Immobilized metal affinity chromatography experiments

Summary

Summarizes the application areas of IMAC, including protein hierarchical separation and proteomics, protein fixation and detection, as well as some special applications, such as immunoglobulin purification and the Chelex method.

Authors: Burgess et al, Translator: Chen Wei, This experiment is from "Protein Purification Guide".

Operation method

Immobilized metal affinity chromatography experiments

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IMAC applications 1. Detection and immobilization

In the quest to utilize H i s to label the specificity and high affinity of proteins for immobilized metal ions, IMAC ligands have been applied to protein-protein interactions, in which proteins must be stably immobilized on surfaces. Two applications including enzyme-linked immunosorbent assay (ELISA) and microarray technology are briefly described below.
ELISA is used as a diagnostic tool and microarray technology is used for functional analysis.

Ni-NTA ligands are adsorbed onto the surface of microtiter plates to immobilize soluble and structurally complete Hs-labeled antigens for serological studies. Direct fixation using the Hils marker has advantages over standard ELSA. Standard Elisa randomly adsorbs proteins onto plastic surfaces, which causes structural damage to the proteins and hides portions of the protein surface and potential binding sites for antibodies. In contrast, IMAC-based ELISA can screen for conformation-dependent monoclonal antibodies (Padan etal., 1998), increasing the sensitivity of the immunosorbent assay (Jinet al., 2004).

The immobilization of his-tagged proteins on microarray surfaces for the study of interactions with other molecules (e.g., by SPR) is a widely used method for protein identification. "Stability" is an important factor to reduce the "leakage" of immobilized molecules. Based on these binding properties, NTA ligands are often used for immobilization (Knecht etal., 2009; Nieba etal., 1997). However, the interaction of biotinylated proteins with streptavidin-encapsulated carriers is considered to be more robust. According to the concept of the multivalent chelator head, which can contain three NTA groups on only one ligand, the stability of the immobilization function can be guaranteed even at low concentrations for glass-surface immobilized Hs-tagged proteins, which is a great step forward! (tris-NTA; Lata and Piehler, 2005; Zhaohua et al., 2006). This advancement provides an alternative to the streptavidin/biotin protein-based immobilization and allows the use of hils-labeled proteins without biotinylation in the following purification process. We have synthesized the tris-NTA ligand and coupled it to magnetic agarose beads to test whether this method can be applied to the purification of hils-labeled proteins, which may allow for more specific, single-step recovery from complex samples. Preliminary data (Fig. 27.3) suggest that this may indeed be the case. The trisNi-NTA beads were purified to a slightly higher purity after application of two media to AKTl kinase isolated from the cell-free lysis reaction of the meadow nightshade moth source. Whether these findings are generalizable and whether the tris-NTA chromatography method can be commercialized on a large scale remains to be evaluated.

IMAC ligands have also successfully replaced antibodies as indicators for many types of immunoblotting. H is-tagged proteins are transferred onto nitrocellulose membranes and detected by Ni-NTA coupled with alkaline phosphatase or horseradish peroxidase for colorimetric or chemiluminescent detection (Lv et al., 2003) or with quantum dots for fluorescence detection (Kim etal., 2008). This means that it can be a fast and economical alternative to antibody detection when high specificity is not required, and is therefore popular. The availability of tris-N T A couplings has improved the specificity of N T A coupling assays (Lata et al., 2006; Reichel et al., 2007).

2 Purification of protein fractions

Initially, IMAC was developed as a group separation method for proteins containing metal ions and histamine CPorathetaL , 1975). These functions of IMAC are now used in proteomics. In proteomics research, reducing the complexity of the system (proteome) is essential to ensure the sensitivity of low-abundance protein analysis. The reason for this is that
Therefore, proteomics is increasingly using pre-separation methods to concentrate proteins that may be lost during the assay, such as liquid phase, reversed-phase, ion exchange, and affinity chromatography (e.g., IMAOCLOO, 2003; StasykandHuber, 2004). Recently, there has been a review of the application of IAMC to proteomics (Sun et al., 2005), mainly for the concentration of phosphorylated proteins, phosphorylated peptides and metal-bound proteins. When concentrating complex samples such as cell lysates or blood, the sample is flowed through the IMAC medium and then washed, followed by elution of the target fractions by different p H or high concentrations of imidazole, and then concentrated using the IMAC medium.
The eluted components are then analyzed by mass spectrometry (MS), or further separated by two-dimensional gel electrophoresis and analyzed by MS or liquid-mass spectrometry (LC-MS).

