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ScienceDirect New trends and affinity tag designs for recombinant protein purification David W Wood Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, nonchromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Addresses Department of Chemical & Biomolecular Engineering, 435 Koffolt Laboratories, 140 W Nineteenth Avenue, Columbus, OH 43210, United States Corresponding author: Wood, David W ([email protected])
Current Opinion in Plant Biology 2014, 26:54–61 This review comes from a themed issue on New constructs and expressions of proteins Edited by Junichi Takagi and Christopher G Tate For a complete overview see the Issue and the Editorial Available online 12th June 2014 http://dx.doi.org/10.1016/j.sbi.2014.04.006 0959-440X/# 2014 Elsevier Ltd. All rights reserved.
Purification tag methods Although there are many applications related to gene fusions, perhaps the most common involves the addition of a purification ‘tag’, which provides a standardized method to purify the fused target protein [1]. These tags are usually fused to the N-terminus or C-terminus of a target protein, and commonly allow their partner proteins to be selectively captured and purified through association with a tag-specific affinity resin, or by highly selective tag-dependent precipitation or aggregation (Figure 1). Many tags can also provide additional functions unrelated to purification, such as facilitating detection of the target protein or improving its solubility [2]. In cases where a native untagged protein is required, the tag can usually be removed by a variety of methods once the target is purified. Current Opinion in Structural Biology 2014, 26:54–61
Despite the fact that tag-based methods have been used in laboratories throughout the world for decades, no universal tag has been identified that can be applied to any protein expressed in any host. In most cases, the identification of an optimal tag for a given host–target combination requires significant trial and error, and for some target proteins there are no effective tags. These limitations continue to drive the development of new options, and this research has led to ingenious and effective innovations over the past several years. Although a truly universal purification strategy is likely unrealistic, short-term objectives include increasing the variety of proteins that can be effectively purified with tag methods, while simultaneously facilitating the rapid identification of a suitable method for each new target. To accomplish these goals, researchers are increasingly using compound tags made up of multiple small domains, while continuing to develop single-domain tags with increased capabilities. Many new tags bind to highly inexpensive substrates, and are made even more economical through a variety of new approaches for tag removal. Taken together, these strategies can provide greater expression, increased purity and simplified detection for a wide range of fused products, along with inexpensive tag removal via simple self-cleaving or conventional proteolytic methods. A comprehensive discussion of even those tags developed in the last three years is far beyond the scope of this review, so I will highlight some interesting examples of each of these trends while providing a more comprehensive list of recently reported tags along with their basic advantages and recent references in Table 1. Notably, Table 1 lists many well-known conventional tags as well, which are included here due to their continued wide-spread use. Indeed, it is these tags that set the bar for newly developing methods in this field.
Popular tag methods Undoubtedly the most commonly used purification tag in laboratories worldwide is the Polyhistidine tag, or His-tag [3]. The primary characteristics of this tag are that it is small, inexpensive to use, and it typically has minimal or no effect on the target protein structure or function [4]. The small size of the tag allows it to be trivially added to either terminus of a given target protein, and it does not require a specific fold in order to function, making it highly reliable in all major expression systems. Further, several expression vectors are commercially available for fusion to included His-tags, and anti-His antibodies are available for immunoassay detection. Importantly, the His-tag can function under native or denaturing conditions, allowing it to be www.sciencedirect.com
Affinity tags for recombinant protein purification Wood 55
Figure 1
Fusion protein construction strategy
Expressed Fusion proteins (basic purification modalities)
Expression Vector
His tag for purification by IMAC Affinity column
Target Protein Sequence
Epitope tag for purification by Immunoaffinity column
Tag/Linker Sequence
Cloning and strain selection Ligand-binding domain for purification by conventional affinity column
Precipitation domain for purification by non-chromatographic method Fermentation and Harvest
Compound tag for simultaneous detection and purification Current Opinion in Structural Biology
Schematic representation of basic cloning strategies and commonly used purification modalities. Although many novel tags have been developed, the majority of them fit one of the basic modalities shown at right. An important advance has been the construction of complex multidomain compound tags, with a simple example shown at lower right. These tags exhibit optimized combinations of behavior, and can provide highly effective methods for large groups of protein targets.
used in protein refolding protocols [5], and it has also become central to recent efforts on the purification of soluble membrane proteins that are stabilized by lipids or detergents [6–8]. Indeed, this unique combination of strength and reliability has made the His tag ubiquitous in research, where many laboratories simply clone new proteins into His tag vectors before any attempt is made to express or purify the native untagged product. Despite these strengths, the His tag does suffer from a number of limitations. Perhaps the most significant is a tendency for contaminant proteins with external His residues to co-purify with His-tagged targets. The removal of these contaminants can require significant optimization [9], and indeed the Escherichia coli strain, LOBSTR (Low Background Strain), has been recently engineered to eliminate the most abundant contaminating host proteins [10]. Further, in some instances www.sciencedirect.com
the His tag can interfere with proper folding and activity of the target protein [11–13], and it has recently been reported to be incompatible with secretion in Streptomyces expression hosts [14]. In addition, the His-tag is generally ineffective in promoting proper folding of proteins with solubility issues, although it is routinely appended to a variety of solubility-enhancing domains. Some of the problems associated with His tags have been solved through the use of small epitope tags, with the most widely recognized being arguably the FLAG, c-Myc and HA tags [15,16]. These tags are typically eight to twelve amino acids in length and are very strongly and specifically bind to their corresponding immunoaffinity resins [17]. The small size of these tags allows them to retain many of the advantages of the His tag, while providing superior purity and recovery of the fused target. These tags are also compatible with virtually any expression host and facilitate Current Opinion in Structural Biology 2014, 26:54–61
56 New constructs and expressions of proteins
Table 1 Major tag classes with recent references, including examples of commonly used conventional tags. Sizes of tags shown in table are proportional to actual sizes of typically used tags. More recently reported tags are denoted in bold Tag type
