Protein Expression and Purification 85 (2012) 9–17
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Protein Expression and Purification
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Review Simplifying protein expression with ligation-free, traceless and tag-switching plasmids ⇑ Venuka Durani a, Brandon J. Sullivan b, Thomas J. Magliery a,c, a Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA b Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA c Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA article info abstract
Article history: Synthetic biology and genome-scale protein work both require rapid and efficient cloning, expression and Received 17 March 2012 purification. Tools for co-expression of multiple proteins and production of fusion proteins with purifica- and in revised form 1 June 2012 tion and solubility tags are often desirable. Here we present a survey of plasmid vectors that provide for Available online 21 June 2012 some of these features with a focus on tools for rapid cloning and traceless tagging – a setup that facil- itates removal of fusion tags post-purification leaving behind no ‘scar’ on the final construct. Key features Keywords: are reviewed, including plasmid replication origins and resistance markers, transcriptional promoters, Plasmid cloning methods, and fusion tags and their removal by proteolysis. We describe a vector system called Protein expression pHLIC, which assembles features for simple cloning, overexpression, facile purification, and traceless Co-expression Protease cleavage cleavage, as well as flexibility in modifying the vector to exchange fusion tags. Ligation independent cloning Ó 2012 Elsevier Inc. All rights reserved. Traceless tagging
Contents
Introduction...... 9 Origin of replication ...... 10 Antibiotic resistance ...... 10 Cloning regions ...... 10 Transcription promoters ...... 12 Fusion tags...... 13 Protease cleavage sites ...... 13 Seamless cloning and traceless tagging ...... 13 Vectors for traceless tagging – some examples...... 13 pHLIC vectors ...... 14 Conclusion ...... 15 Appendix A. Supplementary data ...... 15 References ...... 15
Introduction often expensive and time-consuming prerequisites, particularly for beginning researchers. Solutions to these problems have High-throughput approaches in modern protein science – emerged [6–9]: recombinogenic (e.g., Gateway [10]) cloning and including structural genomics and proteomics – have necessitated ligation-independent cloning (LIC)1 [11,12]; vectors with very the development of high-throughput methods for cloning, protein expression, and purification [1–5]. Even for laboratories studying a 1 Abbreviations used: MBP, maltose binding protein; GST, glutathione S-transferase; single protein target, such as designed proteins, these steps are CBP, calmodulin binding protein; Trx, thioredoxin; PSI–MR, protein structure initiative–materials repository; LIC, ligation independent cloning; SLiCE, seamless ⇑ Corresponding author at: Department of Chemistry, The Ohio State University, ligation cloning extract; IPTG, isopropyl b-D-1-thiogalactopyranoside; IMAC, immo- Columbus, OH 43210, USA. Fax: +1 614 292 1685. bilized metal-ion affinity chromatography; SUMO, small ubiquitin-like modifier; Ub, E-mail addresses: [email protected] (V. Durani), bsulliva@ ubiquitin; DUBs, deubiquitylating enzymes; TEV, tobacco etch virus; TIM, triose- chemistry.ohio-state.edu (B.J. Sullivan), [email protected] (T.J. Magliery). phosphate isomerase.