^Fe3+, ^A13+ and ^Ga3+ are the preferred ions for the study of phosphorylated proteins and are usually fixed in IDA. Ions suitable for MAC analysis of metalloproteomics are copper, nickel, zinc and iron elements, which are essential for life. The metalloproteome refers to a group of proteins with metal-binding ability, and there have been several recent reviews of this proteomics (Shi and Chance, 2008; SunetaL, 2005). Concentration can be achieved by exploiting the ability of these metal-binding proteins to bind to a certain type of solidified ^Me2+ (e.g., ^Me2+-NTA ), or by exploiting the ability to trap ^Me2+ on an uncharged IMAC ligand (e.g., NTA) and to bind to it to form a ^Me2+-protein.

I M A C microarray analysis of the proteome has also been reported (Slentz et aL, 2003) and can be used as a tool for clinical screening of proteins and peptides containing phosphorylated groups and histidines (S E L D I - I M A C ).

The heterogeneity of affinity interactions between potential binding sites (oxygen in phosphorylated groups, nitrogen and oxygen in bases, and transgroups in nucleotides) and immobilized metal ions is a complex phenomenon that underlies the use of I M A C for the conjugation and isolation of nucleotides in mono-, di-, or more polymeric forms (Hubert and Porath, 1980; 1981).

The affinity interactions of antibodies with immobilized metal ions can be applied to the IM A C separation technique, which is a completely different method of motif-specific separation. The metal ion binding site within the heavy chain is the molecular basis for this interaction (H ale and Beidler, 1994), and the arrangement of histidine residues in antibodies has been analyzed (p orathand Olin, 1983). Many authors have previously reported the adsorption of immunoglobulins from different sources with IM A C mediators (human IgG, Porath and Olin, 1983; humanized mouse IgG, Hale and Beidler, 1 9 9 4 ; goat IgG, Bodene t a l., 1995). Various forms of IMAC purified antibodies have been successfully used, including gels (Hale and Beidler, 1994; Vancanetall, 2002), methyl propionate polymers (Mfeszdrosovd et al., 2003), and hollow-fiber columns (Serpaeaall, 2005). The mild nature of salt elution, the low cost and the durability of the IM A C media, compared to conventional P ro tein A /P ro te in G chromatography, have become advantages of the IM A C method (Serpa et al., 2005).

For a complete overview of the application of IMAC ligands, it is important to mention the Chelex method. The classical IM A C method uses the affinity of transition metal ions to purify peptides/polypeptides by immobilizing metal ions. In contrast to traditional methods, Chelex describes a nucleic acid sample preparation method that utilizes the technique to deplete the sample of P C R metal ion inhibitors prior to use in the following steps (W d s h e t al., 1991). Analogous to IDA, the uncharged ligand is coupled to a commonly used agarose medium, the Chelex resin (e.g., Bio^Rad Chelex 100). The procedure is briefly summarized as follows: blood or tissue samples are incubated with Chelex resin with/without protease K, and the supernatant containing nucleic acids is separated from the resin pellet. The nucleic acid fraction obtained is not as pure, but the metal ions have been removed from the sample, making it suitable for use in PCR amplification, where high temperatures might otherwise catalyze the cleavage of DNA and inhibit the PCR reaction. The Chele x method is fast and inexpensive, and is therefore mainly used for the amplification of small fragments of DNA from biopsies and small amounts of samples obtained by puncture (GarciaGonzAlez et al., 2004; Gill etal., 1992).

