1. Introduction

2. Materials and Methods

3. Results and Discussion

3.1 Functionalization of purified corundum

We created a new corundum-based platform acting similarly to an ion metal affinity chromatography (IMAC) support. The first functionalization step was the covalent attachment of the common amino silane APTES to obtain an amine function on the corundum surface. Previous experiments with this silane indicated that 10 % of APTES at room temperature and a reaction time of 16 hours led to higher binding yields compared to 1 % (data not shown). After silanization, a solution of 1 % EDTA dianhydride in dry DMF was added. This fast reaction will lead to an amide bond between the amine group of the APTES and a ring opening of the anhydride, resulting in the covalent attachment of EDTA to the corundum surface. When incubated with a solution of nickel ions, this ligand will form a complex with Ni²⁺, which will later interact with the Histidine residues of the His-tagged protein that is desired to be purified. The described functionalization scheme is shown in Figure 1.

Figure 1. Functionalization of corundum with APTES, EDTA dianhydride, and Ni²⁺ to purify His-tagged proteins (Schematic representation).

Figure 1. Functionalization of corundum with APTES, EDTA dianhydride, and Ni²⁺ to purify His-tagged proteins (Schematic representation).

To verify the surface modification described above we applied the Kaiser test. This is a very sensitive assay for the qualitative determination of primary amine groups [24,25] frequently applied in peptide synthesis. First, the Kaiser test can confirm that the silanization with APTES was successful. In the next step, the reaction with EDTA dianhydride, the faint blue color indicates that the derivatization is still incomplete. Figure 2 illustrates the Kaiser test with unfunctionalized corundum, which does not show any blue color (a). After a successful reaction of corundum with APTES, a strong blue color was observed with the Kaiser test (b), confirming the amino silanization of the surface. After incubation with 1 % of EDTA dianhydride in dry DMF for 30 min (c), the blue color was weaker, indicating that some non-reacted primary amine groups were still present. After a second incubation with 1 % EDTA dianhydride solution (d), no blue color could be detected with the subsequent Kaiser test meaning that the derivatization is completed.

Figure 2. Kaiser test (ninhydrin-based) for the detection of primary amine groups on the corundum surface. (a) Unmodified corundum showing no blue color, (b) strong blue color after functionalization with APTES, (c) a weaker blue color after the first incubation with EDTA dianhydride (EDTAD), (d) no blue color after the second incubation with EDTA dianhydride indicating that all primary amines have been derivatized.

Figure 2. Kaiser test (ninhydrin-based) for the detection of primary amine groups on the corundum surface. (a) Unmodified corundum showing no blue color, (b) strong blue color after functionalization with APTES, (c) a weaker blue color after the first incubation with EDTA dianhydride (EDTAD), (d) no blue color after the second incubation with EDTA dianhydride indicating that all primary amines have been derivatized.

3.2 Determination of nickel content via ICP-MS

In order to quantify the nickel capacity of the modified material, one gram of functionalized and nickel-loaded corundum as well as 1 gram of Ni-unmodified corundum as negative control were treated with concentrated HNO₃ to release the metal ions into the supernatant. This direct approach can be considered very reliable and highly quantitative [26]. Figure 3 illustrates the working scheme.

Figure 3. Workflow for the nickel determination via ICP-MS. (a) Unmodified and modified corundum particles were treated with 65 % HNO₃ for 1 hour at 100 °C. (b) The strong acid was diluted 1:33 with pure lab water. (c) ICP-MS determination was performed. (d) An external calibration was used to quantify the nickel concentration.

Figure 3. Workflow for the nickel determination via ICP-MS. (a) Unmodified and modified corundum particles were treated with 65 % HNO₃ for 1 hour at 100 °C. (b) The strong acid was diluted 1:33 with pure lab water. (c) ICP-MS determination was performed. (d) An external calibration was used to quantify the nickel concentration.

ICP-MS results are shown in Table 1. The nickel content of fully functionalized corundum particles was more than 3500 times higher than in the blank samples. This underlines the successful functionalization and proves the high nickel capacity of the surface.

Table 1. Nickel determination via ICP-MS of loaded corundum and EDTA-functionalized corundum as a negative control. The nickel calibration curve is shown in the Supplementary Material (Fig. S1).

