A modified Gateway cloning strategy for overexpressing tagged proteins in plants
© Dubin et al; licensee BioMed Central Ltd. 2008
Received: 19 November 2007
Accepted: 22 January 2008
Published: 22 January 2008
Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale.
Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations.
This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.
The flood of information resulting from the sequencing of a number of plant genomes and other recent developments have led to a huge increase in the number of studies carried out using plants transformed with DNA fragments to overexpress or modulate the expression of a gene of interest. While the process of Agrobacterium tumefaciens-mediated plant transformation has improved over the last 15 years or so , the binary vectors used in this method are large (typically 10 to 19 kb) and often contain only a few unique restriction sites that are useful for cloning. As cloning constructs into these vectors is often an awkward and time-consuming process, some investigators have shifted to the Gateway site-specific recombination (SSR) cloning system commercialized by Invitrogen (Carlsbad, CA). In this system the construct of interest is first cloned into a minimal Entry vector (pENTR) and is then transferred to a Destination vector (pDEST) by site-specific recombination. This is done using the Int and Xis recombination proteins that recognize the att L and att R sites in Entry and Destination vectors, respectively, and that recombine them to generate att B sites . The gene of interest is inserted into the Entry vector either using SSR with BP clonase or by cloning into the multiple cloning site (MCS) contained between the att L1 and att L2 sites by restriction enzyme and ligase (REaL) cloning. The entry clone can then be easily transferred to several Destination vectors by simple SSR reactions.
Gateway binary vectors have proved to be particularly useful for high-throughput plant transformation studies [3–6] where traditional REaL cloning methods would have been prohibitively time consuming. However, the use of these vectors has a number of drawbacks. For example, the presence of the att B recombination site between the tag and the gene of interest in the recombined Destination vectors results in a long (at least 8 and sometimes up to 20 amino acids) and often highly charged sequence which has the potential to result in the synthesis of a non-functional or insoluble protein-tag fusion and may also affect the expression levels of the protein of interest .
Here we report an alternative Gateway-based strategy that has been developed to create N- or C-terminally tagged protein overexpression constructs that lack att B-derived linker amino acids between the protein of interest and the tag. The att B sequences are avoided because the tag is contained within the Entry cassette instead of being in the Destination vector, and so the entire open-reading-frame (ORF) encoding the tagged protein of interest is generated within the Entry vector. In this system REaL cloning is first used to generate the fusion of the gene of interest with the tag, and SSR cloning is then used to transfer the ORF into a plant binary Destination vector containing a promoter and resistance cassette of choice, without concerns about reading frame. A total of twelve modified pENTR vectors have been generated, comprising six different tags for both N- and C-terminal fusions, and we show that these constructs can be used to study proteins of interest following introduction of these vectors into Arabidopsis or following transient Agrobacterium-mediated transformation and co-transformation in Nicotiana benthamiana leaves .
Oligonucleotides used to generate epitope tags. Restriction sites are in bold and linker sequences are underlined.
GATCC GCCGCTGCCGCC TTTTTCGAACTGCGGATGGCTCCACATG
GAAGGATCC TCCACTGCTAGC GGCGTAG
GAAGGATCC CGGGCTTCCGGA ATTCAAGTCCTCTTC
ACGGGATCC ACGCGGAACCAG ATC
GGCCGC AAGCGGCGGA TGGAGCCATCCGCAGTTCGAAAAATAGC
AGCGGCCGC AAGCGGAGGCCTGTCGCGA TAC
GAAGCGGCCGC ACAAGCT ATGGAGCAAAAGC
AGCGGCCGC AAGCGGAGGC ATGTCCCCTATACTAGG
Tests of functionality of the newly generated constructs.
REal – derived pENTR constructs
SSR – derived pDEST construct
Expression in N. benthamiana
Expression in Arabidopsis
Table 2 summarizes the extent to which the constructs described herein have been tested and shown to be functional, confirming the utility of the system we describe.
The hybrid cloning system reported here, based both on REaL and SSR approaches, enables the straightforward generation of protein-tag fusions of a protein of interest with a number of N- or C-terminal tags. The REaL-generated cassettes can then be recombined by SSR into vectors containing the appropriate promoter and resistance combinations suited to the particular experimental situation. The major advantage of our system with respect to conventional Gateway-based cloning systems for plant genes [5, 6], is that the att-derived linker sequences, which may adversely affect protein functionality, are avoided in the resultant protein fusions. A further advantage is that a sequence of interest can be fused to all of the six tags, either as N- or C-terminal fusions, using the same strategy because all the vectors use the same reading frame and because stop codons are present in all the modified pENTR vectors. Disadvantages with respect to conventional plant-based Gateway cloning are that all the pENTR vectors are designed for cloning with only Bam H I and Not I (additional restriction sites can be added, although this will obviously modify the linker sequence between the protein and the tag), and that a REaL cloning step is also required. Consequently it should be viewed as being complementary rather than as a substitute for the more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag.
