Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants
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- Shah, K.H., Almaghrabi, B. & Bohlmann, H. Plant Mol Biol Rep (2013) 31: 1529. doi:10.1007/s11105-013-0614-z
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Production of recombinant proteins in plants is of increasing importance for practical applications. However, the production of stable transformed transgenic plants is a lengthy procedure. Transient expression, on the other hand, can deliver recombinant proteins within a week, and many viral vectors have been constructed for that purpose. Each of them is reported to be highly efficient, robust and cost-effective. Here, a variety of expression vectors which were designed for transient and stable plant transformation, including pPZP3425, pPZP5025, pPZPTRBO, pJLTRBO, pEAQ-HT and pBY030-2R, was compared for the expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana by Agrobacterium-mediated transient expression. Our results show that pPZPTRBO, pJLTRBO and pEAQ-HT had comparable expression levels without co-infiltration of a RNA-silencing inhibitor. The other vectors, including the non-viral vectors pPZP5025 and pPZP3425, needed co-infiltration of the RNA-silencing inhibitor P19 to give good expression levels.
KeywordsTransient expressionRecombinant proteinAgroinfiltrationViral vectorpPZP vector family
Cauliflower mosaic virus
Days post infiltration
Tomato bushy stunt virus
Tobacco mosaic virus
Total soluble protein
The production of recombinant proteins is of major interest for basic and applied research. There are several well-established systems in use for this purpose, e.g. Escherichia coli, yeast, mammalian cells and plant cells. Among these systems, plant cell factories have many advantages. Large quantities of plants can be grown easily at low cost, and when compared to mammalian cell cultures, there is no risk of contamination with retroviruses. Plant cells have an endomembrane system and secretory pathway similar to mammalian cells which allows post-translational modification of recombinant proteins (Pogue et al. 2010; Popescu et al. 2007). However, plants have the limitation that it usually takes a long time to produce stable transformed transgenic plants as compared to other expression systems, but this problem can be solved by transient expression methods. Transient expression in plant leaves enables the small- to medium-scale production of recombinant proteins at a timescale that is comparable to other prokaryotic and eukaryotic expression systems. The preferred host plant for transient expression is Nicotiana benthamiana (Conley et al. 2011; Goodin et al. 2008; Sheludko et al. 2007). It is very susceptible to plant viruses, particularly due to a defective form of a RNA-dependent RNA polymerase found in its genome (Yang et al. 2004). This viral susceptibility allows external viral replicases to replicate the delivered genes. The combination of ease of infection with bacterial and viral components and a long history of experimental use have made N. benthamiana a common laboratory host for the expression of recombinant proteins. This expression platform is based on leaves, and it significantly reduces the potential for gene leakage into the environment through pollen or seed dispersal. Furthermore, N. benthamiana is a non-food, non-feed crop, which minimizes the risk of recombinant proteins entering the food chain (Rymerson et al. 2002; Twyman et al. 2003). It has, for instance, been used to produce the haemagglutinin antigen from avian influenza virus A (Kanagarajan et al. 2012) and for the production of monoclonal antibodies (Castilho et al. 2011). Furthermore, it should be mentioned that transient expression, especially in N. benthamiana, is also intensively used to analyse the function of proteins (for instance, Hashimoto et al. 2012).