3. purification of h i s-labeled proteins

H is tagging and its effect on protein expression

The most important application of IMAC is the purification of H is-tagged recombinant proteins, where the fusion-expressed H is tag is a fragment containing six or more histidine residues (Fig. 27.1). The H is tag has fairly high affinity and specificity, so in most cases, IMAC-step purification is sufficient to prepare target proteins of a certain purity for many applications. The structure of the tag (i.e., position, order, and length) can affect protein production at several levels: expression rate, binding to the IMAC, and the ability of the tag to bind to the IMAC.
The structure (i.e., position and length) of the tag can affect protein production at a number of levels: expression rate, ability to bind to an IMAC ligand, three-dimensional structure of the protein, formation of protein crystals, etc., and has a role, albeit minor, in solubility and activity. The most common form of the H is tag consists of six consecutive histidine residues (H 6) that provide six metal binding sites. In most cases, the higher the binding/dissociation equilibrium conversion capacity, which tips the equilibrium in the direction of binding, the more likely it is to have stable binding capacity (Table 27. l ; K nech t e t a L , 2009). Biacore assays showed that the dissociation rate of hexameric histidine-labeled proteins from Ni-N T A was I X l ^O6 ~ 1.4X ¹⁰⁸ at pH 7. 0 ~ 7. 4 (K n e c h te ta l., 2009; N ie b a e ta l., 1997). However , the mobility, ligand density, and protein concentration of planar chip surfaces are very different from those of porous agarose particles. In addition, the stability of the interaction of H is-tagged proteins with IM A C ligands is influenced by the accessibility of the tag and the number of all chelating residues (histidine, cysteine, aspartate, and glutamate) on the surface of the proteins (Bolanos-Garcia and Davies, 2006; Jensen et al., 2004), and thus to a large extent this interactions are independent. That is, usually even under harsh conditions, if the H is label is accessible (which is the case in most cases), then the affinity (or more appropriately activity) of the protein for Ni-T N A is sufficiently high for column chromatography. Table 27. 1 lists the labeling sequences reported in the literature, as well as some unpublished labeling sequences recently tested in our laboratory.

In contrast to the "standard" purification of soluble proteins, a different situation is encountered with recovery membrane proteins in the presence of surface-active agents (see Section 2.10 of this chapter), as the surface-active particles may partially or completely cover the HiS label. In this case, a longer labeling sequence or a linker is required to bind the IMAC resin to the protein (M0hanty and Wiener, 2004). Table 27 . 1 shows the various lengths of H is labels and H is labels with other optional sequences that are recommended and have been improved for binding to IMAC resin. However, the applied value of these improved labels does not exceed that of the classical Hn label, either for us or for other laboratories (Knecht et al., 2009). Studies have shown that more important than the label sequence itself is the binding position (N- or C-terminal) and the amino acid sequence at the N-terminal. It has been reported that the role of N-terminal amino acids adjacent to methionine is to prevent N-terminal methionine processing, which has a positive effect on the protein expression rate of Escherichia coli in general (Dalb0ge et al., 1990; Hillel, 1989). Evaluation of these reports by us and other researchers confirms that lysine and arginine have this effect when they are located in the second position of the N-terminus of proteins (Pedersen et al., 1999; SchMer et al., 2002 a; Svensson etal., 2006). However, the high success rate of the N-terminal HiS(Strep n) signature is not only due to the stimulatory effect of the second amino acid on expression, but also because the labeling sequence at this position seems to consistently affect the structure of the mRNA in the start region of the translation. Comparison of N- and C-terminal HiS (Strep II) signature positions was performed for several proteins expressed by bacteria and eukaryotes by analyzing their expression levels and solubility (Fig. 27.4). In most cases, the N-terminal labeling was more favorable for protein expression.