1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.06.007 10 V. Durani et al. / Protein Expression and Purification 85 (2012) 9–17
that includes the origin of replication determines its copy number. Usually for large scale protein purification we turn to high copy number plasmids that can maintain tens to hundreds of copies of plasmid per cell. Production of recombinant proteins in Escherichia coli often results in degradation, aggregation or misfolding of pro- teins, and co-expression [22] of molecular chaperones can provide a solution to this problem [23–25]. Co-expression of proteins is also desirable for purification of multi-subunit complexes and studying interacting proteins [26–28]. However, plasmid incom- patibility, a mechanism that prevents the stable co-existence of two similar plasmids in the same bacterial cell, can cause problems for these kinds of applications, and having access to plasmids with different origins of replication that are compatible with each other is important. The best developed class of plasmid vectors for many manipulations including protein expression is based on the ColE1/ pMB1 [29] replicons including the pBR322 [30,31], pUC [32,33] and pET (Novagen) vector systems. Vectors containing ColE1/pMB1 de- rived origins of replication fall into the same incompatibility group [34,35]. On the other hand, plasmids like pACYC177 and pACYC184 [36,37] with origins of replication derived from p15A [38], although incompatible with other vectors with p15A derived ori- gins, are compatible with ColE1/pMB1 derived plasmids. These are two compatible vector groups that are commonly used for co-expression of proteins. Since ColE1 based plasmids are very common for production of target proteins, several chaperone- encoding plasmids are available on vectors with p15A derived rep- licons [23]. For experiments in which the population of three or more different plasmids needs to be maintained in a cell, other Fig. 1. Schematic plasmid map (not drawn to scale) showing various important plasmids with pSC101 [39], CloDF13 [40,41], ColA [42,43], regions of plasmid vectors that are discussed in this review. RF1030 [44] and pEC [45] replicons can be used [22]. strong promoters such as T7 and cspA; and the use of fusion tags Antibiotic resistance such as hexahistidine (6 His) [13], maltose binding protein (MBP) [14,15], glutathione S-transferase (GST) [16,17], calmodu- In order to ensure that a plasmid vector is taken up and main- lin-binding peptide (CBP) [18,19] and thioredoxin (Trx) [20] for tained in a cell, genes encoding resistance to antibiotics are ex- solubility and/or purification. Many useful vectors are available pressed from plasmids. Moreover, if populations of multiple through the Protein Structure Initiative Material Repository (PSI– vectors are to be maintained in a cell, then not only do their repli- MR), and information about these vectors is available in a search- cator regions need to be compatible, their antibiotic resistance able database called DNASU [7]. markers also need to be orthogonal to ensure co-transformation. While affinity tagging results in convenient purification, many The most common antibiotic resistance markers are ampicillin, applications demand removal of the fused tag post-purification, kanamycin, chloramphenicol and tetracycline. Table 1 has exam- typically by proteolysis. However, the recognition sequences of ples of some vectors with spectinomycin, zeocin and erythromycin restriction enzymes for cloning and of proteases for freeing the fi- resistance. Ampicillin is especially susceptible to degradation nal protein typically put constraints on one or more amino acids in when present the bacterial culture and this effect can be observed the protein, leaving a ‘scar’ that may not be desirable. This problem as ‘satellite’ colonies on agar plates. This effect can be alleviated to has initiated work on seamless cloning and traceless tagging strat- some extent by using carbenicillin [46], a less degradation- egies, and plasmid vectors that simplify such manipulations are susceptible analog of ampicillin. desirable. Plasmid vectors require various components to carry out their functions (Fig. 1). The origin of replication determines Cloning regions the copy number of a plasmid; antibiotic resistance provides a selection marker; promoter regions facilitate gene transcription; Most plasmid vectors that are commercially available have and careful engineering of cloning sites can provide for features multiple unique restriction enzyme sites arranged in tandem in a like ligation-independent cloning and fusion tags to facilitate solu- polylinker cloning region. These can be used for traditional liga- bility and purification of the expressed protein. Vectors with un- tion-dependent cloning methods (Fig. 2a) where a target gene is ique combinations of these functionalities are especially suited amplified by PCR using primers containing unique restriction sites, for specific applications in structural genomics and synthetic biol- followed by restriction enzyme digest and ligation into a vector ogy. Here we present a brief survey of plasmid vectors useful for containing compatible cohesive ends. These traditional methods protein expression with a focus on vectors that facilitate quick often produce ‘background’ due to incomplete digestion and cloning and traceless tagging of proteins. re-ligation of the digested vector and require unique restriction en- zyme sites in the vector that do not occur in the DNA to be cloned. Origin of replication These limitations and others such as difficulty to manipulate very large inserts have led to research in recombinogenic cloning meth- The origin of replication is the smallest region of the plasmid ods especially for applications in structural genomics. Homologous that is able to replicate on its own and is also the site where plas- recombination occurs through stretches of DNA shared by the two mid replication is initiated [21]. The replicator region of a plasmid molecules. Because the sequence of the homology regions can be V. Durani et al. / Protein Expression and Purification 85 (2012) 9–17 11
Table 1 List of plasmid vectors.
