Protein Expression and Purification 85 (2012) 9–17 Contents lists available at SciVerse ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 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 (6ÂHis) [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