Protein purification

Purified proteins are required for many experimental applications, including structural studies and in vitro biochemical assays. Proteins can be obtained from a tissue or, more often, by their overexpression in a model organism, such as bacteria, yeast, or mammalian cells in culture. Protein purification involves isolating proteins from the source, based on differences in their physical properties. The objective of a protein purification scheme is to retain the largest amount of the functional protein with fewest contaminants. The purification scheme of a protein must be optimized to complete this process in the least number of steps. Figure 1. A Tris-buffered solution contains Tris base and its conjugate acid. The pKa of Tris at 25°C is 8.06, indicating that at pH = 8.06, 50% of the Tris is protonated (in its acidic form) and 50% is deprotonated (in its basic form). The article reviews four types of column chromatography that are commonly used in protein purification and discusses the advantages, disadvantages, and potential problems of each. This article also reports a Labome survey on 98 publications. The survey indicates that affinity column chromatography, mainly that based on HIS, GST, and FLAG tags, and size exclusion chromatography are the main methods cited in the publications. GE Healthcare is the major supplier of reagents and instruments used in protein purification. Developing a Protein Purification SchemeThe most important consideration in the development of a protein purification scheme is the downstream application of the purified protein. Both the quantity and purity of the protein must be sufficient for experimental analysis. Additionally, information about the behavior of the protein must be taken into consideration, as well-folded and functional protein is required for downstream studies. During purification and subsequent storage, many processes can occur that affect protein quality: protein unfolding, aggregation, degradation, and loss of function. Careful planning to purify protein as quickly as possible and under the most stabilizing conditions will maximize the chance of a successful purification scheme. Figure 2. An Aktaprime plus system for the automated chromatographic separation of proteins. From GE. Buffering componentThe solution conditions of a protein at each step of the

Their solubilization from the lipid bilayer while retaining their functional integrity. The typical approach involves the isolation of intracellular membranes by centrifugation followed by detergent solubilization of integral membrane proteins and high-speed centrifugation to remove insoluble membrane residues [55-57]. A wide range of detergents have been employed for membrane protein solubilization [58-60] and, in the absence of any literature or laboratory precedent, the investigator will need to determine the best detergent for their particular protein empirically. The solubilized membrane protein may then be purified by column chromatography in essentially the same way as for soluble proteins. However, the purification buffers will need to contain detergents to maintain the protein in a soluble state [61, 62]. Purification of membrane proteins is often extremely challenging due to loss of protein functional integrity and aggregation following initial removal from the lipid bilayer and through the various purification steps. Nonetheless, several groups have successfully purified membrane proteins in sufficient quantities for structure determination (see for example, Structural Genomics Consortium). Recently, nanodiscs have been successfully employed in the affinity purification of a Family B GPCR (see Labome article on nanodiscs) [63].Labome Survey of the Literature Citing Protein PurificationLabome surveyed publications citing protein purification methods. Table 10 lists the major suppliers and purification methods. Affinity and size exclusion methods are the most commonly used approaches in the literature. GE Healthcare is the predominant supplier for all methods, and Qiagen is the significant provider of HIS tag-based protein purification, and MilliporeSigma, of FLAG-based tag. typesuppliermajor brandnumsample referencesaffinityHISQiagenNi-NTA agarose/resin63 [64, 65] GE HealthcareNi Sepharose, HisTrap FF/HP37 [66, 67] ClontechTALON metal affinity23 [68, 69] Thermo Fisher / InvitrogenHisPur Cobalt/Ni-NTA resin9 [70, 71] Roche/ MilliporeSigmacOmplete His-Tag Purification Resins1 [72] Cube BiotechNi-NTA resin1 [73] GSTGE Healthcareglutathione Sepharose 4B66 [66, 74] MilliporeSigmaglutathione agarose3 [75, 76] FLAGMilliporeSigmaFLAG M2 affinity17 [77, 78] DNA binding proteinGE Healthcareheparin16 [67] maltose-binding protein / MBPNew England Biolabsamylose resin10 [73, 79] Other cited affinity-based protein purification systems include Strep-tactin resin from IBA Lifesciences for SBP-fused proteins [73], Streptactin Sepharose® High Performance from GE Healthcare (28-9355-99) [80]. size exclusionGE-HealthcareSuperdex / Superose / Sephacryl104 [81] ion exchangeanionGE HealthcareMono Q column11 [79,