Transient expression of foreign proteins in plants has been mediated via simple tissue infiltration using Agrobacterium tumefaciens (Kapila et al. 1997) or by vectors based on the genomes of a variety of plant viruses. Furthermore, viral vectors have also been integrated into binary vectors, such that the replicating vector is delivered and released into the host tissues by infiltration with recombinant agrobacteria (Gleba et al. 2005; Van der Hoorn et al. 2000; Vaquero et al. 1999). The simple integrative plant expression vector approach has the disadvantage that expression levels are usually low and decline after a few days. However, ectopic expression of RNA-silencing suppressor proteins such as P19 from tomato bushy stunt virus or HC-Pro from tobacco etch virus results in significantly increased expression of the desired proteins (Voinnet et al. 2003). The virus-based vectors offer advantages over simple T-DNA vectors by exploiting the cytoplasmic replication cycle of the virus. A large number of viral vectors have been designed which can be classified as gene substitution vectors, gene insertion vectors, modular or deconstructed vector systems and peptide display vectors (Lico et al. 2008). The first generation virus vectors were gene substitution vectors or gene insertion vectors that were inoculated as virus particles or viral RNA and used virus-encoded cell-to-cell and systemic movement activities to infect host plants (Gleba et al. 2007; Scholthof 2004). In substitution vectors, a viral gene, usually the coat protein gene, is replaced by a gene encoding the protein to be expressed. The loss of the coat protein usually limits the cell-to-cell and systemic movement in the plant, thus leading to low expression levels if the inoculation depends on virus particles or viral RNA. This problem can be solved if the viral vector is introduced by agroinfiltration into the majority of plant cells, thereby bypassing the need for cell-to-cell and systemic movement. The gene insertion vectors are complete (functional) viruses with an additional coding sequence which is transcribed from a viral promoter (Lico et al. 2008). Since the packaging capacity of most virus envelops is close to the size of the wild-type genome, these vectors are not useful for the production of large proteins. In modular vector systems, different parts of the virus vector are placed on separate binary vectors. As these viral vectors do not show systemic movement in inoculated plants, agroinfiltration is used to transfer the viral constructs to the tissues of host plants to launch the infection process. The best known example of these vectors has been developed by the company Icon Genetics (Halle, Germany). The system has, for instance, been used to produce different antibodies (Giritch et al. 2006; Gils et al. 2005). It is reported that it gives expression levels of up to 5 g recombinant proteins per kg fresh weight of infiltrated tissues (Gleba et al. 2005). In deconstructed vector systems, some unnecessary parts of the viral genome are removed such as deletion of coat protein gene. The vector pEAQ-HT was reported as an efficient transient expression vector which is based on a deleted version of the Cowpea mosaic virus that permits high-level and rapid production of proteins (0.2–0.5 g per kg fresh weight (FW) of infiltrated tissues) without viral replication (Sainsbury et al. 2008, 2009). The vector pBY030-2R is a bean yellow dwarf virus single-stranded DNA-based vector (Huang et al. 2009; Mor et al. 2003) which harbours a long intergenic region (LIR), a short intergenic region (SIR) and two open reading frames (C1 and C2), encoding replication-associated proteins Rep and RepA, respectively, within the T-DNA (Liu et al. 1997). This vector was reported to produce 0.5 mg of recombinant proteins per g FW of the infiltrated tissues (Huang et al. 2009).
Another example of the degenerated viral vectors is the pJLTRBO (tobacco mosaic virus (TMV) RNA-based overexpression) vector which is an improved agroinfection-compatible TMV vector that lacks the TMV coat protein gene and is therefore unable to form virus particles during the infection process (Lindbo 2007). It was reported to have high agroinfection efficiency and a high recombinant protein expression level of 3–5 mg of recombinant proteins per g FW of infiltrated tissues.
In this study, a variety of viral and binary plant transformation vectors were compared for their ability to produce recombinant proteins in transient expression in N. benthamiana. In addition to the above-mentioned viral vectors, the binary vectors pPZP3425 (Szakasits et al. 2007) and pPZP5025 (Ali et al. 2012) were included.
Materials and Methods
The GFP coding sequence was cut out as a NcoI-BamHI fragment from an intermediate cloning vector (pUC-HB3-GFP) and used to replace the GUS coding sequence in pPZP3425 to produce pPZP3425-GFP, and it was inserted as a NotI-PacI fragment into pPZPTRBO and pJLTRBO to produce pPZPTRBO-GFP and pJLTRBO-GFP, respectively (Fig. 1). The intron containing GUS sequence from pPZP3425 was cloned as a NcoI-BamHI fragment into pUC-HB3 (Bohlmann, unpublished) from which it was cut out as a NotI-PacI fragment and cloned into pPZPTRBO and pJLTRBO to produce pPZPTRBO-GUS and pJLTRBO-GUS, respectively. The vectors pBY030-2R-GUS and pEAQ-HT-GUS were produced as follows: the GUS fragment was amplified from pUCHB3-GUS with the oligos P-XhoI for (5′-CAGCTCGAGCATGGTCCGTCCTGTAGAAACC-3′) and P-SacI rev (5′-AGTGAGCTCCCCACCGAGGCTGTAG-3′), digested with XhoI and SacI and cloned into pBY030-2R digested with XhoI and SacI. Similarly, the GUS fragment was also amplified from pUC-HB3-GUS with the oligos P-AgeI for (5′-CTTAACCGGTCCATGGTCCGTCCT-3′) and P-XhoI rev (5′-GACTCTCGAGCGGCCGCGGAT-3′), digested with AgeI and XhoI and cloned into pEAQ-HT digested with AgeI and XhoI. The vector pBin61-P19 (Voinnet et al. 2003) was used to express the P19 RNA-silencing suppressor.