Vector name Fusion tagsa Cleavage sites ‘Scar’ residues post-cleavageb Cloning method Promoter region Ori Res. Ref. Origins of replication and antibiotic resistance markers: pET11a T7 None 13 amino acids Ligation T7 ColE1 Amp [99] pURI2-Ery 6 His EK protease M LIC lpp ColE1 Ery [100] pACYCDuet-1 6 His None 13 amino acids Ligation T7 p15A Cmp [101] pT7LICK 6 His (Ct) None DHHHHHH (Ct) LIC T7 pSC101 Kan [12]
pEC None N.A. N.A. Ligation PBAD pEC Kan [45] pMCSG21 6 His TEV protease SNA LIC T7 Clo-DF13 Spec [102] pCOLADuet-1 6 His None 14 amino acids Ligation T7 ColA Kan [43] Cloning methods: pET32a Trx-6 His EK protease AMA Ligation T7 ColE1 Amp [99] pVP16 8 His-MBP TEV protease S Gateway T5 ColE1 Amp [8] pNIC28-Bsa4 6 His TEV protease SM LIC T7 ColE1 Kan [4] pBL None N.A. N.A. SLiCE T7 ColE1 Amp [52] pCR-BluntII- None N.A. N.A. TOPO T7, SP6 ColE1 Kan, [55,56] TOPO Zeo Transcription promoters: pVP33A 8 His-MBP 3C and TEV AIA Ligation T5 ColE1 Amp [8] pURI2 6 His EK protease M LIC lpp ColE1 Amp [13] pURI2-TEV 6 His TEV protease GM LIC lpp ColE1 Amp [100] pURI3 6 His EK protease M LIC T7 ColE1 Amp [13]
pBAD/His 6 His EK protease DRWGSELE Ligation PBAD ColE1 Amp [103] pCKSP6 None N.A. N.A. Ligation SP6 ColE1 Amp [104] PinPoint Xa1 Biotin Factor Xa None Ligation tac ColE1 Amp [105] pCOLD I 6 His Factor Xa HM Ligation cspA ColE1 Amp [72] Tag-switching plasmids: pHLIC 6 His TEV protease None LIC T7 ColE1 Amp [98] pHLIC-MBP 6 His-MBP TEV protease None LIC T7 ColE1 Amp pMCSG7 6 His TEV protease SNA LIC T7 ColE1 Amp [11] pMCSG9 6 His-MBP TEV protease SNA LIC T7 ColE1 Amp [95] pMCSG10 6 His-GST TEV protease SNA LIC T7 ColE1 Amp [102] pMCSG11 6 His TEV protease SNA LIC T7 p15A Cmp [102] pMCSG13 6 His-MBP TEV protease SNA LIC T7 p15A Cmp [97] pMCSG14 6 His-GST TEV protease SNA LIC T7 p15A Cmp [97] pLIC-His 6 His TEV protease GAAS LIC T7 ColE1 Amp [2] pLIC-MBP 6 His-MBP TEV protease GAAS LIC T7 ColE1 Amp [2] pLIC-GST 6 His-GST TEV protease GAAS LIC T7 ColE1 Amp [2] pOPINF 6 His 3C protease GP LIC T7 ColE1 Amp [1] pOPINM 6 His-MBP 3C protease GP LIC T7 ColE1 Amp [1] pOPINJ 6 His-GST 3C protease GP LIC T7 ColE1 Amp [1] Protease cleavage sites: pMCSG19 MBP-6 His TVMV and TEV SNA LIC T7 ColE1 Amp [95] pVP33K 8 His-MBP 3C and TEV AIA Ligation T5 ColE1 Kan [8] pCAL-n-EK CBP EK protease None LIC T7 ColE1 Amp [19] pHUE 6 His-Ub Usp2-cc None Ligation T7 ColE1 Amp [77] pHLIC-smt3 6 His-SUMO SUMO protease None LIC T7 ColE1 Amp C-terminal His-tags: pT7LICB 6 His (Ct) None DHHHHHH (Ct) LIC T7 ColE1 Amp [12] pT7LICC 6 His (Ct) None DHHHHHH (Ct) LIC T7 p15A Cmp [12] pT7LICK 6 His (Ct) None DHHHHHH (Ct) LIC T7 pSC101 Kan [12] pURI2-Cter 6 His (Ct) None HHHHHH (Ct) LIC lpp ColE1 Amp [100]
Ori, origin of replication; Res, antibiotic resistance marker; Ref, reference; Amp, ampicillin; His, histidine tag; EK, enterokinase; LIC, ligation independent coning; Ery, erythromycin; Cmp, chloramphenicol; Ct, C-terminal; Kan, kanamycin; N.A., not applicable; TEV, Tobacco Etch Virus; Spec, spectinomycin; Trx, thioredoxin; MBP, maltose binding protein; Zeo, zeocin; 3C, human rhinovirus 3C protease site; GST, glutathione S-transferase; SUMO, Small Ubiquitin-like Modifier; TVMV, tobacco vein mottling virus protease; CBP, calmodulin binding protein. a All the fusion tags are on the N-terminus unless otherwise specified as Ct (C-terminus). b Extra N-terminal (unless otherwise specified) residues that remain after protease cleavage due to the design of the cloning site are listed in one letter amino acid codes. In some cases more residues may be required based on efficiency of protease cleavage. For vectors that have a tag but no cleavage site, the length of the tag is mentioned. chosen freely, any position on a target molecule can be specifically drawbacks of traditional cloning methods [51]. By taking advan- altered (for reviews on recombinogenic cloning see references tage of the controlled exonuclease activity of T4 DNA polymerase, [47–49]). Gateway cloning (Fig. 2b) vectors have several features the LIC procedure creates long single-stranded overhangs of DNA required for high-efficiency and accurate cloning using recombino- in the vector and the insert. These overhangs hybridize on mixing genic methods [10]. Once an insert is cloned into an entry vector the vector and insert together. When transformed into E. coli, the for Gateway cloning, it can be easily swapped into other Gateway nicks are sealed by the cellular machinery. Hence no use of ligase vectors using clonase