Chromatography is one of the most powerful techniques for protein purification. It allows for the separation of proteins based on their unique physicochemical properties. Here are the main types of chromatography used in protein purification:
Gel filtration chromatography
One of the most effective methods discovered in the 1960s to separate and purify various proteins based on molecular size is gel filtration chromatography, also known as molecular sieve chromatography. It is also the gold standard for separating protein polymers from their monomers. During elution, large-molecular-weight proteins are allowed to exit the gel through the interparticle space, but smaller-molecular-weight proteins are allowed to enter the gel mesh, which causes a blockage of the flow and a sluggish exit. It is possible to gather the necessary proteins by following the elution time sequence. Dextran, agarose, and polyacrylamide are some examples of simple substances that can make up the gel filtration matrix. Other possible combinations include dextran polyacrylamide and dextran-agarose. Proteins and peptides have been extensively isolated and purified using gel filtration chromatography due to its straightforward operation, quick separation, and lack of impact on biological activity. In addition to its usage in desalination, dextran gel has other potential applications after salting out. Nevertheless, there are a few issues with gel filtration chromatography as well. Because of its length, the chromatography column necessitates a greater column pressure, more fillers, a slower flow rate, and a longer elution time compared to other separation columns. It is common practice to combine gel filtration chromatography with other chromatography technologies. The structural basis for the development of a new coronavirus vaccine was provided by Zhang’s efforts to purify the SARS-CoV-2 spike protein carrying D614 or G614 with detergent DDM, further purify it through gel filtration chromatography, and then use it for structural analysis. This allowed them to further investigate the effect of D614G substitution on the structure of SARS-CoV-2 spike protein. Using elution buffer consisting of 70% methanol, Ye’s was able to isolate four flavone C-glycosides and a high concentration of isoorientin from flavones in bamboo leaf extract using gel filtration chromatography. As an additional purification step, gel filtration chromatography can be employed for low molecular weight hyaluronic acid. In their pursuit of pure serotonin 2A receptor (5-HT2AR) for use in depression treatment research, Cao’s team employed TALON IMAC metal chelation chromatography in conjunction with gel filtration chromatography. With the help of a combination of Strep-Tactin affinity chromatography and gel filtration chromatography, Zhang Y’s group was able to produce highly pure NMDA receptors (Superose six Increase). By analyzing its three-dimensional structure using cryo-EM, they uncovered new information that might be used to build antidepressants. When it comes to medical examinations, gel filtration chromatography is second to none when it comes to detecting macroprolactin. The presence of macro-TSH can be detected using gel filtration chromatography as an evaluation index of proper thyroid function when the recovery rate of thyroid stimulating hormone (TSH) does not decrease.
Ion exchange chromatography
By employing an ion exchanger as the stationary phase, ion exchange chromatography is able to separate and purify compounds based on the relative reversibility of their binding strengths to the mobile phase’s components and the exchanger’s equilibrium ions. To create an ion exchanger, a polymer (the parent body) is insoluble and then treated with multiple dissociable groups (the active groups). Chromatography relies on the many charged groups and convertible ions attached to the parent body through covalent bonds. To accomplish the goal of separation and purification, the binding strength of the protein is determined by adjusting the concentration of buffer salts or pH, which in turn affects the interaction strength between the proteins and ion exchangers. Ion exchangers can be categorized into anion exchangers and cation exchangers based on the features of the active groups. There are three main types of ion exchangers, distinguished by the parent material: resin, cellulose, and gel. Ion exchange chromatography entails the following fundamental steps: balancing, loading, washing, elution, and rebalancing. In most cases, a buffer solution with a high concentration of salt ions is used for elution after the sample loading is finished in a low salt ion concentration environment. Protein and ion exchanger charges, medium dielectric constant, ion exchanger and other ion competition, temperature, properties, solvent additives of special ions, non-electrostatic interaction, hydrogen bond, and other factors all play a role in how an ion exchanger and protein interact with one another.