Examples
Advantages
Limitations
Hisn [1,3,10,28,43,63–66]
Minimal impact on target expression and folding Many commercial systems Well established
Contaminants can co-purify Can interfere with target function
FLAG [15,67] c-Myc [15] GM-CSF [68] Twin StrepII [69]
Minimal target impact Exceptional purity Encoded on PCR primer Enable immunodetection
Very expensive resins Limited re-use cycles
Can enhance target solubility Highly specific binding Less expensive resins On-column cleaving possible
Can decrease expression yields Can leach from column
Folded domain tags
MBP [4,45,70] GST [4,64] Starch [35] Fluoroapetite [36] Diatomite [38] bGRP [37]
Very inexpensive to use Non-chromatographic High expression yields with aggregation tags
Precipitation tags are usually large in size Aggregation tags can require refolding protocols
Precipitation/ Aggregation tags
ELP [58,59,71,72] RTX [73] ELK16 [74,75] Fh8 [76] 4AaCter [77] PagP [78,79] eGFP [9,80] Heme [32,49] PYP [33]
Direct visual observation of target protein Can be highly quantitative Excellent for trouble-shooting expression
Larger tags can interfere with expression
NusA [15,22] SUMO [24–27,28,31] Trx [47,48,81] XTEN [82] FATT [39]
Can enhance solubility of target during expression Can aid in refolding of target if needed
Most are large and can decreases target yields
His tag
Peptide/Epitope tags
Detection Tags
Solubility/Folding tags
highly sensitive detection via immunoaffinity methods, making them especially attractive for cases where the target protein is difficult to overexpress and tag removal is unnecessary [18–20]. Easily the most significant drawback for the use of small epitope tags is the cost of use, where commercially available immunoaffinity resins are orders of magnitude more expensive than most ligandbased affinity resins. Thus, the convenience and selectivity of these tags make them excellent choices for very smallscale applications, such as tandem affinity purification and some structural studies. For larger scale experiments, however, they quickly become cost prohibitive. A third major class of conventional affinity tag includes the ligand-binding domain tags. These tags are typically much larger than His and epitope tags, and consist of fully folded protein domains with highly specific affinities for small molecule ligands immobilized on commercially available affinity resins. Two of the most widely known and commonly used tags are the Maltose Binding Domain (MBD) and Glutathione S-Transferase (GST) tags, which not only enhance expression in microbial hosts, but can Current Opinion in Structural Biology 2014, 26:54–61
also increase the solubility of their fusion partners [1,21]. The most significant drawbacks of these tags arise from their relatively large size and the resulting impact on their fusion partners, which occasionally necessitates inconvenient tag removal protocols. These tags can also decrease the overall yield of smaller fusion partners, due to their high metabolic demands on protein synthesis. Many common tags do not directly participate in any purification methods, but instead provide accessory functions such as enhanced folding and detection of the target. For example, NusA fusions can improve target solubility [22], while thioredoxin (Trx) fusions can facilitate proper disulfide bond formation in proteins with multiple cysteine residues [23]. The SUMO (small ubiquitinrelated modifier) tag has also been used to enhance expression and folding of recombinant proteins in prokaryotic and eukaryotic hosts [24–26]. The SUMO tag is particularly attractive due to its small size and the availability of a highly specific SUMO protease for tag removal, and for these reasons it has been combined with a number of other conventional tags in a variety of www.sciencedirect.com
Affinity tags for recombinant protein purification Wood 57
configurations [27,28,29–31]. While some tags can improve the expression of their fusion partners, others can allow direct visual detection of the target protein during expression and purification. Recent examples include small heme-binding proteins and motifs that give the fused target a bright red color [32], as well as additional fluorescent and chromogenic proteins [33,34]. Although these tags may not aid in purifying the expressed target, they are often instrumental in overall process troubleshooting and optimization.
New tags for purification, folding and detection Many recently reported tags have been developed for inexpensive substrates that can be used in non-chromatographic configurations, making them economical for larger scale methods in simple laboratory settings. One of the most promising of these is a novel starch-binding domain (SBD) tag, which binds tightly and specifically to raw corn starch as well as a variety of other vegetable starches [35]. In demonstrations with four target proteins, the SBD tag compares favorably to the His tag in delivering highly purified proteins in a single affinity step, where the purification method relies only on simple centrifugation. Moreover, starch is inexpensive, renewable and biodegradable, suggesting the use of this method for various products at large scale. Three additional tags have recently been developed that bind to exceptionally inexpensive affinity substrates. These include a heptamer tag derived from phage display that binds to ceramic fluorapatite [36]; a 112 residue silkworm b-1,3-glucan recognition protein that binds to inexpensive curdlan [37]; and a 70 amino acid segment of the E. coli ribosomal protein L2 that binds to diatomaceous earth [38]. All three of these tags have demonstrated similar performance to conventional tags in delivering pure products, and all three can be used with inexpensive non-functionalized base materials. This aspect not only contributes to the lower price of the affinity substrates, but also eliminates the risk of ligands leaching from the resin into the purified target protein. A notable additional tag is the Flag-Acidic-Target Tag, or FATT [39]. This tag includes a Flag tag module for easy detection, along with a hyperacidic segment from the human amyloid precursor protein (APP) extracellular region. The hyperacidic segment expresses well in E. coli, and because it is highly charged, it can be easily purified in a single step on a conventional anion exchange chromatography resin. More importantly, however, this tag has been shown to significantly increase proper folding of fused target proteins during expression, and can facilitate the proper refolding of misfolded fusion partners that contain disulfide bonds. These capabilities are hypothesized to arise from the disordered structure of the FATT hyperacidic segment, which is thought to act www.sciencedirect.com
as a shield-like non-specific chaperone for the target protein during expression or refolding in vitro. Reported results from this tag are very impressive, and suggest that it may become an important new option for expression of difficult targets.
Compound tags Complex compound tags are now commonly constructed from multiple linked domains, where different combinations can provide highly efficient orthogonal purification methods, as well as additional functions related to target expression and detection (Figure 2). Various compound tags have enabled the characterization of protein–protein interactions through tandem affinity purification (TAP) and mass spectrometry [40–42], and these powerful methods are now being used for the purification of recombinant target proteins for structural studies as well.