Systematic studies of the mRNA 5' region have shown that the hairpin structure formed by the normally translated start region leads to low expression by preventing ribosomes from binding to the mRNA (Cdbe and Geiser, 2006). Destabilization of the hairpin structure using sequence optimization can improve expression. Similar results can be obtained when the N-terminus of protein is expressed with the Hf 5 label. As shown in Figure 27.4, this effect is due to the fact that the kinetochore function, which translates the mRNA secondary structure in the start region, can be protected. Similar results have been observed by other researchers (Busso et al_, 2003; Svensson et al., 2006), which may explain the attractiveness of the N-terminal H is tag. In some cases, however, the C-terminal H is tag has a more pronounced effect on protein expression rate and solubility, as in the case of IRAK4 (Fig. 27. 4). A similar effect was observed in the recently reported expression of entomotoxin in E. coli, where the C-terminal H is tagged expression form had a higher solubility and thermal stability (Xu
e t a l . 2008). The authors concluded that the C-terminal tag stabilizes the overall structure of the protein. Other research teams have found that H is tagging causes a slight decrease in solubility compared to untagged proteins, but improves protein yield when fused to the C-terminus (W oestenenketal., 2004). In conclusion, these data suggest that tagging mutants at the N- and C-termini of proteins, while not necessarily leading to rational expression of proteins and increased amounts of recombinant proteins, may at least increase the chances of doing so. Placing the tag at the C-terminus avoids interfering with membrane transport and ensures that the protein is secreted for expression.

4. Basic Considerations for Protein Purification with I M A C

The purification of H is-tagged proteins by IMAC has several advantages over other affinity chromatography techniques, which is why IMAC is the most widely used chromatographic technique (Biocompare, 2006; Derewenda, 2004). In addition to its low cost and ease of use, the durability of IMAC is undoubtedly its most outstanding feature: (i) the H is tag interacts with the ligand under nondenaturing or denaturing conditions such as 8 mol/L urea or 6 mol/L guanidinium hydrochloride (Hochuli, 1988) and folds in situ (Jungbauer et al., 2004); (ii) the redox conditions are also favorable; and (iii) the protein folds in situ (Jungbauer et al., 2004). (ii) redox conditions; (iii) protein binding is widely resistant to various types of chemicals (Table 27.1 summarizes the chemical compatibility and limitations of Ni-NTA IM AC); (iv) the high affinity and specificity of MAC can improve the capture rate even at high protein concentrations; and (v) the purification process can be scaled up.

Although IMAC has a wide range of compatibility, it has its limitations. Obviously, chelating reagents are a disadvantage in themselves and should be avoided. For example, EDTA, a highly potent metalloproteinase inhibitor, can only be applied at low concentrations. Also, potentially chelating groups such as T ris, ammonium salts, and certain amino acids need to be avoided (Table 27.2).

Until recently, there was a concern that the strong reducing agents used in IMAC (e.g., D TT), would reduce nickel and thus could lead to an increase in nickel concentrations in protein preparations. However, we found that moderate concentrations of D TT were perfectly suited for NTA purification. As shown in Figure 27.5A, the purification of HIV-1 reverse transcriptase (RT) at DTT concentrations up to 10 mmol/L (data not provided) showed no inhibitory effect of high concentrations of heavy metal ions. Although D TT may reduce nickel ions and change the color of the gel column bed, the leaching of nickel ions from the ligands does not increase (Fig. 27. 5A), and treatment of the resin under reducing conditions allows for reuse and regeneration (data not provided). The results suggest that D TT reduces nickel and causes a color change in the resin, but the resin retains its functionality. Nowadays, TCEP is increasingly used for MAC purification instead of D TT and hydrophobic ethanol (β-ME), which is an odorless, sulfhydryl-free reductant, more selective for reducing disulfide bonds, and more stable in aqueous solution. We recommend the use of TCEP in Ni-NTA column chromatography at a concentration of 1~5 mmol/L.