Affinity chromatography
One of the most selective technologies that came out of the 1960s is affinity chromatography (AFC), which is also called liquid chromatography. Affinity chromatography works on the premise that distinct proteins have varied binding strengths to different ligands, allowing for the selective and reversible combination of one or more proteins with specific ligands and the subsequent separation of target proteins. The foundation for affinity column separation or purification of complementary targets is often affinity ligands with specific structures that are fixed in the column as stationary phase carriers. Some of the proteins in the mixture will bind to the stationary phase carriers as they move through the chromatographic column. Proteins lacking affinity, on the other hand, will undergo direct expulsion. By adjusting the binding conditions and using the right elution buffer, the adsorbed protein can be extracted. When studying interactions between biological macromolecules or isolating substances with specific tags, affinity chromatography is a common method due to its speed, simplicity, and efficiency. Proteins and ligands bind to one another through electrostatic gravity, hydrogen bonding, van der Waals forces, molecular hydrophobicity, and other non-covalent interactions.
While gene fusions have numerous potential uses, one of the most prevalent is the incorporation of a purification “tag” that allows for the uniform purification of the fused target protein. Proteins having these tags attached to their N-or C-termini can be isolated and purified using tag-specific affinity resins, precipitation, or aggregation, or by being bound to a tag-specific affinity ligand. Besides aiding in purification, many tags have ancillary purposes, such as making the target protein easier to detect or making it more soluble. After target purification, there are usually a number of options for removing the tag, making the protein native and untagged if that is what is needed.
Located within the N terminus of the target protein—which is essentially a protein or peptide—affinity tags are crucial recognition structures for affinity chromatography. Protein folding, solubility, and yield can all be improved with the use of certain affinity tags. There are a variety of affinity tags available now. The standard that is Small, inexpensive, and hardly perceptible to the target proteins, his tag consists of six or more tandem histidine residues. The human soluble cyclooxidative hydrolase was purified by Abis’s team using a combination of nickel ion chelation chromatography and BTS (Benzylthio-Sepharose) affinity chromatography. SDS-PAGE examination confirmed that the target proteins were very pure.
His-tag protein purification
The polyhistidine tag, more widely known as the His-tag, is by far the most used purification tag used in laboratories across the globe. Small, cheap, and usually having little to no effect on the structure or function of the target protein are the main properties of this tag [4]. This tag is short enough to be easily attached to either end of a target protein, and it works reliably in all the main expression systems because it doesn’t require a special fold. In addition, anti-His antibodies can be purchased for use in immunoassay detection, and there are a number of expression vectors that can be fused to included His-tags. Protein refolding techniques make use of the His-tag since it functions under native or denaturing conditions; also, it has recently played a pivotal role in attempts to purify soluble membrane proteins stabilized by lipids or detergents. The His tag is so common in research because of its exceptional reliability and strength; in fact, many labs just clone novel proteins into His tag vectors before trying to express or purify the native untagged product.
The His tag has several constraints, though, so it’s not without its strengths. The fact that histidine-tagged targets and contaminating proteins with exterior histidine residues tend to co-purify is one of the most important. Eliminating these contaminants can be a challenging task that requires optimization. For example, the LOBSTR (Low Background Strain) strain of Escherichia coli has recently been designed to remove the most abundant contaminating host proteins. Moreover, the His tag may sometimes impede the target protein’s correct folding and function, and there have been reports of it being incompatible with secretion in Streptomyces expression hosts. Also, although it is commonly attached to many solubility-enhancing domains, the His-tag is usually not very good at encouraging the correct folding of proteins that have solubility difficulties.
GST tag protein purification
Proteins coupled to a GST (glutathione S-transferase) tag can be efficiently purified in a single step using Glutathione Affinity. A affinity chromatography on immobilized glutathione followed by competitive elution with excess reduced glutathione can be used to purify GST protein from different sources, whether it is native or recombinantly generated in Escherichia coli or other host cells. Based on this finding, Smith and Johnson (1988) produced a protein tagged with the GST gene from Schistosoma japonicum, purified it using Glutathione Affinity chromatography, and then used it in their study. In the cytoplasm of E. coli, GST can be produced in large quantities as a soluble protein with complete enzyme activity. And it turns out that many eukaryotic proteins that are insoluble in E. coli are partially soluble when synthesized as a GST fusion protein. The ability to catalyze reactions is typically maintained when attached to the N-terminus of an additional protein. Protein GST from S. japonicum has a molecular weight of 26,000. Resolving the crystal structure of the recombinant protein revealed that it undergoes dimerization, just like the naturally occurring protein. Using conventional cloning procedures, one can insert the protein-coding sequences into commercially available vectors like the pGEX (GE Healthcare) or pET (Novagen) series of plasmids to create constructs that express GST fusion proteins. The so-called pull-down experiments that examine protein-protein interactions also make use of the GST tag, which is commonly used for affinity purification. An in vitro technique for determining the physical interaction between several proteins is the pull-down experiment. You can employ pull-down experiments for two purposes: first, to validate the presence of a protein-protein interaction that has been anticipated by other research methods (such co-immunoprecipitation), and second, to find protein-binding partners that have never been identified before. A pure and labeled protein (the bait) must be readily available in order to conduct a pull-down experiment. The bait is then utilized to capture and ‘draw down’ a protein-binding partner (the prey). For GST pull-down tests, you can use any of the Glutathione Affinity resins that are mentioned in the Materials section.