Figure 2
Hyperacidic region
(a)
-
-
-
-
Target Protein Linker
FLAG tag
Functional domain tag (e.g., NusA, eGFP, Trx)
(b)
Target Protein His tag
Linkers
(c)
Heme binding tag SBP tag
Target Protein His tag Linkers
TEV Protease recognition sequence Current Opinion in Structural Biology
Some of the more interesting compound tags described in the text. (a) The FATT tag, which combines a FLAG tag for simple detection, along with a hyperacidic segment of the human amyloid precursor protein [39]. (b) A multifunctional His tagged protein, which includes an additional domain for improving expression or quantitative detection. In at least one case, the His tag is used primarily as a method to easily remove the cleaved functional domain after the target has been purified. (c) The Multitag, which combines two orthogonal purification tags with a convenient detection domain and protease recognition sequence [49]. Current Opinion in Structural Biology 2014, 26:54–61
58 New constructs and expressions of proteins
The simplest examples of compound tags generally consist of a small peptide purification tag, such as a His tag, fused to a functional domain, such as the solubilityenhancing NusA tag [22]. Taken together, this compound tag provides enhanced expression and solubility of the target, along with a simple purification method, and is typical of an initial strategy used for the production of uncharacterized targets in early studies. Additional examples of binary compound tags include the CHiC tag (His-Choline binding domain) for two orthogonal purification methods [43]; His-MBD for enhanced soluble expression and/or orthogonal purification methods [44,45]; His-Trx for proper disulfide bond formation and to simplify removal of the cleaved Trx tag [46–48]; and His-GFP for simple monitoring and optimization of expression and purification in Saccharomyces [9]. More recent binary tags now commonly include a smaller SUMO tag segment, which can significantly increase soluble expression of target proteins in a variety of hosts [27–30]. A final, somewhat more complex example is the recently reported ‘Multitags’ method based on a four-component compound tag. Specifically, this tag includes a His10 tag, a streptavidin-binding peptide (SBP), a heme-binding domain, and a TEV protease sequence for tag removal [49]. The His and SBP tags provide orthogonal purification methods, while the heme binding domain allows simple visual tracking and quantification of the target protein through the process. Notably, the tag is removed by a SBP-tagged TEV protease, thus removing the tag and protease in a single operation. This tag was shown to be effective in delivering highly purified Pfu DNA polymerase and Myosin-VIIa- and Rab-Interacting protein (MyRIP), and is likely to have general applicability to additional targets. Notably, this highly functional tag has a molecular weight of only 23 kDa, or slightly more than half the molecular weight of the commonly used MBD tag. The combination of function and size makes this tag very attractive for medium-scale production methods, and increasing simplicity of recombinant DNA methods facilitates rapid prototyping of new designs. Most importantly, the increasing availability of small functional domains will inevitably assist the construction of additional complex and highly functional tags, and will continue to increase the popularity of compound tags in the future.
Tag removal methods In many cases, the tags used for expression and purification of the target protein must be removed before the target can be characterized or applied. Conventional methods generally include tag removal through the addition of highly specific endopeptidases, where the endopeptidase target sequence is engineered into the fusion protein between the tag and the target [15]. Research on basic tag removal strategies has led to several recent additions, and an increasing trend is the on-column Current Opinion in Structural Biology 2014, 26:54–61
cleavage of the target protein directly from an immobilized tag, often using an immobilized protease [49]. In addition to new process configurations, significant research has focused on the identification of still faster and more specific endopeptidases for tag removal. Successes include a group of four highly specific and orthogonal enzymes with applications in the sequential assembly of protein complexes [50], as well as a detergent-resistant West Nile Virus protease with applications in cleaving tags from detergent-stabilized membrane proteins [51]. A particularly interesting example is the recently reported glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL protease), which has been shown to cleave to over 99% completion in only 15 min under physiological conditions [52]. In addition to new proteolytic enzymes, a number of selfcleaving modules have been developed over the past 20 years [53,54]. The first of these were based on selfsplicing inteins, which were re-engineered into self-cleaving protein elements [55–57]. A highly effective purification method was generated by combining inteins with reversibly precipitating elastin-like protein (ELP) tags, and more recent research has expanded this basic approach to additional configurations [58–61]. Major drawbacks of intein methods include the uncontrolled cleaving of the inteins during expression of the tagged target, and the requirement for reducing agents to trigger the N-terminal cleaving reaction. Issues with premature intein cleaving have been partially addressed through the recent development of natural and engineered split inteins, which are inactive when expressed as separate segments, but can be activated after the purification protocol is complete. A particularly promising embodiment of this strategy is the Split Intein Mediated Ultra-Rapid Purification of Tagless Protein (SIRP) system, which involves the assembly and rapid cleaving of the naturally split Nostoc punctiforme DnaE intein in the presence of dithiothreitol [62]. This system has been shown to completely abolish premature cleavage of the target protein during expression, but provides almost complete tag removal in less than 30 min at room temperature once the cleavage reaction is triggered. Further, the intein segment that is joined to the target protein is quite small, and thus exhibits limited interference in the solubility and expression of the target. For these reasons, this split intein is one of the most promising tag removal methods that has yet been reported, and it will likely be combined with an array of purification, detection and expression tags in future systems.
Conclusion A comprehensive review on tags and tag removal methods would include scores of ligand binding domains, epitopes, precipitation and aggregation tags, as well as close to a www.sciencedirect.com
Affinity tags for recombinant protein purification Wood 59
dozen different flavors of the His tag. These tags would be combined with well over a dozen tag removal methods, utilized in countless mechanical configurations on target proteins of all shapes and sizes. Ultimately, the ease with which tags can be designed, redesigned, combined, recombined and cleaved has led to literally thousands of potential methods for the purification of a single protein. Yet despite this enormous diversity, no truly universal tag method has been developed. Further, many proteins, and some classes of proteins, remain stubbornly recalcitrant to even the most creative and carefully planned strategies. These frustrations have driven relentless research in this area, where most discoveries provide only incremental advances over established methods. The most significant advances of the past few years would arguably be the now widespread use of the SUMO tag for improved expression and solubility in a variety of hosts, along with the development of a number of novel methods for tag removal. On-column tag cleavage is now routine, and several configurations are now commercially available. Self-cleaving tag methods are continuing to evolve, and hold promise for extending affinity tag methods to large-scale manufacturing processes. Several popular protease enzymes can now be produced in recombinant E. coli, substantially decreasing their cost and increasing their availability. Coupled with simple and inexpensive precipitation tags, these advances promise to democratize the use of affinity methods in protein purification, making these methods available to a much wider segment of the scientific community.