Table 1. Nickel determination via ICP-MS of loaded corundum and EDTA-functionalized corundum as a negative control. The nickel calibration curve is shown in the Supplementary Material (Fig. S1).

sample	Ni µmol/g corundum	Ni2+ µg/g corundum
Nickel-loaded corundum	5.2 ± 0.1	304 ± 6
Blank material	0.0015 ± 0.0001	0.09 ± 0.01

If a 2:1 molar binding ratio of Ni²⁺ with a His-tagged protein of ca. 66 kDa (like BSA) is assumed [27], the nickel value would theoretically allow a bound protein mass of about 170 mg protein/g corundum. This means that the amount of immobilized nickel is in vast excess relative to the protein and does not limit the capacity. This result also suggests that polyhistidines may bind to two or more nickel ions [28], without the need for multivalent chelators. It is impossible to exploit the full capacity of the nickel ions with much bigger molecules, such as a protein, due to steric reasons. This corundum material has a surface area of around 4.5 m²/g [11]. According to [29], a side-on interaction of BSA on 1 cm² corresponds to 223 ng BSA. Therefore, for a complete protein monolayer on 1 g corundum, about 10 mg of BSA would be needed. Therefore, this would be the maximum theoretical capacity for BSA and other proteins of similar size.

3.3 Optimization of imidazole concentration in binding buffer

In order to examine the potential of the new corundum platform for the purification of proteins, His-tagged protein A/G (His6-PAG) was separated from a mixture with bovine serum albumin (BSA). Having 17 histidines in its structure [30], BSA can be a potential candidate to interact with the Ni²⁺ on the corundum surface to a certain extent. In general, it can be assumed that the higher the imidazole content during the binding step, the higher the selectivity will be, with the potential drawback that the capacity may decrease and some nickel leaching might occur. BSA and His6-PAG were mixed in a mass ratio of 5:1 to explore the selectivity of this method. Figure 4 shows the SDS-PAGE separation of the eluates obtained after the elution with 500 mM imidazole buffer. The used BSA was of technical quality; hence, it was no surprise that several protein bands were visible in the reference lane (lane 1). The recombinant His6-PAG also shows some impurities and heterogeneities, most prominent in the double bands with the highest intensities (lane 2). When imidazole was used in the binding buffer, the potential interaction of BSA and its impurities should be inhibited in a concentration-dependent fashion. This becomes evident with increasing imidazole content in the binding (and washing) buffers. When 5-15 mM imidazole was used (lanes 3-5), some protein bands from BSA were still visible. This effect is reduced when 20 mM or higher concentrations of imidazole are used (lanes 6-8).

Figure 4. Variation of the imidazole concentration in the binding and washing buffers (TBS); SDS-PAGE electrophoresis of separated His6-PAG from a BSA-containing solution. An imidazole concentration of 20 mM and higher led to the purest preparations, with slightly decreasing yields.

Figure 4. Variation of the imidazole concentration in the binding and washing buffers (TBS); SDS-PAGE electrophoresis of separated His6-PAG from a BSA-containing solution. An imidazole concentration of 20 mM and higher led to the purest preparations, with slightly decreasing yields.

For further experiments, the imidazole content of 20 mM was chosen as a binding buffer since the eluate showed few impurities with high protein capacity. Therefore, when having real-life samples, a binding buffer with 40 mM imidazole was prepared and then diluted with the sample 1:2 to have a final imidazole concentration of 20 mM. This TBS40 was also used as a washing buffer. When the highest protein binding capacities are desired, a lower imidazole content could be beneficial. For these applications, a 10 mM imidazole binding buffer (TBS10) was also tested in further experiments.

3.4 BCA assay for the determination of protein binding capacity in TBS20 and TBS40

The binding capacity of nickel-loaded corundum for His6-PAG was determined with 100 mg aliquots. First, 100 mg of functionalized corundum was incubated with different amounts of His6-PAG ranging from 0 to 600 µg. The incubation took place for one hour at RT in an overhead rotator before the supernatants were collected and measured with the BCA assay [31]. Figure 5 shows the amount of PAG bound to the surface when the amount in the supernatant is subtracted from the initial PAG content. When incubated with more than 400 µg PAG, around 300 µg can be bound to the surface. This means 1 g of functionalized corundum can bind up to 3 mg of His6-PAG (52 nmol). No significant difference in capacity was obtained when different binding buffers with even higher amounts of imidazole up to 40 mM were used.

Figure 5. Determination of the protein binding capacity (binding buffer TBS20 dark blue, TBS10 light blue). The amount of bound His6-PAG is calculated by subtraction of the residual amount from the amount added. A His6-PAG calibration curve (BCA) is shown in the Supplementary Material (Fig. S2). In addition, it could be shown that the use of 40 mM imidazole instead of the concentration of 20 mM does not significantly reduce the protein binding capacity of the material.

Figure 5. Determination of the protein binding capacity (binding buffer TBS20 dark blue, TBS10 light blue). The amount of bound His6-PAG is calculated by subtraction of the residual amount from the amount added. A His6-PAG calibration curve (BCA) is shown in the Supplementary Material (Fig. S2). In addition, it could be shown that the use of 40 mM imidazole instead of the concentration of 20 mM does not significantly reduce the protein binding capacity of the material.