Protein A antibody purification
Protein A, a crucial component isolated from the harmful Staphylococcus aureus (S. aurous) bacteria, is attached to the bacterial cell wall and, in a different state, is released into the culture supernatant. Hjelm and colleagues made the initial effort to purify Protein A using an affinity chromatography technique in the 1970s; they discovered that Protein A binds strongly to the Fc region of IgG but rather weakly to its Fab region. It is now understood that these inclinations differ across different types of animals and subclasses; for example, Protein A is not able to attach to human IgG3. Protein G is another streptococcal binding protein; it binds to every subclass of IgG found in the vast majority of mammalian species. Protein A’s superior binding characteristics, such as lesser IgG binding and softer elution conditions, provide it commercially significant dominance. In order to separate IgG from its impurities, pure protein A must first form a covalent link with the sorbent. Based on the specific and non-specific interactions, various approaches can be used to purify protein A. Due to the high cost of specific purification procedures, non-specific techniques based on anion exchange and gel filtration chromatography were first deployed in 1966 and continued to rise in popularity. Recent reports indicate that researchers have successfully isolated Protein A while preserving its capacity to bind to IgG using a simple and inexpensive chromatography column called Per Aqueous Liquid Chromatography (PALC). Protein A was also purified by Denizli and colleagues using crygel beads that had an imprinted polymer surface. This study utilized wet cryogel beads to selectively extract 18.1 mg of Protein A/G. Purifying Protein A using immobilized-metal affinity chromatography (IMAC) with six histidine residues as an affinity fusion tag on the Protein A chain was a method first described by Hjelm and colleagues.
Magnetic beads protein purification
A paper detailing the usage of magnetic beads with an exterior surface modified with streptavidin to collect biotinylated peptides was published in the mid-1990s by the team of Girault S. This was one of the earliest reports regarding this method. After eluting the peptides, they were combined with the matrix and evaluated using MALDI-TOF. According to the authors, this method offers a great opportunity to investigate the interactions between peptides and proteins. Subsequently, it was documented an instance of this method’s use in biomarker discovery, still centered around magnetic beads. The anti-PSA-IgG-biotin was utilized to enrich and purify PSA from serum by binding to streptavidin-coated beads. Since there was a significant decrease in the co-elution of non-specifically bound proteins, the scientists concluded that the purification process was satisfactory. Before the success of SELDI technology at the turn of the century prompted their increased usage in clinical investigations (as seen by the increase in the number of publications), activities remained relatively quiet.