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60 New constructs and expressions of proteins
proteins by SUMO fusion in Escherichia coli. J Struct Funct Genomics 2005, 6:103-111. 27. Zhang J, Ma L, Zhang SQ: Expression and purification of soluble human APRIL in Escherichia coli using ELP-SUMO tag. Protein Expr Purif 2014, 95:177-181. 28. Liew OW, Ang CX, Peh YP, Chong PC, Ng YX, Hwang LA, Koh XY, Yip YM, Liu W, Richards AM: A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies. J Immunol Methods 2014, 403:37-51. Excellent example of a SUMO application with improved expression and simple purification. 29. Lu W, Cai X, Gu Z, Huang Y, Xia B, Cao P: Production and characterization of hirudin variant-1 by SUMO fusion technology in E. coli. Mol Biotechnol 2013, 53:41-48. 30. Truong L, Hevener KE, Rice AJ, Patel K, Johnson ME, Lee H: Highlevel expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion. Protein Expr Purif 2013, 88:98-106. 31. Wang Z, Li N, Wang Y, Wu Y, Mu T, Zheng Y, Huang L, Fang X: Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification. Protein Expr Purif 2012, 82:174-178. 32. Asher WB, Bren KL: A heme fusion tag for protein affinity purification and quantification. Protein Sci 2010, 19:1830-1839. 33. Kim Y, Ganesan P, Ihee H: High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label. Protein Sci 2013, 22:1109-1117. 34. Hsieh JM, Besserer GM, Madej MG, Bui HQ, Kwon S, Abramson J: Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini. Protein Sci 2010, 19:868-880. 35. Guillen D, Moreno-Mendieta S, Aguilera P, Sanchez S, Farres A, Rodriguez-Sanoja R: The starch-binding domain as a tool for recombinant protein purification. Appl Microbiol Biotechnol 2013, 97:4141-4148. Describes the starch binding protein and its use in a non-chromatographic affinity method. 36. Islam T, Aguilar-Yanez JM, Simental-Martinez J, Ortiz-Alcaraz CI, Rito-Palomares M, Fernandez-Lahore M: A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags. J Chromatogr A 2014, 1339:26-33. 37. Horiuchi M, Takahasi K, Kobashigawa Y, Ochiai M, Inagaki F: A low-cost affinity purification system using beta-1,3-glucan recognition protein and curdlan beads. Protein Eng Des Sel 2012, 25:405-413. 38. Li J, Zhang Y, Yang Y: Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag. Appl Microbiol Biotechnol 2013, 97:2541-2549. 39. Sangawa T, Tabata S, Suzuki K, Saheki Y, Tanaka K, Takagi J: A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein. Protein Sci 2013, 22:840-850. Describes the FATT system, and it ability to improve the folding of fused partner proteins. 40. Li Y: The tandem affinity purification technology: an overview. Biotechnol Lett 2011, 33:1487-1499. 41. Li Y: Commonly used tag combinations for tandem affinity purification. Biotechnol Appl Biochem 2010, 55:73-83. 42. Xu X, Song Y, Li Y, Chang J, Zhang H, An L: The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification. Protein Expr Purif 2010, 72:149-156. 43. Stamsas GA, Havarstein LS, Straume D: CHiC, a new tandem affinity tag for the protein purification toolbox. J Microbiol Methods 2013, 92:59-63. Current Opinion in Structural Biology 2014, 26:54–61
44. Cao H, Chapital DC, Howard OD Jr, Deterding LJ, Mason CB, Shockey JM, Klasson KT: Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2. Appl Microbiol Biotechnol 2012, 96:711-727. 45. Li Y, Wang J, Yang J, Wan C, Wang X, Sun H: Recombinant expression, purification and characterization of antimicrobial peptide ORBK in Escherichia coli. Protein Expr Purif 2014, 95:182-187. 46. Farkas D, Franzen LG, Hansson O: Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding. Protein Expr Purif 2011, 78:156-166. 47. Nespovitaya N, Barylyuk K, Eichmann C, Zenobi R, Riek R: The production of recombinant N, C-labelled somatostatin 14 for NMR spectroscopy. Protein Expr Purif 2014 http://dx.doi.org/ 10.1016/j.pep.2014.03.011. 48. Wu M, Zhao L, Zhu L, Chen Z, Li H: Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli. Protein Expr Purif 2013, 88:7-12. 49. Miladi B, Dridi C, El Marjou A, Boeuf G, Bouallagui H, Dufour F, Di Martino P, Elm’selmi A: An improved strategy for easy process monitoring and advanced purification of recombinant proteins. Mol Biotechnol 2013, 55:227-235. Provides an excellent example of an effective, rationally designed multidomain tag. 50. Frey S, Gorlich D: Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. J Chromatogr A 2014, 1337:106-115. 51. Huang Q, Li Q, Chen AS, Kang C: West Nile virus protease activity in detergent solutions and application for affinity tag removal. Anal Biochem 2013, 435:44-46. 52. Ye W, Wang H, Ma Y, Luo X, Zhang W, Wang J, Wang X: Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli. J Biotechnol 2013, 168:40-45. 53. Li Y: Self-cleaving fusion tags for recombinant protein production. Biotechnol Lett 2011, 33:869-881. 54. Wood DW: Non-chromatographic recombinant protein purification by self-cleaving purification tags. Sep Sci Technol 2010, 45:2345-2357. 55. Southworth MW, Amaya K, Evans TC, Xu MQ, Perler FB: Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques 1999, 27:110-114 116, 118–120. 56. Wood DW, Wu W, Belfort G, Derbyshire V, Belfort M: A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol 1999, 17:889-892. 57. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA et al.: Singlecolumn purification of free recombinant proteins using a selfcleavable affinity tag derived from a protein splicing element. Gene 1997, 192:271-281. 58. Shi C, Meng Q, Wood DW: A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification. Appl Microbiol Biotechnol 2013, 97:829-835. 59. Liu F, Tsai SL, Madan B, Chen W: Engineering a high-affinity scaffold for non-chromatographic protein purification via intein-mediated cleavage. Biotechnol Bioeng 2012, 109: 2829-2835. 60. Tian L, Sun SS: A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform. PLoS ONE 2011, 6:e24183. 61. Banki MR, Feng L, Wood DW: Simple bioseparations using selfcleaving elastin-like polypeptide tags. Nat Methods 2005, 2:659-661.