We have demonstrated the utility of this vector system for both stable and transient Agrobacterium-mediated plant transformation, although the flexibility of the system allows a number of other possibilities to be explored. For example, these cassettes could also be used for direct (biolistic bombardment or poly-ethylene-glycol (PEG)-mediated) transformation of plant cells or protoplasts either using the binary vectors themselves or by recombining them into Gateway minimal overexpression vectors optimized for direct transient transformation, available from the Dept of Plant Systems Biology at the University of Ghent (Belgium) .
Alternatively, although strong viral promoters such as the 35S promoter have been widely and successfully used for expression of transgenes in plants, such high levels of expression can lead to unwanted side effects and lead to misleading results. In many cases it is therefore more desirable to express transgenes from their endogenous promoters. To this end, an improved "MultiSite Gateway" system has recently been developed  and incorporated into plant binary vectors . As its name suggests, the MultiSite Gateway system makes use of multiple unique att sequences (att 1–4) which allow two or three separate fragments to be simultaneously transferred to a Destination vector via the Gateway recombination reaction. In addition, the desired promoter fragment can be cloned into a separate Entry vector containing the att L4 and att R1 sites or may already be available as part of the SAP project (Systematic Analysis of Promoters) . The promoter fragment can then be recombined together with the existing tag-protein Entry cassette into a Gateway multi-site binary Destination vector (containing a transcriptional terminator and the att R2 and att R4 sites) to yield the protein fused to the desired N- or C-terminal tag under the control of its own promoter. Although not yet tested by us, these Destination vectors add a further level of versatility that can be exploited in combination with the Entry vectors described here.
While expression of recombinant proteins in planta has many advantages, it is currently not suitable for generating the large amounts of recombinant protein needed for in vitro investigations or antibody generation. To address this difficulty our tag-protein fusion cassettes can be recombined into vectors for in vitro expression or for overexpression in bacterial (pDEST14), yeast, insect (pDEST8) or mammalian cell systems (pDEST32). We have not directly tested this, but it should nonetheless be feasible to try several expression systems in parallel to determine which system is most suited for expression of the protein of interest because the only requirement is a Gateway Destination vector adapted to a particular expression system.
The modified pENTR vectors described here are highly complementary to the more conventional SSR-based Gateway systems in that they expand the range of possibilities for experimentation, e.g., when combined with different promoter and antibiotic resistance cassettes offered by Gateway Destination vectors. They also overcome some of the disadvantages of previously described Gateway systems, but because the hybrid system we report is somewhat more labor intensive than classical SSR cloning, we consider it best suited to low-medium throughput applications. For example, we believe it can be of great utility for studying protein complexes because it can allow studies of protein:protein interactions and subcellular localisation in a variety of systems and combinations, without the presence of bulky att-derived linker sequences that may interfere with protein activity.
Construction of modifed Gateway Entry cassettes
pENT-Stop was created by annealing the two oligonucleotides ENT-StopFw 5'-GATCCGGTACAGATTTCGGACATGCGGCCGCATAAGTAGCTGAC-3' and ENT-StopRev 5'-TCGAGTCAGCTACTTATGCGGCCGCATGTCCGAAATCTGTACCG-3' and ligating the resultant fragment into Bam H I – Xho I digested pENTR-1A vector (Invitrogen; Carlsbad, CA). This introduces a Not I site followed by a stop codon and removes the ccdB toxicity gene. This plasmid has been used for cloning the different epitope tags. The N-terminal STREP tag was created by annealing two oligonucleotides (see Table I) and ligating the resultant Sal I – Bam H I fragment into the pENT-Stop. The other N-terminal tags were PCR amplified with primers that introduce a 5' Sal I site and a 3' Bam H I site.
The C-terminal STREP tag was created by annealing two oligonucleotides (see Table I) and ligating the resultant Not I – Xho I fragment into the pENT-Stop. The other C-terminal tags were PCR amplified with primers that introduce a 5' Not I site and a 3' Xho I site. Oligonucleotides used for PCR amplifications are listed in Table I. The PCR products were ligated into the pCR2.1 vector using the Topo-TA cloning system (Invitrogen; Carlsbad, CA) and sequenced. The tags were then excised from pCR2.1 using the appropriate restriction enzymes and cloned into the pENT-Stop vector. Genes of interest were amplified by PCR from pre-existing plasmids with gene-specific primers introducing a 5' Bam H I site and a 3' Not I site.
Tag-ORF fusions were transferred from the newly generated Entry vectors to a plant binary Destination vector with the desired promoter and selective marker using the Gateway LR reaction and the Recombinase enzymes (Invitrogen; Carlsbad, CA). The identity of recombined vectors was verified by PCR screening and restriction enzyme analysis.