On temperature High buffering capacity at physiological pH HEPES6.8-8.2Cannot be autoclaved pH is somewhat dependent on temperature Can form radicals under certain conditions [2] Tris7.5-9.0pH is dependent on temperature and dilution Inexpensive Can interfere with the activity of some enzymes [3], [4] Transparent in the UV rangeTable 1. Most commonly used biological buffers for protein purification. Buffers maintain their buffering capacity within a specific pH range, and characteristics of some buffering components could interfere with particular chromatographic procedures or analysis. Summarized from Promega, MilliporeSigma, Applichem, Embl.de.Solution additivesIn addition to an appropriate buffering system, solutions used during protein purification from lysis to storage often contain many other components that play a role in facilitating protein purity, stability, and function.Protease inhibitors are often added to the lysis buffer and in early steps of the purification scheme to prevent degradation of the target protein by endogenous proteases. These are generally not needed toward later stages of the purification, as most or all of the contaminating proteases have been separated from the protein of interest. Metal chelating reagents, such as EDTA or EGTA, are often added to the storage buffer. These metal chelators bind to Mg2+ and, thus, prevent cleavage of the purified protein by contaminating metalloproteases. Other additives are often used to protect proteins against damage and enhance their solubility.TypeFunctionCommonly Used ReagentsReducing AgentsProtect against oxidative damage 2-mercaptoethanol (BME) Dithiothreitol (DTT) Tris (2-carboxyethyl) phosphine (TCEP)Protease InhibitorsInhibit endogenous proteases from degrading proteins Leupeptin (serine and cysteine protease inhibitor) Pepstatin A (aspartic acid protease inhibitor) PMSF (serine protease inhibitor)Metal ChelatorsInactivate metalloproteases EDTA EGTAOsmolytesStabilize protein structure and enhance solubility Glycerol Detergents (e.g., CHAPS, NP-40, Triton X-100, DDM) Sugars (e.g., glucose, sucrose)Ionic StabilizersEnhance solubilitySalts (e.g., NaCl, KCl, (NH4)2SO4Table 2. Additives commonly used in protein purification buffers to increase the stability of proteins. Summarized from Thermo Fisher Pierce and Embl.de.Additives should only be used if necessary. Trial and error are often required to determine the specific additives that are beneficial to a particular protein purification scheme.Other factors to considerOther factors also contribute to protein stability during a purification scheme. The least manipulation of a protein during its purification is

Chromatographic separation of proteins. Summarized from GE.Types of Column ChromatographyThe four major types of column chromatography include affinity chromatography, ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC). Most purification schemes require the use of more than one of these types of chromatographic procedures to yield the necessary purity for downstream applications. Choosing the most appropriate chromatographic method(s) and the order of these methods is essential in optimizing a protein purification scheme.Type of ChromatographySeparates Proteins ByBind WithElute WithAffinityA specific interactionNo competing ligandCompeting ligand (specific); conditions that disrupt protein/protein interactions (non-specific)Ion ExchangeNet surface chargeLow ionic strengthHigh ionic strength; Increased (cation exchange) or decreased (anion exchange) pHHydrophobic InteractionHydrophobicityHigh ionic strengthLow ionic strengthSize ExclusionHydrodynamic radiiTable 4. The four most common types of column chromatography used in protein purification.By analyzing the sequence of a protein, unique characteristics can be identified that might assist in its purification. The size and charge of a protein (at a specific pH) can be determined, along with the identification of large stretches of hydrophobic residues.Affinity chromatographyAffinity chromatography relies on the specific and reversible binding of a protein to a matrix-bound ligand. The ligand can bind directly to either the protein of interest, for example, cAMP-resin for cAMP-binding PKA RIα and RIIβ proteins [19], or a tag that is covalently attached to the protein. Affinity chromatography is often the most robust purification procedure and is typically used in the early stages of the purification scheme. Depending on the downstream application, affinity purification might be the only chromatographic step required to achieve adequate purity. Figure 5. The net charge on a protein is influenced by the pH of its solvent. At pH=pI, the protein has zero net charge and, therefore, will not bind to a cation exchange or an anion exchange stationary phase. Adjusting the pH above or below the pI of the protein will lead to a net charge, and protein binding to either an anion exchange (pH > pI) or a cation exchange (pH The stationary phase for affinity chromatography is made of an inert matrix covalently attached to a ligand that specifically binds to