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62. Guan D, Ramirez M, Chen Z: Split intein mediated ultra-rapid purification of tagless protein (SIRP). Biotechnol Bioeng 2013, 110:2471-2481. Describes the purification of recombinant proteins using a rapid splitintein system for suppressing premature cleaving during expression.
73. Shur O, Dooley K, Blenner M, Baltimore M, Banta S: A designed, phase changing RTX-based peptide for efficient bioseparations. Biotechniques 2013, 54:197-198 200, 202, 204, 206.
63. Nishimura Y, Takeda K, Ishii J, Ogino C, Kondo A: An affinity chromatography method used to purify His-tag-displaying bio-nanocapsules. J Virol Methods 2013, 189:393-396.
74. Xing L, Xu W, Zhou B, Chen Y, Lin Z: Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli. Protein Expr Purif 2013, 88:248-253.
64. Maity R, Pauty J, Krietsch J, Buisson R, Genois MM, Masson JY: GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins. J Vis Exp 2013 http:// dx.doi.org/10.3791/50320:e50320.
75. Xing L, Wu W, Zhou B, Lin Z: Streamlined protein expression and purification using cleavable self-aggregating tags. Microb Cell Fact 2011, 10:42.
65. Kimple ME, Brill AL, Pasker RL: Overview of affinity tags for protein purification. Curr Protoc Protein Sci 2013, 73 Unit 9 9.
76. Costa SJ, Almeida A, Castro A, Domingues L, Besir H: The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology. Appl Microbiol Biotechnol 2013, 97:6779-6791.
66. Randolph TW: The two faces of His-tag: immune response versus ease of protein purification. Biotechnol J 2012, 7:18-19. 67. Schmidt PM, Sparrow LG, Attwood RM, Xiao X, Adams TE, McKimm-Breschkin JL: Taking down the FLAG! How insect cell expression challenges an established tag-system. PLoS ONE 2012, 7:e37779.
77. Hayashi M, Iwamoto S, Sato S, Sudo S, Takagi M, Sakai H, Hayakawa T: Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli. Protein Expr Purif 2013, 88:230-234.
68. Perotti N, Etcheverrigaray M, Kratje R, Oggero M: A versatile ionic strength sensitive tag from a human GM-CSF-derived linear epitope. Protein Expr Purif 2013, 91:10-19.
78. Hwang PM, Pan JS, Sykes BD: Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli. FEBS Lett 2013 http://dx.doi.org/ 10.1016/j.febslet.2013.09.028.
69. Schmidt TG, Batz L, Bonet L, Carl U, Holzapfel G, Kiem K, Matulewicz K, Niermeier D, Schuchardt I, Stanar K: Development of the Twin-Strep-tag(R) and its application for purification of recombinant proteins from cell culture supernatants. Protein Expr Purif 2013, 92:54-61.
79. Hwang PM, Pan JS, Sykes BD: A PagP fusion protein system for the expression of intrinsically disordered proteins in Escherichia coli. Protein Expr Purif 2012, 85:148-151.
70. Salema V, Fernandez LA: High yield purification of nanobodies from the periplasm of E. coli as fusions with the maltose binding protein. Protein Expr Purif 2013, 91:42-48.
80. Kaldis A, Ahmad A, Reid A, McGarvey B, Brandle J, Ma S, Jevnikar A, Kohalmi SE, Menassa R: High-level production of human interleukin-10 fusions in tobacco cell suspension cultures. Plant Biotechnol J 2013, 11:535-545.
71. Hassouneh W, MacEwan SR, Chilkoti A: Fusions of elastin-like polypeptides to pharmaceutical proteins. Methods Enzymol 2012, 502:215-237.
81. Li Y: A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein. Protein Expr Purif 2012, 81:201-210.
72. Meyer DE, Chilkoti A: Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 1999, 17:1112-1115. The first paper describing the use of the non-chromatographic ELP tag for recombinant protein purification.
82. Haeckel A, Appler F, Figge L, Kratz H, Lukas M, Michel R, Schnorr J, Zille M, Hamm B, Schellenberger E: XTEN-Annexin A5: XTEN allows complete expression of long-circulating proteinbased imaging probes as recombinant alternative to PEGylation. J Nucl Med 2014, 55:508-514.
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Current Opinion in Structural Biology 2014, 26:54–61
ScienceDirect New trends and affinity tag designs for recombinant protein purification David W Wood Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, nonchromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Addresses Department of Chemical & Biomolecular Engineering, 435 Koffolt Laboratories, 140 W Nineteenth Avenue, Columbus, OH 43210, United States Corresponding author: Wood, David W ([email protected])
Current Opinion in Plant Biology 2014, 26:54–61 This review comes from a themed issue on New constructs and expressions of proteins Edited by Junichi Takagi and Christopher G Tate For a complete overview see the Issue and the Editorial Available online 12th June 2014 http://dx.doi.org/10.1016/j.sbi.2014.04.006 0959-440X/# 2014 Elsevier Ltd. All rights reserved.
