Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in ∼6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.
Plants have emerged as a viable alternative to microbial fermentation and mammalian cell culture for industrial production of biopharmaceuticals; they offer rapid scale-up potential, lower production costs and are free of human pathogens and toxins. For example, it is possible to produce up to 360 million doses of an anthrax vaccine in one acre of tobacco1. The combination of maternal inheritance of transgenic chloroplast genomes in most crops2,3,4 with cytoplasmic male sterility5 minimizes the risk of cross-pollination with non-genetically modified crops or their relatives in the wild. Indeed, chloroplast transgenic plants producing human therapeutic proteins have been grown and tested in the field6.
When stably integrated into the chloroplast genome, transgenes express large amounts of foreign proteins7. This is due to the presence of up to 10,000 copies of the chloroplast genome in each plant cell8,9. Transgenes integrate at a precise location in the genome by homologous recombination, which is mediated by flanking chloroplast DNA sequences present in the chloroplast vector; this eliminates position effects frequently observed in nuclear transgenic lines9. Although techniques have been developed for targeted integration into the nuclear genome10, these have not yet been used to develop useful traits. Other advantages of transplastomic plants over nuclear transgenic plants include lack of transgene silencing, despite 169-fold higher levels of transgene transcript than in nuclear transgenic plants11,12, and foreign protein levels up to 46% (wt/wt) of total leaf protein7. Multivalent vaccines can be engineered in a single transformation step because polycistrons are translated without processing into monocistrons; several heterologous operons have been expressed in transgenic chloroplasts13.
It is important that foreign proteins expressed in chloroplasts are post-translationally modified and processed like native proteins. Several human therapeutic proteins have been successfully expressed in transgenic chloroplasts; appropriate functional assays on a range of foreign proteins expressed in chloroplasts have been shown to form disulfide bonds and to be folded correctly6,14,15,16. Similarly, several chloroplast-derived vaccine antigens have been shown to form the correct disulfide bonds and assemble as heterodimers, pentamers (or other suitable configurations)17,18,19 or to contain the appropriate lipid modifications20. These observations confirm that the chloroplasts have the machinery to fold complex proteins in the chloroplast stroma and form disulfide bonds or other post-translational modifications required for their functionality. Expression of glycoproteins with glycosylation identical to that occurring in humans has been a major challenge in nuclear transformation of plant cells, when targeting such proteins to the endoplasmic reticulum. However, glycosylation does not occur in chloroplasts, which provides a unique opportunity to express therapeutic proteins free of glycosylation.
The tobacco plant is often chosen because of its large biomass, yielding ∼170 metric tons of biomass per hectare21,22. In addition, it is easy to engineer the tobacco chloroplast genome and regenerate transgenic lines within a few months. Each transgenic plant is capable of producing 1 million seeds and, therefore, it is possible to scale-up from a single transgenic plant to 100 acres within 1 year. Tobacco is a nonfood and nonfeed crop and is self-pollinated, which minimizes transgene escape. Most important, the expression of foreign protein in leaves facilitates their harvest before appearance of any reproductive structures, offering total biological containment of transgenes.
Design of chloroplast vectors
A chloroplast transformation vector contains several critical elements; a schematic diagram of the standard vector design used in our lab is shown in Figure 1. Two distinct components are required to construct the final transformation vector: a vector containing the flanking (targeting) sequence and the sequences required for efficient transgene expression (expression cassette). Guidance on how to construct these components is provided in the CONSTRUCTING THE TARGETING VECTOR section. While this overall design is effective across most species, the specific sequences should be derived from the chloroplast genome being targeted to ensure that the transgene is integrated and expressed with maximum efficiency. Fully annotated crop plastid genome sequences (Table 1) can be found at http://www.bch.umontreal.ca/ogmp/projects/other/cp_list.html, http://www.ncbi.nlm.nih.gov/genomes/static/euk_o.html and http://chloroplast.cbio.psu.edu/cgi-bin/organism.cgi. GenBank (http://www.ncbi.nlm.nih.gov) is also a very useful source of information for designing and constructing plastid transformation vectors. For the purposes of the procedure detailed in this protocol, it is assumed that the required sequence components have been previously assembled; the starting point is the cloning of the expression cassette into the targeting vector to produce the final transformation vector.