activity. This makes Gateway cloning very is required, and since the vector has two fairly large overhangs useful for applications where the target construct is required in which can be designed to be completely non-complementary to various contexts like different solubility tags or different origins each other, the chances of re-sealing the vector are very small. of replication. Moreover, since this scheme does not require digestion of the in- Ligation-independent cloning (Fig. 2c) [50] adds flanking se- sert with any restriction enzymes, there are no complications quence extensions to the target gene that are shorter than those regarding multiple occurrences of the preferred cloning enzymes used in recombinogenic methods while still avoiding some of the within the sequence to be cloned. A new cloning method called 12 V. Durani et al. / Protein Expression and Purification 85 (2012) 9–17
Fig. 2. Cloning schemes for (a) ligation-based cloning; (b) ligation-independent cloning; (c) recombinogenic cloning (Gateway); (d) SLiCE cloning; and (e) TOPO cloning.
SLiCE (Seamless Ligation Cloning Extract) relies on in vitro recom- to degradation of the host chromosome [57,58]. Ligation of an in- bination in bacterial cell extract (Fig. 2d) [52]. Extracts from the sert into the vector disrupts the ccdB gene and enables the recom- two recA strains DH10B and JM109 yielded the highest cloning binant colonies to grow. This method eliminates background from efficiencies indicating that SLiCE is facilitated by a RecA-indepen- self-ligation of the vector. dent mechanism. SLiCE facilitates seamless cloning by recombining short end homologies (>15 bp) with or without flanking heterolo- gous sequences. Seamless cloning using SLiCE has been demon- Transcription promoters strated using plasmid vector pBL [52]. Other recently-reported methods of cloning that fall into this category include Gibson clon- Promoter regions affect the level of gene expression in E. coli, and ing (enzymatic assembly of DNA molecules using T5 exonuclease, the suitability of a promoter for protein expression depends on Phusion polymerase and Taq ligase) [53] and CPEC (Circular Poly- three main factors. First, the promoter must be strong and have merase Extension Cloning that involves extension of the overlap the capacity to over-produce proteins to the extent that they be- between single stranded vector and insert to form a complete plas- come a significant percentage of the total expressed protein in the mid) [54]. cell. Second, it is desirable to have a highly tunable (or at least highly TOPO cloning (Fig. 2e) is a method of cloning where a vector and repressible) promoter particularly for proteins that have adverse ef- insert with single nucleotide overhangs are ligated together by tak- fects on the growth of the host cell. Third, the promoter should be ing advantage of topoisomerase activity [55]. The single nucleotide easily inducible typically by chemical or thermal means. Several overhang in the insert is created upon amplification by Taq poly- promoter systems used for protein production in E. coli, their regu- merase and this technique, like LIC, negates the use of restriction lation mechanisms, and methods of induction can be found in refer- enzyme digest for the insert. The vector pCR-BluntII-TOPO (Invitro- ences [59,60]. Vectors with SP6 (phage promoter, mainly used for gen) uses TOPO cloning in conjunction with the ccdB (control of cell cell free expression [61,62]), T5 (phage promoter, induced by IPTG, death) gene for zero background cloning [56]. The CcdB protein very high level of expression, tight regulation possible [63]), hybrid causes cell death by poisoning bacterial DNA gyrase, which leads lpp (E. coli promoter, induced by IPTG, very high level of expression V. Durani et al. / Protein Expression and Purification 85 (2012) 9–17 13