Carboxyl Magnetic Beads
| Catalog No. | Bead Size | Concentration | Package Size |
| CMB-50N | 50 nm | 10 mg/mL | 5 mL |
| CMB-100N | 100 nm | 10 mg/mL | 5 mL |
| CMB-200N | 200 nm | 10 mg/mL | 5 mL |
| CMB-500N | 500 nm | 10 mg/mL | 5 mL |
| CMB-1U5 | 1.5 μm | 10 mg/mL | 5 mL |
| CMB-3U | 3 μm | 10 mg/mL | 5 mL |
Amino Magnetic Beads
| Catalog No. | Bead Size | Concentration | Package Size |
| AmMB-50N | 50 nm | 10 mg/mL | 5 mL |
| AmMB-100N | 100 nm | 10 mg/mL | 5 mL |
| AmMB-200N | 200 nm | 10 mg/mL | 5 mL |
| AmMB-500N | 500 nm | 10 mg/mL | 5 mL |
| AmMB-1U5 | 1.5 μm | 10 mg/mL | 5 mL |
| AmMB-3U | 3 μm | 10 mg/mL | 5 mL |
Tosyl Magnetic Beads
| Catalog No. | Bead Size | Concentration | Package Size |
| TsMB-100N | 100 nm | 10 mg/mL | 5 mL |
| TsMB-200N | 200 nm | 10 mg/mL | 5 mL |
| TsMB-500N | 500 nm | 10 mg/mL | 5 mL |
| TsMB-1U5 | 1.5 μm | 10 mg/mL | 5 mL |
| TsMB-3U | 3 μm | 10 mg/mL | 5 mL |
Epoxyl Magnetic Beads
| Catalog No. | Bead Size | Concentration | Package Size |
| EpMB-50N | 50 nm | 10 mg/mL | 5 mL |
| EpMB-100N | 100 nm | 10 mg/mL | 5 mL |
| EpMB-200N | 200 nm | 10 mg/mL | 5 mL |
| EpMB-500N | 500 nm | 10 mg/mL | 5 mL |
| EpMB-1U5 | 1.5 μm | 10 mg/mL | 5 mL |
| EpMB-3U | 3 μm | 10 mg/mL | 5 mL |
Azide Magnetic Beads
| AzMB-50N | 50 nm | 10 mg/mL | 5 mL |
| AzMB-100N | 100 nm | 10 mg/mL | 5 mL |
| AzMB-200N | 200 nm | 10 mg/mL | 5 mL |
| AzMB-500N | 500 nm | 10 mg/mL | 5 mL |
| AzMB-1U5 | 1.5 μm | 10 mg/mL | 5 mL |
| AzMB-3U | 3 μm | 10 mg/mL | 5 mL |
Hydrophobic chromatography
The surface of the majority of proteins contains hydrophobic side chains of certain amino acids, including phenylalanine, tryptophan, methionine, and others. Protein characteristics are defined by the amino acid sequence, which includes their quantity, size, and distribution. A protein-based purification technique, hydrophobic chromatography takes advantage of the fact that different proteins have different hydrophobicities. Salt primarily regulates the hydrophobicity balance. In most cases, the self-aggregation or self-interaction that occurs in samples as a result of hydrophobic contact can be successfully eliminated by adjusting the quantity of salt ions. Another method for ensuring the purity of the target proteins and removing contaminants is hydrophobic chromatography. When purifying influenza A and B viruses, Weigel’s team utilized hydrophobic chromatography to reversibly mix virus particles, therefore removing leftover contaminated DNA and proteins.
HPLC protein purification
In the last quarter of a century, advancements in high-performance liquid chromatography (HPLC) packings and equipment have radically improved the speed and efficiency of peptide separation, among other molecule types. Because of this advancement, there has been an explosion of literature on HPLC of peptides, which may make deciding how to tackle a given separation challenge appear insurmountable to someone new to or even familiar with HPLC. The good news is that the basics of chromatographic techniques are the same whether high-performance approaches are used for regular peptide separations or for cutting-edge fields like proteomics, capillary methods, biospecific interactions, etc.
Traditional peptide separation methods using HPLC include size-exclusion HPLC (SEC), ion-exchange HPLC (IEX), and reversed-phase HPLC (RP-HPLC), all of which take use of peptide size variations. In these modes, you can adjust the mobile phase settings to make the most of your HPLC column’s separation capabilities. Hydrophilic interaction chromatography (HILIC)/cation-exchange chromatography (CEX), a new mixed-mode method for peptide separation, has recently demonstrated great promise as an adjunct to RP-HPLC. The major modes of HPLC continue to make extensive use of silica-based packings, since the high flow rates of mobile phases are made possible by the stiffness of microparticulate silica. Furthermore, quick studies are made possible by the excellent mass transfer properties. Organic polymers having a broad pH tolerance, such as cross-linked polystyrene-divinylbenzene, are being used more and more in high-performance column packings because silica-based packings can only be used in eluents with a pH range of 2.0-8.0.
Typically, commercial columns were intended for protein separations and had a fractionation ability that was lower than what was needed for peptides (~100-6,000 Da). Although these columns are no longer manufactured, they are still useful for peptide/protein separations and for the initial steps of multi-step purification protocols. Recent years have seen an increase in the use of this HPLC mode for peptide analysis, thanks to the release of a size-exclusion column tailored for peptide separations (molecular weight range 100-7000).