N. benthamiana plants were grown in a growth chamber at 25 ± 2 °C temperature, 16-h photoperiod and ~65 % humidity. Binary plasmids were transformed into agrobacteria by the freeze-thaw method (Holsters et al. 1978). In most experiments, the strain GV3101 (Koncz and Schell 1986) was used. In addition, some experiments were performed with LBA4404 (Hoekema et al. 1983). Agrobacteria were grown overnight in a YEB liquid medium with appropriate antibiotics (35 μg/ml rifampicin for both agrobacteria and 25 μg/ml gentamicin for GV3101, 200 μg/ml spectinomycin for pPZP5025 and pPZPTRBO and 50 μg/ml kanamycin for the rest of the plasmids) to an optical density (OD)600 of 0.8 in an incubator/shaker at 28 °C. Bacteria were harvested by centrifugation at 5,000 rpm for 6 min in a table top centrifuge at room temperature and suspended in an infiltration medium (10 mM MES, pH 5.6; 10 mM MgCl2 and 100 μM acetosyringone) to obtain bacterial suspensions of the required OD600 (0.4 and 1.0). After incubation for 2 h, agrobacteria were infiltrated into the abaxial side of leaves by using a 1-ml syringe without needle. Infiltrated plants were kept under the same growth conditions as mentioned above. For co-infiltration of RNA-silencing inhibitor P19, an equal volume of a bacterial suspension harbouring pBin61-P19 (Voinnet et al. 2003) was added prior to infiltration.
In one experiment, two leaves (leaf numbers 3 and 4) were infiltrated. Leaf samples were taken after 6 days unless otherwise indicated. One sample consisted of 20–30 leaf disks of 10 mm diameter that were cut out from 1 leaf and immediately frozen in liquid nitrogen for later analysis. All samples were analysed in triplicate. Three independent experiments were performed.
GUS Staining and GFP Imaging
GUS staining of N. benthamiana leaves was done by overnight incubation in X-Gluc solution (50 mM sodium phosphate buffer, pH 7.0; 10 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0; 0.1 % (v/v) Triton X-100 and 0.5 mg/ml X-Gluc) at 37 °C (Jefferson et al. 1987) followed by several washes with 70 % ethanol to clear the chlorophyll from leaf tissues. The GFP fluorescence imaging was done by illuminating the infiltrated leaves under UV light with a handheld UV lamp. Images were taken with the Nikon digital camera.
Total soluble proteins (TSP) were extracted from 200 to 400 mg FW of the infiltrated leaf tissues. Leaf disks (20–30) were frozen in the Eppendorf tubes in liquid nitrogen, ground in a mixer mill (Retsch MM 400) and immediately dissolved in 1 ml of extraction buffer consisting of 50 mM Tris–HCl, pH 7.25; 150 mM NaCl; 2 mM EDTA; 0.1 % (v/v) Triton X-100 for GFP quantification (Sainsbury 2009) and 50 mM NaH2PO4, pH 7.0; 10 mM EDTA; 0.1 % (v/v) Triton X-100; 0.1 % sodium lauryl sarcosine and 10 mM β-mercaptoethanol for GUS quantification (Jefferson et al. 1987). The extracts were centrifuged (13,000 rpm at 4 °C) for 10 min. The supernatant was then centrifuged again for 10 min to clarify it from leaf tissues debris. The 1:10 dilutions of the extracts were made for quantification.