Purification tag methods Although there are many applications related to gene fusions, perhaps the most common involves the addition of a purification ‘tag’, which provides a standardized method to purify the fused target protein [1]. These tags are usually fused to the N-terminus or C-terminus of a target protein, and commonly allow their partner proteins to be selectively captured and purified through association with a tag-specific affinity resin, or by highly selective tag-dependent precipitation or aggregation (Figure 1). Many tags can also provide additional functions unrelated to purification, such as facilitating detection of the target protein or improving its solubility [2]. In cases where a native untagged protein is required, the tag can usually be removed by a variety of methods once the target is purified. Current Opinion in Structural Biology 2014, 26:54–61
Despite the fact that tag-based methods have been used in laboratories throughout the world for decades, no universal tag has been identified that can be applied to any protein expressed in any host. In most cases, the identification of an optimal tag for a given host–target combination requires significant trial and error, and for some target proteins there are no effective tags. These limitations continue to drive the development of new options, and this research has led to ingenious and effective innovations over the past several years. Although a truly universal purification strategy is likely unrealistic, short-term objectives include increasing the variety of proteins that can be effectively purified with tag methods, while simultaneously facilitating the rapid identification of a suitable method for each new target. To accomplish these goals, researchers are increasingly using compound tags made up of multiple small domains, while continuing to develop single-domain tags with increased capabilities. Many new tags bind to highly inexpensive substrates, and are made even more economical through a variety of new approaches for tag removal. Taken together, these strategies can provide greater expression, increased purity and simplified detection for a wide range of fused products, along with inexpensive tag removal via simple self-cleaving or conventional proteolytic methods. A comprehensive discussion of even those tags developed in the last three years is far beyond the scope of this review, so I will highlight some interesting examples of each of these trends while providing a more comprehensive list of recently reported tags along with their basic advantages and recent references in Table 1. Notably, Table 1 lists many well-known conventional tags as well, which are included here due to their continued wide-spread use. Indeed, it is these tags that set the bar for newly developing methods in this field.
Popular tag methods Undoubtedly the most commonly used purification tag in laboratories worldwide is the Polyhistidine tag, or His-tag [3]. The primary characteristics of this tag are that it is small, inexpensive to use, and it typically has minimal or no effect on the target protein structure or function [4]. The small size of the tag allows it to be trivially added to either terminus of a given target protein, and it does not require a specific fold in order to function, making it highly reliable in all major expression systems. Further, several expression vectors are commercially available for fusion to included His-tags, and anti-His antibodies are available for immunoassay detection. Importantly, the His-tag can function under native or denaturing conditions, allowing it to be www.sciencedirect.com
Affinity tags for recombinant protein purification Wood 55
Figure 1
Fusion protein construction strategy
Expressed Fusion proteins (basic purification modalities)
Expression Vector
His tag for purification by IMAC Affinity column
Target Protein Sequence
Epitope tag for purification by Immunoaffinity column
Tag/Linker Sequence
Cloning and strain selection Ligand-binding domain for purification by conventional affinity column
Precipitation domain for purification by non-chromatographic method Fermentation and Harvest
Compound tag for simultaneous detection and purification Current Opinion in Structural Biology
Schematic representation of basic cloning strategies and commonly used purification modalities. Although many novel tags have been developed, the majority of them fit one of the basic modalities shown at right. An important advance has been the construction of complex multidomain compound tags, with a simple example shown at lower right. These tags exhibit optimized combinations of behavior, and can provide highly effective methods for large groups of protein targets.
used in protein refolding protocols [5], and it has also become central to recent efforts on the purification of soluble membrane proteins that are stabilized by lipids or detergents [6–8]. Indeed, this unique combination of strength and reliability has made the His tag ubiquitous in research, where many laboratories simply clone new proteins into His tag vectors before any attempt is made to express or purify the native untagged product. Despite these strengths, the His tag does suffer from a number of limitations. Perhaps the most significant is a tendency for contaminant proteins with external His residues to co-purify with His-tagged targets. The removal of these contaminants can require significant optimization [9], and indeed the Escherichia coli strain, LOBSTR (Low Background Strain), has been recently engineered to eliminate the most abundant contaminating host proteins [10]. Further, in some instances www.sciencedirect.com
the His tag can interfere with proper folding and activity of the target protein [11–13], and it has recently been reported to be incompatible with secretion in Streptomyces expression hosts [14]. In addition, the His-tag is generally ineffective in promoting proper folding of proteins with solubility issues, although it is routinely appended to a variety of solubility-enhancing domains. Some of the problems associated with His tags have been solved through the use of small epitope tags, with the most widely recognized being arguably the FLAG, c-Myc and HA tags [15,16]. These tags are typically eight to twelve amino acids in length and are very strongly and specifically bind to their corresponding immunoaffinity resins [17]. The small size of these tags allows them to retain many of the advantages of the His tag, while providing superior purity and recovery of the fused target. These tags are also compatible with virtually any expression host and facilitate Current Opinion in Structural Biology 2014, 26:54–61
56 New constructs and expressions of proteins
Table 1 Major tag classes with recent references, including examples of commonly used conventional tags. Sizes of tags shown in table are proportional to actual sizes of typically used tags. More recently reported tags are denoted in bold Tag type
Examples
Advantages
Limitations
Hisn [1,3,10,28,43,63–66]
Minimal impact on target expression and folding Many commercial systems Well established
Contaminants can co-purify Can interfere with target function
FLAG [15,67] c-Myc [15] GM-CSF [68] Twin StrepII [69]