[64]), PBAD (E. coli promoter, induced by L-arabinose, variable level of after purification. Common proteases for this purpose include expression from low to high, tight regulation possible [65]), tac TEV protease [79–81] and related 3C protease [82], enterokinase (E. coli hybrid promoter, induced by IPTG, moderately high level of [83–85], Factor Xa [86], thrombin [87,88] and SUMO protease expression, high basal level of expression [66]), cspA (E. coli pro- [76]. TEV protease, the capsid protease of the tobacco etch virus, moter, cold shock induction at temperatures below 20 °C, low level is among the most robust of the proteases in common use [89]. of expression [67]) and T7 (phage promoter, induced by IPTG, very It is more specific than enterokinase [85] or Factor Xa [90], and it high level of expression, tight regulation possible [68]) promoters is active under a wide range of conditions. It is commercially avail- are listed in Table 1. The pET vector system that has the promoter able and can also be overexpressed and purified from E. coli using a system from bacteriophage T7 has been very widely used due to standard protocol [91]. The general form of its recognition se- the orthogonality of this system to E. coli promoters, leading to ease quence is taken to be E-X-X-Y-X-Q-(G/S), with cleavage after the of cloning toxic genes and also its capacity to overproduce proteins Gln residue; the most commonly used sequence is ENLYFQG. Re- at very high levels constituting up to 50% of the total cell protein cently, Kapust et al. demonstrated that the TEV protease will cleave [69–71]. The T7 promoter, along with some of the other promoters with greater than 90% efficiency with 12 different amino acids at listed above, is indirectly induced by IPTG in that T7 RNA polymer- the P10 site (C-terminal to Gln) and greater than 50% efficiency ase is expressed from the lac promoter-operator. One of the for all other amino acids except proline [92]. Effectively, TEV pro- limitations of the T7 promoter system, however, is that rapid tease can be used to quantitatively produce mature proteins with over-expression of relatively ill-behaved proteins can lead to pro- any N-terminal amino acid except Pro under reasonable reaction tein misfolding, aggregation and segregation into inclusion bodies. conditions. However, proteins with structured N-terminal regions Although using a cold shock promoter like cspA [67] (pCold vectors sometimes do not display very efficient cleavage with TEV protease by Takara Bio Inc. [72]) is sometimes a solution to this problem, a presumably due to inaccessibility of the protease recognition and major disadvantage of that system is that it becomes repressed 1– cleavage site. For such cases, having an alternative option is desir- 2 h after lowering the temperature, and this time frame might not able, such as Factor Xa, which cleaves I-(E/D)-G-R-X between R and be sufficient for a high level accumulation of all proteins. X, where X can be any amino acid except proline [93]. Another op- tion is the SUMO protein fusion. In this case, the 100 residue SUMO protein serves as the recognition site for SUMO protease, which Fusion tags cleaves after the C-terminal Gly of SUMO. It has been shown that recombinant SUMO–GFP fusions are efficiently cleaved when any Besides cold shock, there are several approaches to the expres- amino acid except proline is in P1´ position of the cleavage site sion of proteins that end up in the insoluble fraction, including [76]. Similar to SUMO, the ubiquitin tag also serves as a recognition the co-overexpression of chaperones [23–25], periplasmic expres- site for deubiquitylating enzymes (DUBs), however most DUBs that sion of proteins [73] and the use of solubilizing fusion tags. Protein have been isolated from various species are relatively large and dif- fusions are efficient tools for protein purification, detection and ficult to express and purify. No stable DUB known with general immobilization. One of the most commonly used fusion tags for activity against a range of fusion proteins was known. Recently, a protein purification is the hexahistidine (6 His) [13] tag that facil- mouse DUB, Usp2, was engineered to provide a minimal catalyti- itates purification by immobilized metal-ion affinity chromatogra- cally active deubiquitylating domain (Usp2-cc) [77]. It was ex- phy (IMAC) purification with Ni–NTA agarose. This method is pressed and purified with a polyhistidine tag that allowed the inexpensive; the resin has very high capacity and can be reused in vitro cleavage of Ub from the desired protein as well as its selec- many times. It is less expensive than other resins such as streptavi- tive removal from the cleavage reaction. din agarose used for purification of biotin-tagged proteins, and it has a higher capacity than the less-expensive amylose resin used to purify proteins with an MBP tag [59,74]. Although fusion tags Seamless cloning and traceless tagging were originally developed for purification purposes, it was found that many tags lead to better solubilization of proteins fused at Generation of the