In a high-throughput format, such as proteases. For some proteins, activity assays provide a fast and reliable method for protein detection. Activity measurement is often ideal for enzymes because protein that has lost activity can be excluded from subsequent use.Purified Protein StorageWhen proteins are deemed pure enough for use in experimental studies, they should be stored appropriately. The selection of a final storage buffer is just as important as the selection of buffers used during the purification scheme and should depend on the stability of the protein and conditions required for the downstream application of the purified protein. Often, size exclusion chromatography is selected as a final step in the purification scheme, as the storage buffer can be used in this chromatographic step to exchange the buffer effectively. The pure fractions can be pooled for immediate storage. Alternatively, the final pooled fractions can be dialyzed into the selected buffer before storage. Protein storage conditions depend on the protein of interest and should be optimized, so the protein maintains structural and functional stability over long periods of storage. Additives are often included in the storage buffer to enhance the lifetime of purified proteins under storage conditions, and trial and error are often required to determine optimum conditions, as every protein behaves differently.High-throughput Protein PurificationIn this post-genomics era, there is much interest in high throughput purification of proteins both for structural determination and for drug discovery/compound screening purposes. To screen a wide range of constructs and select the optimal one(s) for the desired end use investigators have utilized a range of commercially available automated/robotic systems that enable the rapid, parallel, (semi-) automated purification of affinity-tagged proteins [52, 53]. The basic principles of protein still apply; liquid handling robotics / automated platforms are simply used to enable to streamline and accelerate the purification process.Membrane proteinsSome 20 - 30% of the proteins produced by cells are integral membrane proteins, and some 50% of small molecule drugs act on membrane proteins [54]. Thus, there is great interest in solving the structure of membrane proteins. A crucial step in the purification of integral membrane proteins is

Always the best. Designing a purification scheme that uses the minimum number of steps in the shortest amount of time ensures the highest yield of functional protein. Additionally, it is often best to keep the protein cold throughout the purification. Typically, purification is performed at 4°C, as this lower temperature both slows down the rate of proteolysis (in the event of contaminating proteases) and promotes structural integrity of proteins. Impact of expression system on protein purificationBefore one can proceed to purify the protein of interest an initial crude sample must be prepared. The first consideration, which takes place well in advance of performing the actual purification, is the source of the protein of interest. This could be a native source such as liver, muscle or brain tissue, though in the post-genomic area it is now relatively rare for investigators to purify proteins from native sources. However, there is still sometimes the need if the investigator wishes to link a catalytic activity to a specific protein sequence.Nowadays it is far more common for proteins to be purified from recombinant sources. Important decisions need to be made in advance to optimize the subsequent purification. The investigator needs to consider the end-use of the protein (e.g., enzyme assay, structural studies, antibody generation) as this will dictate the quantities and purity required of the final protein preparation. An important consideration is the choice of expression system (see Labome article Recombinant Protein Expression: Vector-Host Systems) for a discussion of the most widely used expression systems based on an unbiased survey of the literature. While a detailed discussion of protein expression is outside the scope of this article a few basic points are worth considering at this point as they directly impact upon the subsequent purification of the protein.Which expression system will give the highest level of expression? As a general rule, the higher the expression level, the easier it will be to obtain large quantities of highly purified protein.If opting for E.coli expression, is the ultimate aim soluble expression or insoluble expression in the form of inclusions bodies? Isolation of inclusion bodies can in itself