Minimal target impact Exceptional purity Encoded on PCR primer Enable immunodetection
Very expensive resins Limited re-use cycles
Can enhance target solubility Highly specific binding Less expensive resins On-column cleaving possible
Can decrease expression yields Can leach from column
Folded domain tags
MBP [4,45,70] GST [4,64] Starch [35] Fluoroapetite [36] Diatomite [38] bGRP [37]
Very inexpensive to use Non-chromatographic High expression yields with aggregation tags
Precipitation tags are usually large in size Aggregation tags can require refolding protocols
Precipitation/ Aggregation tags
ELP [58,59,71,72] RTX [73] ELK16 [74,75] Fh8 [76] 4AaCter [77] PagP [78,79] eGFP [9,80] Heme [32,49] PYP [33]
Direct visual observation of target protein Can be highly quantitative Excellent for trouble-shooting expression
Larger tags can interfere with expression
NusA [15,22] SUMO [24–27,28,31] Trx [47,48,81] XTEN [82] FATT [39]
Can enhance solubility of target during expression Can aid in refolding of target if needed
Most are large and can decreases target yields
His tag
Peptide/Epitope tags
Detection Tags
Solubility/Folding tags
highly sensitive detection via immunoaffinity methods, making them especially attractive for cases where the target protein is difficult to overexpress and tag removal is unnecessary [18–20]. Easily the most significant drawback for the use of small epitope tags is the cost of use, where commercially available immunoaffinity resins are orders of magnitude more expensive than most ligandbased affinity resins. Thus, the convenience and selectivity of these tags make them excellent choices for very smallscale applications, such as tandem affinity purification and some structural studies. For larger scale experiments, however, they quickly become cost prohibitive. A third major class of conventional affinity tag includes the ligand-binding domain tags. These tags are typically much larger than His and epitope tags, and consist of fully folded protein domains with highly specific affinities for small molecule ligands immobilized on commercially available affinity resins. Two of the most widely known and commonly used tags are the Maltose Binding Domain (MBD) and Glutathione S-Transferase (GST) tags, which not only enhance expression in microbial hosts, but can Current Opinion in Structural Biology 2014, 26:54–61
also increase the solubility of their fusion partners [1,21]. The most significant drawbacks of these tags arise from their relatively large size and the resulting impact on their fusion partners, which occasionally necessitates inconvenient tag removal protocols. These tags can also decrease the overall yield of smaller fusion partners, due to their high metabolic demands on protein synthesis. Many common tags do not directly participate in any purification methods, but instead provide accessory functions such as enhanced folding and detection of the target. For example, NusA fusions can improve target solubility [22], while thioredoxin (Trx) fusions can facilitate proper disulfide bond formation in proteins with multiple cysteine residues [23]. The SUMO (small ubiquitinrelated modifier) tag has also been used to enhance expression and folding of recombinant proteins in prokaryotic and eukaryotic hosts [24–26]. The SUMO tag is particularly attractive due to its small size and the availability of a highly specific SUMO protease for tag removal, and for these reasons it has been combined with a number of other conventional tags in a variety of www.sciencedirect.com
Affinity tags for recombinant protein purification Wood 57
configurations [27,28,29–31]. While some tags can improve the expression of their fusion partners, others can allow direct visual detection of the target protein during expression and purification. Recent examples include small heme-binding proteins and motifs that give the fused target a bright red color [32], as well as additional fluorescent and chromogenic proteins [33,34]. Although these tags may not aid in purifying the expressed target, they are often instrumental in overall process troubleshooting and optimization.
New tags for purification, folding and detection Many recently reported tags have been developed for inexpensive substrates that can be used in non-chromatographic configurations, making them economical for larger scale methods in simple laboratory settings. One of the most promising of these is a novel starch-binding domain (SBD) tag, which binds tightly and specifically to raw corn starch as well as a variety of other vegetable starches [35]. In demonstrations with four target proteins, the SBD tag compares favorably to the His tag in delivering highly purified proteins in a single affinity step, where the purification method relies only on simple centrifugation. Moreover, starch is inexpensive, renewable and biodegradable, suggesting the use of this method for various products at large scale. Three additional tags have recently been developed that bind to exceptionally inexpensive affinity substrates. These include a heptamer tag derived from phage display that binds to ceramic fluorapatite [36]; a 112 residue silkworm b-1,3-glucan recognition protein that binds to inexpensive curdlan [37]; and a 70 amino acid segment of the E. coli ribosomal protein L2 that binds to diatomaceous earth [38]. All three of these tags have demonstrated similar performance to conventional tags in delivering pure products, and all three can be used with inexpensive non-functionalized base materials. This aspect not only contributes to the lower price of the affinity substrates, but also eliminates the risk of ligands leaching from the resin into the purified target protein. A notable additional tag is the Flag-Acidic-Target Tag, or FATT [39]. This tag includes a Flag tag module for easy detection, along with a hyperacidic segment from the human amyloid precursor protein (APP) extracellular region. The hyperacidic segment expresses well in E. coli, and because it is highly charged, it can be easily purified in a single step on a conventional anion exchange chromatography resin. More importantly, however, this tag has been shown to significantly increase proper folding of fused target proteins during expression, and can facilitate the proper refolding of misfolded fusion partners that contain disulfide bonds. These capabilities are hypothesized to arise from the disordered structure of the FATT hyperacidic segment, which is thought to act www.sciencedirect.com
as a shield-like non-specific chaperone for the target protein during expression or refolding in vitro. Reported results from this tag are very impressive, and suggest that it may become an important new option for expression of difficult targets.
Compound tags Complex compound tags are now commonly constructed from multiple linked domains, where different combinations can provide highly efficient orthogonal purification methods, as well as additional functions related to target expression and detection (Figure 2). Various compound tags have enabled the characterization of protein–protein interactions through tandem affinity purification (TAP) and mass spectrometry [40–42], and these powerful methods are now being used for the purification of recombinant target proteins for structural studies as well.