Since the introduction of HPLC packings that can hold both basic (net positive charge) and acidic (net negative charge) peptides, IEX has shown to be highly effective for peptide separations. Peptide and protein separations have both made use of anion-exchange (AEX) and CEX HPLC, with the corresponding ion-exchange modes retaining negatively charged and positively charged solutes, respectively. Most anion-exchange packings include one or more amine groups attached to a support, either covalently or adsorbed. These groups might be weak, tertiary, or quaternary. The positively charged packings will come into contact with the negatively charged aspartic and glutamic acid residues in the peptide (above ~pH 4.0) and the negatively charged α-carboxyl group at the C-terminus. Standard cation-exchange packings include a support matrix bound to carboxyl (weak CEX) or sulfonate (strong CEX) groups. Histidine and other basic residues are positively charged, and these negatively charged packings will interact with them.
RP-HPLC is still the gold standard when it comes to peptide separations using HPLC. In comparison to other HPLC modes, it is typically faster and more efficient. It is also great for preparative and analytical separations because volatile mobile phases are available. Most studies have used acidic volatile mobile phases, such as aqueous trifluoroacetic acid [TFA]/acetonitrile, to run HPLC at pH levels lower than 3.0. When the HILIC column was first developed, the phrase “hydrophilic interaction chromatography” was used to describe separations that relied on the hydrophilicity of the solutes. In this method, the opposite of the elution pattern observed in RP-HPLC, the solutes were eluted from the column in order of increasing hydrophilicity. Our group advanced this idea further by capitalizing on the hydrophilic nature of ion-exchange columns, more especially strong cation-exchange columns. Therefore, HILIC/CEX integrates the best features of two quite distinct separation methods, namely, a net charge separation on top of a separation based on hydrophilicity/hydrophobicity disparities between peptides. Hydrophilic interactions between the solute and the hydrophilic/charged cation-exchange stationary phase are enhanced in HILIC/CEX separations by the addition of a high concentration of organic modifiers, most often acetonitrile. A salt gradient is subsequently used to elute the peptides from the column. It is common practice to elute peptides in ascending net positive charge order. The elution order of peptides within these categories is based on their hydrophilicity. In fact, HILIC/CEX is essentially CEX with 50–80% acetonitrile content. HILIC/CEX works well with RP-HPLC in many cases. For certain peptide mixes, it has been as effective as, if not more so than, RP-HPLC.
FPLC protein purification
One method for analyzing or purifying protein mixtures is fast protein liquid chromatography (FPLC). Similar to other types of chromatography, the moving fluid (mobile phase) and the porous solid (stationary phase) allow for separation due to the fact that various components of the mixture possess distinct affinities for the two substances. An aqueous buffer solution is used as the mobile phase in FPLC. A positive displacement pump maintains a steady buffer flow rate, allowing for the modification of the buffer’s composition through the drawing of fluid from two or more external reservoirs in various amounts. A cylinder-shaped glass or plastic column contains the resin beads that make up the stationary phase. These beads are typically made of cross-linked agarose. Different FPLC resins with different bead sizes and surface ligands are available for different applications. FPLC has been around since 1982. Although FPLC is typically reserved for protein applications, its versatility stems from the many resins and buffers available. In contrast to HPLC, this method typically employs a buffer pressure of less than 5 bar and a flow rate of 1–5 ml/min. By simply extending FPLC, it is possible to analyze mixtures of milligram grade in columns with a total volume of 5 ml or less, and to industrially generate pure proteins of kilogram grade in columns with a volume of several liters. It is common practice to collect eluents in increments of 1–5 ml when using them to examine mixtures. Only two containers are required for protein purification: one to hold the finished product and another to hold the effluent.
Chemicals used in Protein Purification
| Classification | Name | CAT |
| Buffer system | Tromethamine | 77-86-1 |
| Buffer system | Instant premixed granules of Tris-HCl | BFORMU20-5 |
| Buffer system | Disodium hydrogenorthophosphate | 7558-79-4 |
| Buffer system | Potassium dihydrogen phosphate | 7778-77-0 |
| Detergent | Nonidet P-40 | 9016-45-9 |
| Detergent | Sodiumdodecylsulfate(SDS) | 151-21-3 |
| Detergent | Triton X-100 | 9002-93-1 |
| Reducing agent | DL-Dithiothreitol | 27565-41-9 |
| Affinity ligands and tags | Imidazole | 288-32-4 |
| Affinity ligands and tags | Glutathione | B0005-464855 |
| Affinity ligands and tags | Glutathione S-Transferase from equine liver | 50812-37-8 |
| Other | Blood serum albumins | 9048-46-8 |
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