overhang for cloning into a vector requires their C-termini. It is believed that the general mechanism for this restriction enzyme scission, which places a limitation on the se- effect involves the fast and efficient folding of the N-terminal fusion quence of the overhang and usually results in a particular protein tag upon exiting the ribosome immediately after translation, thus sequence at the site of fusion. A way to get around this problem pulling the C-terminally fused protein into solution. The most com- and accomplish ‘seamless’ cloning [94] is to digest with a type IIs monly used fusion tags for solubility enhancement are maltose restriction enzyme which cuts outside of its recognition sequence binding protein (MBP) [14,15], calmodulin-binding peptide (CBP) and therefore in any sequence. (GeneArt by Invitrogen and CloneEZ [18,19], glutathione S-transferase (GST) [16,17], and thioredoxin by GenScript are kits that can be used for seamless cloning.) Even (Trx) [20]. More recently it has been shown that the N-terminal fu- for routine protein expression and purification, it would be useful sion of SUMO protein [75,76], ubiquitin [77], and HaloTag7 [78] also to have a very flexible plasmid vector for high-level E. coli overex- enhance expression levels and solubility of ill-behaved proteins. pression of proteins using an N-terminal 6 His tag that could be Several fusion tags can have multiple functionalities; for instance, removed tracelessly – which is to say, with no residues left behind MBP and HaloTag7 can be used for both solubility enhancement and and few or no constraints on the amino acid next to the scissile purification. Similarly, SUMO and ubiquitin (Ub) protein fusions can bond. To circumvent some issues with ligation-dependent cloning, be used as both solubility tags and also protease cleavage tags. Some- a ligation-independent method is desirable, but with minimal ex- times finding the optimum tag for an application requires some trial pense, and with the flexibility to use ligation-dependent methods, and error and also some applications may require the same protein as well. Several vectors which nearly fit these criteria are available, with different tags. Hence, tag switching vectors where the same in- perhaps most notably the pMCSG7 vector [11] and the pHLIC vec- sert can be used to clone with various tags are desirable. tor system (described here).
Protease cleavage sites Vectors for traceless tagging – some examples
Even though fusion tags often solubilize proteins and provide Today several plasmid vectors are available that have cloning options for easy purification, it is often desirable to remove them sites designed to attach fusion tags and also provide for protease 14 V. Durani et al. / Protein Expression and Purification 85 (2012) 9–17
optionally C-terminal) 6 His tag to a target protein cloned by LIC under the control of the T7 promoter. Variants of this vector that allow for TEV cleavable MBP and GST fusions are also available and this series has some ColE1 derived vectors and others with p15A derived replicons for co-expression [97]. However, the LIC cloning scheme designed for these vectors requires that the cleaved N-terminal sequence begin with Ser-Asn-Ala. We have constructed a related vector in which there are no constraints on the N-terminal sequence, and in which the protease recognition se- quence can be removed or changed by a simple standard cloning step. We call the resulting vector pHLIC (plasmid for 6 His tagging and Ligation Independent Cloning, Fig. 3 and Supplementary Fig. 3. Schematic maps of the pHLIC and pHLIC-MBP plasmids. The AlwNI site is not material). unique in the pHLIC-MBP vector. To achieve high-level expression and simple, inexpensive purifi- cleavage sites to remove the fusion tags later. These vectors have cation, we selected a pET vector system with a T7 promoter for combinations of different methods of cloning, different fusion tags overexpression in DE3-lysogenized cells, and we selected an N-ter- and different protease cleavage sites (several examples are listed in minal 6 His-tag. It would be difficult to achieve ‘‘traceless’’ scission Table 1). For instance, vectors pURI [13] and pCAL-n-EK [19] can be with a C-terminal tag, since virtually all useful (highly specific and used for ligation-independent cloning, and use enterokinase, which active) proteases have their recognition elements upstream of or is not as specific as TEV and also more expensive. Vectors