Proteins in the extracts were quantified using the Pierce® BCA Protein Assay Kit. Twenty-five microlitres of a 1:10 dilution of the extract and 200 μl of the working reagent were added to a transparent 96-well microtiter plate (Greiner). After incubation for 30 min, OD600 was measured at 562 nm with the FLUOstar Omega micro-plate reader (BMG Labtech). A standard curve was produced by using BSA standards in the range of 25–2,000 μg/ml.
Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis
TSP samples were resolved on tricine–sodium dodecyl sulphate (SDS) polyacrylamide gels (16 % separating and 4 % stacking gel) according to Schägger (2006).
GFP Fluorescence Measurement
GFP relative fluorescence produced by different vectors was measured at 400 nm excitation and 510 nm emission with the FLUOstar Omega micro-plate reader (BMG Labtech) using 100 μl of a 1:10 dilution of extracts (prepared as described in ‘Protein Extraction’) in black 96-well microtiter plates (Greiner).
GUS Activity Assay
The GUS activity was measured according to Jefferson et al. (1987). The 3 mM solution (50 μl) of the GUS substrate 4-methylumbelliferyl-β-d-glucuronide (4-MUG) was added to 100 μl of protein extract (prepared as described in ‘Protein Extraction’) in a black 96-well microtiter plate (Greiner) to obtain a final concentration of 1 mM 4-MUG in 150 μl of reaction volume. The reaction was carried out by incubation at 37 °C for 5 min, and then, 50 μl of 0.5 M Na2CO3 was used to stop the reaction. Fluorescence was measured at 355 nm excitation and 460 nm emission in the FLUOstar Omega micro-plate reader (BMG Labtech) using 4-MU standards (10 mM stock in ethanol and diluted in GUS extraction buffer) in the range of 1–100 μM.
The goal of this work was to compare the efficiency of different viral and binary vectors for transient expression in N. benthamiana leaves using the GUS and GFP reporters. They have a different size (GUS has a monomeric molecular weight of 68 kDa, while that of GFP is of 27 kDa), and both can be measured and quantified using either the enzymatic activity of GUS or the fluorescence of GFP as described in experimental procedures. The vectors included the viral vectors pPZPTRBO (this study), pJLTRBO (Lindbo 2007), pEAQ-HT (Sainsbury et al. 2009) and pBY030-2R (Huang et al. 2009) and the binary vectors pPZP3425 (Szakasits et al. 2007) and pPZP5025 (Ali et al. 2012) (Fig. 1). The vector pJLTRBO was reported to be a high efficiency vector; however, it has a low copy number. To remove this limitation, the TMV cassette was transferred from its T-DNA to our previously designed high-copy-number vector pPZP500, thus generating pPZPTRBO (Figs. 1 and S1). The vector pPZP3425 contains a kanamycin-selectable marker and the GUS coding sequence (interrupted by a single intron) driven by a doubled CaMV 35S promoter with a TMV omega translational enhancer. The vector pPZP5025 is a streamlined version of pPZP200 (Hajdukiewicz et al. 1994) without a plant-selectable marker, but containing the CaMV expression cassette from pPZP3425.
Transient expression in plants has become a widely used technique in basic research and for the production of proteins to be used in applied research. Since the first report of Agrobacterium-mediated transient expression of a GUS binary vector in bean leaves (Kapila et al. 1997), interest has shifted to viral vectors which are delivered by agroinfiltration and which can replicate in the infiltrated plants. Here, we have compared the efficiency of several viral vectors together with simple binary vectors. The magnifection vectors (Gleba et al. 2005, 2007; Marillonnet et al. 2005) were excluded because these are not freely available. This TMV system consists of different pro-vectors which are delivered by agrobacteria to the plant cell where they recombine to produce the final expression vector.