3.5 Purification from E. coli cell lysates spiked with His6-PAG

In order to test matrices of high complexity, the soluble fraction of E. coli cell lysates of two different strains, NiCo21(DE) and BL21(DE) pLysS, containing endogenous histidine-rich proteins, were spiked with His6-PAG. E. coli cytoplasm can contain up to 320 mg/mL of total protein [32] and is a demanding matrix for the selective purification of a single protein First, the spiked NiCo21(DE) sample was diluted 1:2 in TBS20 or TBS40 leading to a final imidazole concentration of 10 mM or 20 mM in the extraction solution, respectively. After incubation, three washing steps with a buffer containing either 20 mM (for the 10 mM sample) or 40 mM (for the 20 mM sample) imidazole were performed. Finally, the bound His6-PAG was dissociated from the corundum with 500 mM imidazole for 30 min for both samples. Figure 6 shows the obtained SDS-PAGE gels with reference and sample lanes.

Figure 6. Isolation of His6-PAG from spiked E. coli NiCo21(DE) cell lysate (CL). For both binding buffers (TBS10 and TBS20) the isolation of His-tagged PAG was successful with high purity. A silver-stained gel is shown in the Supplementary Material (Fig. S3).

Figure 6. Isolation of His6-PAG from spiked E. coli NiCo21(DE) cell lysate (CL). For both binding buffers (TBS10 and TBS20) the isolation of His-tagged PAG was successful with high purity. A silver-stained gel is shown in the Supplementary Material (Fig. S3).

Both binding and washing buffers show very similar isolation and purification characteristics of His6-PAG from the cell lysate of NiCo21(DE), an E. coli strain with reduced endogenous histidine-rich proteins. In this specific case, GlmS (Glutamine-fructose-6-phosphate aminotransferase) is mutated to reduce co-binding [33]. The purity of both eluates is excellent, and the intensity of bands is similar. Therefore, we concluded that an imidazole concentration of 10 mM in the binding and 20 mM in the washing buffer seems suitable to avoid non-specific binding and does not compromise capacity. Subsequently, we tested this method with an E. coli strain with normal content of endogenous histidine-rich proteins that could interfere with affinity purification, and hence, His6-PAG was spiked into the cytoplasm originating from E. coli BL21(DE3)pLysS. Figure 7 shows the corresponding SDS-PAGE gel. Next to the spiked eluates, a matrix eluate was examined, where no His6-PAG was added. These lanes might show any non-specific interactions of endogenous proteins in more detail.

Figure 7. Isolation of His6-PAG spiked into the cell lysate (CL) of E. coli BL21(DE3)pLysS. The silver-stained gel reveals that the matrix eluate shows fewer impurities when using TBS20 as a binding buffer (

Figure 7. Isolation of His6-PAG spiked into the cell lysate (CL) of E. coli BL21(DE3)pLysS. The silver-stained gel reveals that the matrix eluate shows fewer impurities when using TBS20 as a binding buffer (

The eluate with a binding buffer containing 10 mM imidazole shows a slightly higher number of blank proteins than the buffer with 20 mM. Furthermore, it can be shown that most of the His6-PAG is bound and eluted with TBS10. With higher concentrations of imidazole in the binding and washing buffer, the amount of bound His6-PAG decreased, but the purity was improved. This effect can also be seen in the blank eluates (cytoplasm without His6-PAG).

3.6 Enrichment of His6-PAG from higher volumes

In the next experiment, we investigated if 1 mg PAG can be re-isolated from higher volumes as well, addressing the issue of a one-step recombinant protein purification that is performed on larger scales and is still considered challenging [34]. For this purpose, 1 mg PAG was spiked into 2 mL, 10 mL, 100 mL, and 1000 mL of TBS10 and incubated with 1 g Ni-loaded corundum, each. The incubation took place overnight in an overhead rotator. All samples were washed three times with TBS10 and then eluted with 500 mM imidazole. Figure 8 illustrates the mentioned volumes to visualize the reduction of the sample volume from extraction to elution, resulting in a significant protein enrichment. Figure 9 shows the correspondent SDS-PAGE of extraction and elution solutions.

Figure 8. Reduction of a sample volume between 100 and 1000 mL resulting in a final elution volume down to 1 mL (Schematic representation).

Figure 8. Reduction of a sample volume between 100 and 1000 mL resulting in a final elution volume down to 1 mL (Schematic representation).

Figure 9. Enrichment experiment: 1 mg of His6-PAG was spiked into different volumes (2 mL-1000 mL) of 0.1 % BSA in TBS10. His-tagged protein could be enriched in one step even from highly diluted solutions.

Figure 9. Enrichment experiment: 1 mg of His6-PAG was spiked into different volumes (2 mL-1000 mL) of 0.1 % BSA in TBS10. His-tagged protein could be enriched in one step even from highly diluted solutions.