5. How to implement IMAC for protein co-purification?

In most cases, very pure proteins can be obtained by one-step IMAC purification (Fig. 27. 2 A, D, E, and Fig. 27. 5 A; Bornhorst and Falke, 2000; Schmitt et al., 1993). The amount of IMAC resin used in chromatography correlates with the amount of His-tagged recombinant protein in the sample to be purified, and the closer the relationship, the higher the protein purity. The reason for this is that proteins that naturally have surface motifs that can interact with metal ions may also bind to the resin. The affinity for this binding is weak compared to foldable His tags. As a result, most His-tagged proteins displace proteins with natural or accidental surface motifs. However, there are proteins with such a high density of locally chelated amino acids (e.g., histidine) that binding to immobilized metal ions is almost inevitable. In general, mammalian systems contain naturally occurring continuous histidine in higher abundance than bacterial systems (Crowe etaL, 1994). A very important example is the subunit of the transcription factor TFIIA of human cells, which has seven consecutive surface-exposed histidine residues that can be of natural origin and purified under natural conditions using IM AC (DeJong and Roeder, 1993; Ma et al., 1993). Western Blot detection using an anti-His tag antibody typically detects 55 kDa (αβ precursor) or 35 kDa (a subunit) of TFIIA. Another example is the human transcription factor YYKShi etal. with 11 consecutive histidines (1991). Proteins observed in E. coli that can be co-purified with His-tagged target proteins can be classified into four groups: (i) proteins with natural metal-binding motifs; and (ii) proteins with histidine clusters on their surface;
(iii) proteins that bind to His-tagged proteins expressed heterologously, e.g., through molecular chaperones; and (iv) proteins with affinity for agarose carriers (Bolanos-Garcia and Davies, 2006). It is not easy to predict whether one of the E. coli proteins will be co-purified. For example, the 21 kDa SlyD protein is often reported to be co-purified when Ni-N T A is used. It is a protein belonging to group ②. However, in our laboratory, this protein has never been observed during purification using E. coli BL21 (DE3), DH5a, M15 (PREP4) and other strains. This may be due to the fact that most of these impurities are stress-responsive proteins, and both culture conditions and strain status have an effect on their content, and the result manifests itself as sample contamination during the preparation of the target protein. It is therefore recommended to minimize stress during E. coli culture (e.g. by using baffle-free shake flasks). In addition, some co-purified proteins seem to prefer binding to C o rather than Ni (or other ions), while some do the opposite.

A number of methods for eliminating copurified proteins or preventing adsorption of copurified proteins at an early stage have been evaluated, some of which will be discussed in the following sections. These methods include (i) taking alternative purification steps; (ii) adjusting the ratio of His-tagged protein to resin; (iii) using engineered host bacteria that do not express the protein; (iv) using alternative vectors; and (v) using reverse chromatography after cutting the tag.

Suitable purification steps can be attached, including classical chromatographic techniques such as ion exchange chromatography (IEX) and volume exclusion chromatography (SEC). IEX has a much higher separation capacity, while SEC not only separates and removes ultra-high molecular mass polymers by molecular size, but can also be used for desalting to provide suitable conditions for the next procedure. Therefore, high-throughput laboratories such as the Center for Structural Biology, where protein crystallization or NMR mass spectrometry is required, often use additional SEC as a standardized procedure (Acton et al., 2005; GrSslund et al., 2008). Although IMAC-SEC (which corresponds to IMACIEX) is used as a standardized procedure without regard to the chemical properties of the protein (e.g., pi), the range of separations on a single SEC column is not suitable for all separation purposes, and therefore it may be necessary to have a series of SEC columns. Another problem with IEX and SEC applications is that in order to take full advantage of these techniques, expensive equipment such as automated chromatography systems are required, which usually do not have multiple parallel programs, thus resulting in low throughput. IMAC affinity purification is usually performed using a binding-washing-elution procedure, which can be performed in benchtop/gravity flow mode. By priming the expression structure with another affinity tag (e.g., Strep II, GST, or Flag), a two-step purification can be performed to obtain a high-purity protein preparation, which can be accomplished by a benchtop two-step affinity chromatography procedure (Cass et al., 2005; Prinz et al., 2004).

As mentioned previously, adjusting the amount of His-tagged protein recovered to accommodate the binding capacity of IMAC resin can help improve protein purity by avoiding co-purification of proteins with affinity for IMAC resin. However, the amount of His-tagged protein for a protein is usually unknown unless the amount of the target protein is estimated by preexperimentation. In addition to this, a better option might be to express the target proteins using engineered bacteria that have knocked out the relevant genes to remove these co-purified proteins . However, to the best of our knowledge, this has not been reported and there is very little experience with the production of proteins using knockout strains. It has been reported that E. coli strains can be used to produce proteins in the absence of 17 proteins that bind to the IMAC resin, including some important functional proteins such as superoxide dismutase and iron uptake regulators (Bolanos-Garciaand Davies, 2006), and that such strains can be used to produce proteins under stress conditions such as overexpression of proteins. But this does not seem to be realistic either.