like pOPINJ overlapping the scissile bond. To truly make this traceless, we se- [1], pLIC [2], pNIC28 [4], pVp [8] allow for TEV cleavage of 6 His tag, lected the robust and easy-to-prepare TEV protease, which cleaves but due to the design of the vector they leave behind 2–4 residues on before every amino acid except Pro. To circumvent ligation-depen- the N-terminus after protease cleavage. The vector pT7LIC [12] dent cloning, we designed a sequence for ligation-independent leaves behind an unmodified N-terminus, but uses a C-terminal cloning, which is to say, a sequence that encodes the TEV cleavage 6 His-tag that cannot be removed. pMCSG7 [11] and pMCSG9 sequence but that is devoid of a single nucleotide (dG in the vector; [95,96] are tag-switching vectors where the same insert can be used see Fig. 4) to generate the LIC overhang with T4 polymerase editing. to clone in the context of a 6 His–TEV fusion and also a 6 His–MBP– To remove any restriction on the first amino acid at the cloning TEV fusion. However pMCSG7 and pMCSG9 leave three extra N-ter- junction, we embedded BsaI sites in opposite orientations between minal residues after TEV cleavage and do not use a type IIs restriction the sequences for the TEV site and the terminator. BsaI is a type IIs enzyme for seamless cloning. On the other hand, the use of BsaI en- restriction enzyme that cuts outside of its recognition sequence. zyme for seamless cloning has been used with pX-LacZ vector [3] but Therefore, cleavage with BsaI removes the cassette without leaving not in the context of a His-tag purification scheme. a trace. Finally, we placed an NcoI site between the 6 His tag and TEV site, making it possible to clone without the TEV site (between NcoI and BamHI) by standard means or replace the 6 His tag (be- pHLIC vectors tween NdeI and NcoI). The pHLIC vector can also be used for cloning using enzymes NdeI and BamHI. The result would be equivalent to The pMCSG7 vector of the Midwest Center for Structural using the pET11a vector (Novagen) between those sites and would Genomics appends a TEV protease-cleavable N-terminal (and
Fig. 4. LIC scheme for pHLIC. BsaI digested pHLIC vector (a) is treated with T4 DNA polymerase in the presence of dGTP to create large overhangs (b). The PCR product for the insert (c) is treated with T4 DNA polymerase in the presence of dCTP to create complementary overhangs (d). Then (b) and (d) are mixed together leading to the formation of the desired clone by hybridization of the overhangs of the vector and insert. This product has nicks, at positions indicated by arrows in (e), which are resolved in the cell upon transformation. V. Durani et al. / Protein Expression and Purification 85 (2012) 9–17 15
protein sequence desired, with no ‘scar’ left behind from fusions designed for expression, solubilization and purification. Many fu- sion proteins and proteases are useful in different contexts, and so it is helpful to use systems that allow for simple tag switching. Vectors from the Gateway system, as well as many from structural genomics centers, assemble many of these useful features. The pHLIC system and similar vectors combine these features in a sim- ple plasmid that is easy to construct and modify for the research- er’s specific needs. Fig. 5. Coomassie Blue stained 12% SDS–PAGE gel showing protein purification and TEV cleavage of 6 His–cTIM and 6 His–MBP–cTIM. Lane 1, Marker; lane 2, soluble fraction of the cell lysate for 6 His-cTIM; lane 3, eluate; lane 4, TEV reaction; lane 5, flow-through from Ni–NTA column after TEV cleavage; lane 6, soluble fraction of Appendix A. Supplementary data the cell lysate for 6 His-MBP-cTIM; lane 7, eluate; lane 8, TEV cleavage reaction; lane 9, flow-through from Ni–NTA column after TEV cleavage. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2012.06.007. attach no sequence for a 6 His-Tag or TEV cleavage site to the 50 end of the insert. Also, pHLIC can be easily modified to make other References recombinant forms of proteins using LIC by changing the affinity tag to MBP, GST, Trx or other such fusion tags. For example, we made a [1] N.S. Berrow, D. Alderton, S. Sainsbury, J. Nettleship, R. Assenberg, N. Rahman, pHLIC-MBP vector (Fig. 3) and successfully used it for LIC and pro- D.I. Stuart, R.J. Owens, A versatile ligation-independent cloning method suitable for high-throughput expression screening applications, Nucleic Acids tein expression. 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