Lanes 2-5 in Figure 9 show the corresponding stock solutions of 1 mg His6-PAG in different volumes of a 0.1 % BSA solution as a matrix model. The His6-PAG concentration in the stock solutions decreases with higher volumes to a point where the His6-PAG band in 1000 mL is not even visible, whereas the matrix concentration was kept constant. In contrast, in the eluate of the 1000 mL sample, the His6-PAG band is clearly visible, indicating successful enrichment from a higher volume. The amount of His6-PAG in that sample was 1000 times lower than BSA, which is also corresponding to a selectivity factor of 1000. This is very promising in terms of up-scaling the purification process, which due to the low costs for the corundum material, is considered as realistic. When an enrichment from even higher volumes is desirable, it must be taken into consideration that the affinity of Ni chelates to bind His-tags might not be sufficient. Choosing tags with higher affinities could be a way to counteract this issue.

3.7 Comparison of corundum platform with commercial Ni-NTA agarose beads

Next, we investigated how the corundum particles perform compared to well-established and commercial Ni-NTA agarose beads. Again, His6-PAG was spiked into E. coli BL21(DE3)pLysS cell lysates and the samples were purified with the established corundum method as well as Ni-NTA agarose beads according to the manufacturer’s protocol (Supplementary Material). Different buffers and volumes were used for both protocols to compare both methods under their optimized conditions. For corundum, a TBS20 for binding and a TBS40 for washing were used, and finally, an elution buffer with 500 mM imidazole. The standard agarose protocol was performed with 10 mM imidazole in the binding buffer, 20 mM imidazole in the washing buffer, and elution was performed with 250 mM imidazole. Figure 10 shows the respective SDS-PAGE gel with a Coomassie stain. The eluate of the corundum particles contains fewer impurities than the nickel-NTA-agarose beads (particularly in the lower molecular weight range).

Figure 10. Isolation of His6-PAG from spiked E. coli cell lysate by nickel-loaded corundum particles compared with a commercial Ni-NTA agarose material.

Figure 10. Isolation of His6-PAG from spiked E. coli cell lysate by nickel-loaded corundum particles compared with a commercial Ni-NTA agarose material.

3.8 Purification of recombinant His6-MBP-mSA2 fusion protein from E. coli cytoplasm

In order to show that this new corundum material also performs well with real-life samples, recombinantly expressed His6-MBP-mSA2 was extracted from E. coli cell lysate. This protein, with a mass of around 60 kDa, could be isolated in one step with excellent purity. The respective SDS-PAGE gel is shown in Figure 11. The lane of the supernatant revealed an overload of the capacity of the material. This is a general issue in affinity methods if the approximate concentration of the target is unknown in advance.

Figure 11. Extraction of cytoplasmically expressed His6-MBP-mSA2 (Maltose binding protein and monovalent streptavidin tagged with N-terminal His6) from E. coli BL21(DE3)plysS. For both binding buffers TBS 10 and TBS20, the extraction and purification of the fusion protein were successful. No significant difference could be observed for these two buffers.

Figure 11. Extraction of cytoplasmically expressed His6-MBP-mSA2 (Maltose binding protein and monovalent streptavidin tagged with N-terminal His6) from E. coli BL21(DE3)plysS. For both binding buffers TBS 10 and TBS20, the extraction and purification of the fusion protein were successful. No significant difference could be observed for these two buffers.

3.9 Purification of recombinant SARS-CoV-2-S-RBD-His8 from Expi293F cells

The SARS-CoV-2 pandemic greatly increased interest in virus-related proteins and their recombinant expression. The SARS-CoV-2 Spike protein (S) interacts with a human cell surface receptor and is crucial for infection [35]. It can be expressed in cell culture as a valuable tool for ELISA and LFA assay development or vaccine development [36]. Here, a recombinant SARS-CoV-2 S-RBD (receptor binding domain) was purified with Ni-functionalized corundum from human Expi293F cell culture supernatant. Figure 12 shows the SDS-PAGE gel for the purification process of SARS-CoV-2-S-RBD-His8 from Expi293F cell culture supernatant with a molecular mass of 23.4 kDa.

Figure 12. Purification of recombinant SARS-CoV-2-S-RBD-His8 (23.4 kDa) from Expi293F cell supernatant (CL). TBS20 was used as the binding buffer and TBS500 for elution

Figure 12. Purification of recombinant SARS-CoV-2-S-RBD-His8 (23.4 kDa) from Expi293F cell supernatant (CL). TBS20 was used as the binding buffer and TBS500 for elution

Figure 12 shows the successful isolation and enrichment of a recombinant receptor-binding domain (RBD). No product is visible in the depleted supernatant, indicating that the protein was extracted quantitatively from the cell culture (CL) supernatant. The wash solutions show little to no protein. The eluate consists of the His-tagged RBD domain in high purity.

4. Conclusions

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