A different approach to improve the purity of recovered proteins from IM AC has been reported, i.e. dextran wrapping of agarose vectors. This construction of chromatographic carriers has been widely used (Sepharoses, Superflow, Agaroses) and prevents co-purification of proteins through the binding medium (Mateo etal., 2001). However, dextran-embedded beads do not readily form commercial IMAC resins, and this method only excludes proteins that have an affinity for agarose but not impurities that bind to immobilized metal ions or target proteins. Silica IMAC carriers also prevent affinity adsorption of proteins to agarose and have good pressure stability, making them suitable for high resolution HPLC, but typically silica resins have low binding capacity and limited tolerance to high pH sterilization procedures. Recently, a method called affinity precipitation (Hilbrig and Freitag, 2003), which avoids the use of a solid-phase chromatographic carrier, has been applied to IMAC (Matias-sonetal., 2007). This method involves chemically coupling the IMAC ligand to a reactive polymer that, when bound to His-tagged proteins, agglutinates and can be centrifuged for precipitation once ambient conditions, such as pH or temperature, change. This application is still complex, but once simple and durable commercially available materials are developed, this approach may play an important role in IMAC applications. The use of ligands in solution form overcomes the spatial barrier between His-tagged proteins and immobilized ligands, as well as the limitations of microporous chromatography media for substance transport. Moreover, it meets the requirements of the trend toward the use of disposable materials for production-scale chromatography.

Another method has recently been reported for the separation of proteins in cell-free post-expression lysates (Kim et al., 2006). Proteins with affinity for Ni-NTA can be removed by preloafing E. coli lysates with Ni-NTA affinity agarose magnetic beads prior to addition of template and protein expression. Literature results showed that the expressivity of the S3 0 extracts remained unchanged and the purity of the His-tagged protein fractions purified by this method Ni-NTA was higher than without pretreatment.

Although the pretreatment strategies described above can improve M AC protein purity and have been shown to be useful in most cases, they are not universally applicable. However, one method that is suitable for almost all purity enhancement requirements is reversed-phase IMAC chromatography following His-tag cleavage by His-tagged proteases, which has the added advantage of preserving the natural or near-natural protein structure (Block e ta L , 2008). This strategy overcomes the co-purification problem. Proteins are hydrolyzed with proteases under the same or similar conditions, samples are spiked on the same chromatographic column, and proteins bound to the IMAC resin, such as impurities in the first step of purification, are bound again to the same resin, while the cleaved, label-free target proteins are collected in the flow-through fraction [reverse-phase IMAC or negative-phase IMACXSubtractiv^ IMAC)]. Hydrolysis can be carried out with exogenous or endogenous proteases that themselves carry a (non-cleavable) H is tag (Nilsson et al., 1997; Polayes et al., 2008). The advantage of the treatment method with exogenous proteases is the faster rate and the formation of naturally structured proteins without carrier-derived amino acids (Arnau et al., 2006; Block et al., 2008; Pedersen et al., 1999). This method is particularly suitable for similar downstream applications such as protein crystallization or biopharmaceuticals. Notably, the method requires only a single chromatographic column to achieve very high purity. As shown in FIG. 27. 6, tumor necrosis factor was crystallized in the form obtained by IMAC-step purification (A , B, C, D) or in the form obtained by reverse-phase IMAC as described above (A , E, F, G). Although SDS PAGE and Caumas Brilliant Blue staining showed high purity of His-tagged tumor necrosis factor (TNFa) purified by Ni-N T A (Fig. 27. 6A , Swimming
lane IMAC), but impurities were visible in the two-dimensional gel silver staining analysis (Fig. 27. 6B). The impurities were removed by reversed-phase IMAC, and the protein preparation with high purity was obtained (Fig. 27.6E). Moreover, Figure 27.6 shows that H is tag affects protein crystallization. In SEC chromatography, both labeled and mature naturally structured tumor necrosis factor can be eluted as trime

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