As expected, the binary vectors pPZP3425 and pPZP5025 performed poorly without the co-infiltration of an RNAi inhibitor (Fig. 5). For GFP, expression without P19 reached only 10 % of the expression level of pPZP3425 with P19, while for GUS, both reached 30 % without P19 as compared to expression with P19. It has been shown before that RNAi leads to a downregulation of protein expression in transiently transformed plant leaves after a few days and that this can be suppressed by co-infiltration of RNAi inhibitors (Voinnet et al. 2003). Lombardi et al. (2009) compared the three different RNAi inhibitors (P25 of potato virus X, P19 of either artichoke mottled crinkle virus or tomato bushy stunt virus) for their effect on transient expression of the Nef protein and found that the P19 protein of artichoke mottled crinkle virus gave the highest expression level. Here, the P19 inhibitor from pBIN61-P19 (Voinnet et al. 2003) was used which is also included in the pEAQ-HT vector T-DNA (Sainsbury et al. 2009).
In contrast to the binary vectors, quantification of GUS and GFP expression (Figs. 3 and 5) showed that TRBO and the pEAQ-HT vector gave similar results with and without co-infiltration of P19. pEAQ-HT has an expression cassette for P19 integrated within the T-DNA, while TRBO does not contain any RNAi inhibitor. It has been reported before that TRBO expression does not need an RNAi inhibitor, and it was speculated that this was due to the deletion of the TMV coat protein open reading frame (ORF) and subgenomic promoter (Lindbo 2007). However, there was a small but significant effect of P19 co-infiltration on both GFP and GUS expressions in the case of TRBO. In the case of pPZPTRBO, this effect was only seen with GUS expression, not with GFP. The reason for this difference might be related to the higher copy number of pPZPTRBO. P19 had no effect on pEAQ-HT expression in GV3101 but did result in an increased expression in the LBA4404 strain.
The pBY030-2R vector gave much lower expression levels, both with GFP and GUS, even with co-infiltration of the P19 inhibitor as compared to TRBO and pEAQ-HT. The expression level was approximately 45 % for GFP and 20 % for GUS if used with P19 and approximately 15 and 10 %, respectively, without P19. It also performed less well than the pPZP vectors co-infiltrated with P19 (Figs. 3 and 5). An explanation why this vector gave the lowest expression levels in our experiments might be that it was designed to be used for the production of virion particles for vaccination (Huang et al. 2009). Since it contains the coat protein, this might be reducing the efficiency in the transient expression of other proteins.
The binary vectors pPZP3425 and pPZP5025 were included for comparison with the viral vectors and pEAQ-HT. However, it turned out that their performance in our comparison was similar to that of TRBO and pEAQ-HT if used together with P19. With the GFP marker, pPZP3425 gave 75 % of the TRBO and pEAQ-HT expression level (Fig. 3), while the GUS protein was expressed at comparable levels by pPZP3425 and pPZP5025 (Fig. 5). This is especially interesting, because these vectors could also be used for stable transformation of plants. Thus, different constructs could, for instance, be tested by transient expression in N. benthamiana, and the best ones could then directly be used for stable transformation. This would also be possible with the pEAQ-HT vector, but in that case, the P19 cassette which is included in the T-DNA would also be integrated into the plant genome, leading to a constitutive expression of P19 in the transgenic plants which is certainly not intended.
The TMV RNA-based vector pJLTRBO and its derivative pPZPTRBO show good potential to produce recombinant proteins in plants without using the RNA-silencing inhibitor P19. The advantage of pPZPTRBO is that it is a high-copy-number plasmid, also in E. coli, making cloning easier. Similar expression levels were provided by the pEAQ-HT vector which has an integrated P19 expression cassette. Binary pPZP vectors gave an expression level which was only 10–20 % lower than TRBO or pEAQ-HT and have the advantage that they can also be directly used for the production of transgenic plants.
The authors thank Sabine Daxböck-Horvath and Martina Niese for their technical assistance; Dr. Shahid Masood Siddique, Amjad Abbas, Muhammad Amjad Ali and Stephan Plattner for their assistance in lab work; and Dr. Mason, Dr. Lindbo, Dr. Lomonossoff and Dr. Baulcombe for providing the vectors used in this study. This research was supported by a grant P21896-B16 from the Austrian Science Fund (FWF). Kausar Hussain Shah and Bachar Almaghrabi were supported by Higher Education Commission (HEC) of Pakistan and OEAD, respectively.
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