Plant material and Agrobacterium-mediated transformation
Arabidopsis thaliana (ecotype Columbia) and Nicotiana benthamiana were grown in soil in a growth room at 22°C under long day conditions (16/8 hrs light dark cycle). Arabidopsis plants were transformed by the floral dip method . Transgenic lines were selected with the appropriate resistance agent (50 μg/ml kanamycin, 10 μg/ml hygromycin B or 10 μg/ml Basta) and tested for expression. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens strain GV3101 harbouring recombined pDEST vectors. GV3101 cells were grown in 10 ml LB broth with antibiotic overnight at 28°C and then pelleted at 2,500 g for 10 min. Cells were then washed in 10 ml of infiltration medium (50 mM MES pH 5.7, 0.5% glucose, 2 mM NaH2PO4 and 200 μM acetosyringone), centrifuged again at 2,500 g for 5 min and resuspended in infiltration medium at an OD600 of 0.3. The Agrobacterium solution was infiltrated in the abaxial surface of the leaf using a 1 ml syringe without needle. After infiltration plants were placed under normal light conditions and leaves were collected after 36–48 hours for further analysis.
Protein extraction, western blotting and immunoprecipitation analysis
Rosette leaves (approximately 2 cm2 in total) were collected from individual 3 week old homozygous lines, ground in liquid nitrogen to fine powder and then resuspended in 50 μl 1 × Laemmli buffer (62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 1% (v/v) β-mercaptoethanol, 0.0005% (w/v) bromophenol blue).
Transformed tobacco leaves were ground in liquid nitrogen and then resuspended in Lysis buffer (50 mM Tris pH 7.8, 10 mM EDTA, 300 mM sucrose, 100 mM NaCl, 1 mM DTT, protease inhibitor cocktail (Roche)). Samples were then cleaned by successive centrifugations at 1,000, 2,000 and 10,000 g. For western blotting analysis samples were separated on SDS-PAGE and proteins were transferred onto PVDF membranes by semidry electroblotting. For detection of proteins the antibodies used were: mouse anti-HA (Covance) at 1:3,000 dilution, mouse anti-c-Myc (Upstate) at 1:2,000 dilution, rabbit anti-GFP (Abcam) at 1:10,000 dilution and mouse anti-Strep (IBA, Göttingen, Germany) at 1:5,000 dilution. As secondary antibodies anti-mouse and anti-rabbit (Promega) were used both at 1:10,000 dilution. Blots were developed using an ECL kit (GE Healthcare; Little Chalfont, UK).
For immunoprecipitation analysis 300 μg of total proteins were incubated overnight at 4°C with rabbit anti-Myc antibody (Abcam) at 1:400 dilution in lysis buffer and then incubated with protein-A Sepharose (Amersham) for 2 hours at 4°C. The immunoprecipitates were washed three times with lysis buffer, resolved on 10% polyacrylamide gel and subjected to western blotting analysis as described above.
For Strep-tagged protein purification 200 ng of plant tissue from 3-week old seedlings were ground to fine powder in liquid nitrogen and resuspended in 0.5 ml Ex-strep buffer (100 mM Tris-HCl pH 8.0, 5 mM EGTA, 5 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.5 mM AEBSF, 0.5% (v/v) Triton-X100, 100 μg/ml avidin, protease inhibitor cocktail (Roche)). The lysate was sonicated 3 times for 10 seconds (30% output on a Branson Sonifier 250 (Branson Ultrasonics; Danbury, CT)) on ice with a 50 seconds pause between pulses. The lysate was centrifuged at 21,000 g for 10 min at 4°C and the supernatant was then incubated with 30 μl of Streptactin Sepharose beads (IBA; Göttingen, Germany) for 1 hour at 4°C with gentle rotation. The beads were washed 4 times with 0.75 ml W-strep buffer (50 mM Tris-HCl pH 8.0, 2.5 mM EDTA, 150 mM NaCl, 2 mM DTT and 0.05% (v/v) Triton-X100). Bound proteins were then eluted in 60 μl of 1× Laemmli buffer at 95°C for 10 min and analysed by western blotting as described above.
The twelve pENTR plasmids described herein will be made available for noncommercial research purposes at Addgene . Accessions numbers are as follows: pE1n, (GenBank:EU334816). pE2n, (GenBank:EU334817). pE3n, (GenBank:EU334818). pE4n, (GenBank:EU334819). pE5n, (GenBank:EU334820). pE6n, (GenBank:EU334821). pE1c, (GenBank:EU334822). pE2c, (GenBank:EU334823). pE3c, (GenBank:EU334824). pE4c, (GenBank:EU334825). pE5c, (GenBank:EU334826). pE6c, (GenBank:EU334827).
This work was supported by FIRB (RBNE01CFKB_005 and RBLA0345SF_007) and the EU-SOL project (contract 016214 (Food)).
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