lohmuller, Aydin et al. 2011) with an average particle spacing of 58 nm as determined by scanning electron microscopy. Briefly, 5 mg/ml of polystyrene(501)-b-poly-2-vinylpyridine(323) (Polymer Source, Canada) respectively was dissolved in o-xylene. Subsequently, tetrachloroauric acid to vinylpyridine monomer ratio of 0.5 were added to the solution and stirred for 24 hours. The solution was spin-coated on 20×20 mm N° 1 glass coverslips (Carl Roth, Germany). Afterwards, the substrates were subjected to a plasma procedure (10% H.sub.2/90% Ar, 350 W, 0.4 mbar, 45 min).

[0491] To prevent non-specific adhesion of any proteins to the glass substrate in-between the gold nanostructures, the glass surfaces were passivated according to a procedure as described before (Blummel, Perschmann et al. 2007). Therefore, the nano patterned surfaces were activated in an oxygen plasma (150 W, 0.4 mbar, 10 min) and incubated at 80° C. over night in dry toluene p.a. (Acros Organics, USA) containing 0.25 mM α-methoxy-ω-trimethoxysilyl poly(ethylene glycol) (molecular weight 2000 g/mol) (Iris Biotech, Germany), 5.5 μM water and 20 mM dried trimethylamine (Acros Organics, USA) under nitrogen atmosphere. Finally, the substrates were washed thrice with ethyl acetate (Acros Organics, USA), once with methanol (VWR chemicals, USA) and dried under N.sub.2 flow.

[0492] Functionalization of Surfaces with thiol-PEG-NTA

[0493] After passivation 100 μl of 0.5 mM HS-(CH.sub.2).sub.11-EG.sub.3-NTA (Prochimia; TH007) in 99.8% ethanol or ethanol only for “PEG only” and “PEG/GFP” sample were pipetted on each surface. Following an incubation for 1 h at room temperature surfaces were washed thrice in a ddH.sub.2O bath or for sample “[Co(III)(NTA)(His-GFP)]” in 1 M NaHCO.sub.3.

[0494] Formation of [Co(III)(NTA)(His-GFP)] Complex

[0495] After functionalization the surfaces are covered with 400 μl of 1 mM Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 for sample “[Co(III)(NTA)(His-GFP)]” or ddH.sub.2O for the other samples and incubated for 10 min at room temperature. Subsequently, surfaces were washed thrice in a bath of 1 M NaHCO.sub.3 for sample “[Co(III)(NTA)(His-GFP)]” or ddH.sub.2O for the other samples. Finally, 300 μl 10 μM His.sub.6-GFP in protein binding buffer (50 mM HEPES pH7.2, 150 mM NaCl) were added on top of surfaces and incubated for 30 min at room temperature. After three washes in a bath of protein binding buffer, surfaces were placed in a transparent 6-well plate, covered with protein binding buffer and GFP-fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) as measure of immobilized protein on surfaces was analysed using a plate reader (TECAN, Spark).

[0496] The results of the experiment are presented in FIG. 21. Thereby a much higher fluorescent signal for the surface treated with thiol-PEG-NTA, Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O and His-GFP could be measured compared to the other control samples showing the successful formation of the [Co(III)(NTA)(His-GFP)] complex on the surfaces.

Example 20: Site-Specific Biotinylation of His-GFP with [Co(M)(Biotin-X-NTA)(CO.SUB.3.)] Complexes

[0497] In several examples the immobilization of His-tagged proteins to beads and in example 19 to surfaces functionalized with NTA via to [Co(III)(NTA)(CO.sub.3)].sup.2− complexes is demonstrated. In the following example the in solution biotinylation of His-GFP at its His-tag via [Co(III)(Biotin-X-NTA)(CO.sub.3)] complexes is tested.

[0498] Biotinylation of His-GFP

[0499] 60 μM Biotin-X-NTA (Sigma-Aldrich; 51410) is mixed with 30 μM (sample 1:2), 60 μM (sample 1:1) or 600 μM (sample 10:1) Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O in 1 M NaHCO.sub.3 or only 1 M NaHCO.sub.3 for samples with metal and incubated for 10 min at room temperature on a rotating wheel. The resulting [Co(III)(Biotin-X-NTA)(CO.sub.3)] complexes are subsequently incubated at room temperature with a solution of 6 μM His.sub.6-GFP (SEQ ID NO: 14) in protein binding buffer (50 mM HEPES pH7.2, 150 mM NaCl) for 30 min or 48 h on a rotator wheel.