Figure 2
Hyperacidic region
(a)
-
-
-
-
Target Protein Linker
FLAG tag
Functional domain tag (e.g., NusA, eGFP, Trx)
(b)
Target Protein His tag
Linkers
(c)
Heme binding tag SBP tag
Target Protein His tag Linkers
TEV Protease recognition sequence Current Opinion in Structural Biology
Some of the more interesting compound tags described in the text. (a) The FATT tag, which combines a FLAG tag for simple detection, along with a hyperacidic segment of the human amyloid precursor protein [39]. (b) A multifunctional His tagged protein, which includes an additional domain for improving expression or quantitative detection. In at least one case, the His tag is used primarily as a method to easily remove the cleaved functional domain after the target has been purified. (c) The Multitag, which combines two orthogonal purification tags with a convenient detection domain and protease recognition sequence [49]. Current Opinion in Structural Biology 2014, 26:54–61
58 New constructs and expressions of proteins
The simplest examples of compound tags generally consist of a small peptide purification tag, such as a His tag, fused to a functional domain, such as the solubilityenhancing NusA tag [22]. Taken together, this compound tag provides enhanced expression and solubility of the target, along with a simple purification method, and is typical of an initial strategy used for the production of uncharacterized targets in early studies. Additional examples of binary compound tags include the CHiC tag (His-Choline binding domain) for two orthogonal purification methods [43]; His-MBD for enhanced soluble expression and/or orthogonal purification methods [44,45]; His-Trx for proper disulfide bond formation and to simplify removal of the cleaved Trx tag [46–48]; and His-GFP for simple monitoring and optimization of expression and purification in Saccharomyces [9]. More recent binary tags now commonly include a smaller SUMO tag segment, which can significantly increase soluble expression of target proteins in a variety of hosts [27–30]. A final, somewhat more complex example is the recently reported ‘Multitags’ method based on a four-component compound tag. Specifically, this tag includes a His10 tag, a streptavidin-binding peptide (SBP), a heme-binding domain, and a TEV protease sequence for tag removal [49]. The His and SBP tags provide orthogonal purification methods, while the heme binding domain allows simple visual tracking and quantification of the target protein through the process. Notably, the tag is removed by a SBP-tagged TEV protease, thus removing the tag and protease in a single operation. This tag was shown to be effective in delivering highly purified Pfu DNA polymerase and Myosin-VIIa- and Rab-Interacting protein (MyRIP), and is likely to have general applicability to additional targets. Notably, this highly functional tag has a molecular weight of only 23 kDa, or slightly more than half the molecular weight of the commonly used MBD tag. The combination of function and size makes this tag very attractive for medium-scale production methods, and increasing simplicity of recombinant DNA methods facilitates rapid prototyping of new designs. Most importantly, the increasing availability of small functional domains will inevitably assist the construction of additional complex and highly functional tags, and will continue to increase the popularity of compound tags in the future.
Tag removal methods In many cases, the tags used for expression and purification of the target protein must be removed before the target can be characterized or applied. Conventional methods generally include tag removal through the addition of highly specific endopeptidases, where the endopeptidase target sequence is engineered into the fusion protein between the tag and the target [15]. Research on basic tag removal strategies has led to several recent additions, and an increasing trend is the on-column Current Opinion in Structural Biology 2014, 26:54–61
cleavage of the target protein directly from an immobilized tag, often using an immobilized protease [49]. In addition to new process configurations, significant research has focused on the identification of still faster and more specific endopeptidases for tag removal. Successes include a group of four highly specific and orthogonal enzymes with applications in the sequential assembly of protein complexes [50], as well as a detergent-resistant West Nile Virus protease with applications in cleaving tags from detergent-stabilized membrane proteins [51]. A particularly interesting example is the recently reported glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL protease), which has been shown to cleave to over 99% completion in only 15 min under physiological conditions [52]. In addition to new proteolytic enzymes, a number of selfcleaving modules have been developed over the past 20 years [53,54]. The first of these were based on selfsplicing inteins, which were re-engineered into self-cleaving protein elements [55–57]. A highly effective purification method was generated by combining inteins with reversibly precipitating elastin-like protein (ELP) tags, and more recent research has expanded this basic approach to additional configurations [58–61]. Major drawbacks of intein methods include the uncontrolled cleaving of the inteins during expression of the tagged target, and the requirement for reducing agents to trigger the N-terminal cleaving reaction. Issues with premature intein cleaving have been partially addressed through the recent development of natural and engineered split inteins, which are inactive when expressed as separate segments, but can be activated after the purification protocol is complete. A particularly promising embodiment of this strategy is the Split Intein Mediated Ultra-Rapid Purification of Tagless Protein (SIRP) system, which involves the assembly and rapid cleaving of the naturally split Nostoc punctiforme DnaE intein in the presence of dithiothreitol [62]. This system has been shown to completely abolish premature cleavage of the target protein during expression, but provides almost complete tag removal in less than 30 min at room temperature once the cleavage reaction is triggered. Further, the intein segment that is joined to the target protein is quite small, and thus exhibits limited interference in the solubility and expression of the target. For these reasons, this split intein is one of the most promising tag removal methods that has yet been reported, and it will likely be combined with an array of purification, detection and expression tags in future systems.
Conclusion A comprehensive review on tags and tag removal methods would include scores of ligand binding domains, epitopes, precipitation and aggregation tags, as well as close to a www.sciencedirect.com
Affinity tags for recombinant protein purification Wood 59
dozen different flavors of the His tag. These tags would be combined with well over a dozen tag removal methods, utilized in countless mechanical configurations on target proteins of all shapes and sizes. Ultimately, the ease with which tags can be designed, redesigned, combined, recombined and cleaved has led to literally thousands of potential methods for the purification of a single protein. Yet despite this enormous diversity, no truly universal tag method has been developed. Further, many proteins, and some classes of proteins, remain stubbornly recalcitrant to even the most creative and carefully planned strategies. These frustrations have driven relentless research in this area, where most discoveries provide only incremental advances over established methods. The most significant advances of the past few years would arguably be the now widespread use of the SUMO tag for improved expression and solubility in a variety of hosts, along with the development of a number of novel methods for tag removal. On-column tag cleavage is now routine, and several configurations are now commercially available. Self-cleaving tag methods are continuing to evolve, and hold promise for extending affinity tag methods to large-scale manufacturing processes. Several popular protease enzymes can now be produced in recombinant E. coli, substantially decreasing their cost and increasing their availability. Coupled with simple and inexpensive precipitation tags, these advances promise to democratize the use of affinity methods in protein purification, making these methods available to a much wider segment of the scientific community.
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