[0500] Immobilization of [Co(III)(Biotin-X-NTA)(His-GFP)] to Streptavidin Functionalized Sepharose

[0501] After the incubation the resulting [Co(III)(Biotin-X-NTA)(His-GFP)] complexes were bound in a 30 min or 48 h as indicated incubation step with rotation at room temperature to streptavidin functionalized sepharose (GE Healthcare, 17-5113-01), which was prepared by three washes with 166 bead volumes protein binding buffer. After the incubation the beads were washed thrice with 16 bead volumes of protein binding buffer and resuspended in 16 bead volumes of protein buffer

[0502] Chemical Stability of the [Co(III)(Biotin-X-NTA)(His.sub.6-GFP)] Complex

[0503] After the immobilization process of [Co(III)(Biotin-X-NTA)(His-GFP)] complex to the beads, the amount of protein bound to the beads with or without chemical stress was analyzed. To this end, beads were splitted in two portions and washed once either with 17 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 17 bead volumes protein buffer, beads were resuspended in 17 bead volumes protein buffer. Finally, 10 μl bead slurry was analyzed for its immobilized protein amount via GFP fluorescence (λ.sub.ex=490 nm, λ.sub.em=535 nm) using a plate reader (TECAN, Spark). The experiments were performed in triplicates.

[0504] As presented in FIG. 22A the in solution biotinylation of His-tagged GFP could be demonstrated for all Co/NTA ratios. However, with higher ratios of Biotin-X-NTA to Na.sub.3[Co(III)(CO.sub.3).sub.3]*3H.sub.2O also a better labeling efficiency could be achieved as measured based in the fluorescence of biotinylated protein immobilized on the streptavidin beads. In FIG. 22B it is demonstrated that longer protein incubations yield in a higher amount of stable formed complex as more complex after 48 h of protein incubation is immobilized on streptavidin beads after a stringent imidazole wash as compared to samples with 10 min protein incubation.

Example 21: Complex Formation and Chemical Stability of [Pt(IV)(NTA)(His.SUB.6.-GFP)] using Platinum(IV) Nitrate

[0505] We speculated that not only the carbonate as ligand, but also nitrate at the metal center may facilitate the formation of the [(metal)(NTA)(His-protein)] complex. Additionally, we speculate that beside Co.sup.3+ also other transition metals with low ligand exchange rates may form chemically stable [(metal)(NTA)(His-protein)] complexes. To confirm this finding, the binding efficiency of His.sub.6-GFP to [Pt(IV)(NTA)(NO.sub.3)] was examined.

[0506] Functionalization of NTA Agarose Beads

[0507] NTA functionalized agarose beads (Qiagen; 1022963) were washed 1) with 10 bead volumes ddH.sub.2O, 2) with 10 bead volumes 100 mM EDTA pH8.0, 3) twice with 10 bead volumes ddH.sub.2O and once with 10 bead volumes 1 M nitric acid. Subsequently 16 bead volumes platinum(IV) nitrate solution (44 mg/1 Pt(IV)) (Fisher Scientific; 15407817) in 1 M nitric acid or only 1 M nitric acid for samples without metal were added. After incubating the samples for 10 min in a thermo shaker at 25° C. at 1400 rpm the beads were washed thrice with 16 bead volumes of 1 M nitric acid.

[0508] Immobilization of His.sub.6-GFP to functionalized [Pt(IV)(NTA)(NO.sub.3)] Agarose Beads

[0509] The produced beads were incubated with 16 bead volumes of 10 μM His.sub.6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25° C. shaking at 1400 rpm on a thermo shaker for 30 min. After the incubation of functionalized beads with His.sub.6-GFP (SEQ ID NO: 14), beads were washed once with 16 bead volumes protein buffer and resuspended in 16 bead volumes protein buffer.

[0510] Chemical Stability of the [Pt(IV)(NTA)(His.sub.6-GFP)] Complex

[0511] After the indicated Pt(IV)(NTA)(His.sub.6-GFP)] complex formation process was performed, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, beads were splitted in two portions (each 7.2 bead volumes) and washed once either with 17.8 bead volumes protein buffer or 250 mM imidazole in protein binding buffer, respectively. After a final wash with 17,8 bead volumes protein buffer, beads were resuspended in 17.8 bead volumes protein buffer. Protein amount on 25 μl beads slurry was determined by BCA assay (Thermo, 23227) in microplate based on the manufacturer's guidelines. The experiment was performed in triplicates.

[0512] The results depicted in FIG. 23 clearly demonstrate the platin(IV) mediated immobilization of His.sub.6-GFP to NTA functionalized beads in a chemical stable manner. This proofs that not only cobalt(III), but also platin(IV) can form kinetically inert complexes with NTA and his-tagged protein. Additionally a very fast complex formation could be demonstrated by the use of nitrate as metal binding ligand.

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