Constructing the targeting vector
This vector contains sequence from the chloroplast genome that is homologous to the desired site of integration; it facilitates site-specific recombination and defines the integration site of the transgene. Thus, the sequence must be specific to the plastid genome being targeted and can be PCR-amplified from the relevant genome using suitably designed primers. Attempts have been made to transform potato23 and tomato24 plastid genomes using targeting sequences from tobacco but the efficiency of transformation was significantly lower than that observed in tobacco, although some species are inherently more challenging to transform than others. Targeting sequences are generally 1 kb in size and are located on either side of the expression cassette, which is inserted using a suitable restriction enzyme in the spacer region of the targeting sequence. Transgenes may be integrated into three types of spacer regions. Transcriptionally silent spacer regions are found at sites where chloroplast genes are located on opposite DNA strands. Read-through spacer regions are found between chloroplast genes located on the same strand and where each gene has its own promoter. Transcriptionally active spacer regions are found in chloroplast operons in which a single promoter drives transcription of several genes. The region most commonly used is the transcriptionally active spacer region between the trnI and trnA genes. This region is located within the rRNA operon, where the 16S rRNA promoter drives transcription of six genes and each spacer region within this operon is transcriptionally active (Fig. 1). The trnI gene intron also contains a chloroplast origin of replication, which might facilitate replication of foreign vectors within chloroplasts and enhance the probability of transgene integration25,26. Transcriptionally active spacer regions also offer the unique advantage that transgenes lacking promoters or 5′- or 3′-untranslated regions (UTRs) can be inserted and expressed. However, other spacer regions (transcriptionally silent or read-through) may also be used; some examples of integration sites used in a range of crops are listed in Table 2. A schematic representation of how new targeting vectors are constructed in our lab is provided in Figures 2 and 3.
Constructing the expression cassette
The expression cassette includes a chloroplast operon promoter, ribosome-binding sequences, a selectable marker gene (e.g., antibiotic resistance), a 3′-UTR, gene promoter, a 5′-UTR, the gene of interest (GOI) and its 3′ UTR. The necessary regulatory sequences are PCR-amplified from the genome of interest using primers containing suitable restriction sites, sequenced to identify any errors introduced by PCR and assembled by consecutive rounds of restriction digestion and ligation; the GOI is inserted between the 5′-UTR and the 3′-UTR (Fig. 3a). For speed and ease of future cloning, the regulatory cassette (before insertion of the GOI) can be cloned into a suitable vector and maintained. The entire expression cassette (including the GOI) can be inserted into the appropriate restriction site within the targeting sequence to produce the final transformation vector (Fig. 3b). In the case of the trnI–trnA spacer region, a unique PvuII site is used; this PvuII site is present in most of the sequenced chloroplast genomes.
Gene expression in plastids is regulated at both transcriptional and post-transcriptional levels. Protein levels in chloroplasts depend on mRNA abundance, which is determined by promoter strength and mRNA stability. However, high mRNA levels do not necessarily result in high levels of protein accumulation, as post-transcriptional processes ultimately determine levels of foreign proteins within transgenic chloroplasts. Usually, the strong plastid rRNA operon promoter (Prrn) is used to drive transgene expression. In tobacco, the Prrn promoter has binding sites for both the nuclear- and plastid-encoded RNA polymerases27. The mRNA is stabilized by a 3′ UTR, usually derived from the psbA, rbcL or rps16 genes; using an alternative 3′ UTR will only marginally improve protein accumulation, as it has been shown that transgenic lines with and without 3′ UTRs accumulate the same level of foreign proteins in transgenic chloroplasts28. Therefore, the major focus is on the 5′ UTR to enhance foreign protein accumulation. The most commonly used 5′ regulatory sequences are derived from the psbA and rbcL genes. The use of 5′ psbA UTR is not advised when high-level expression of foreign proteins has deleterious phenotypic effects on transplastomic plants29. In this case, a compromise on the level of gene expression must be reached or an inducible expression system should be used. A lac repressor-based inducible expression system has been reported, but transgene repression in the uninduced state is incomplete30.
The GOI can be expressed with any tag in chloroplasts to facilitate purification. To date, only N-terminal His tags (with protease cleavage sites for removal of tags) have been used to purify chloroplast-derived vaccine antigens or biopharmaceuticals1,6. Other approaches used for purification of proteins expressed from chloroplast vectors include fusion of the foreign protein to a protein-based polymer and utilization of the inverse temperature transition property of this polymer for purification31.
Methods of plastome transformation and plant regeneration
Plastome (plastid genome) transformation was initially thought to be almost impossible due to the physical barrier imposed by the double membrane of the chloroplast envelope. Moreover, there are no viruses or bacteria known to infect chloroplasts that could be used as vectors for gene transfer. Therefore, early attempts to transform plastids involved the introduction of foreign DNA into isolated intact chloroplasts, which could then be re-introduced into protoplasts to obtain transgenic plants32.
The development of gene gun and biolistic technology has enabled the delivery of foreign DNA directly into living cells. Particle bombardment, in which small gold or tungsten particles are coated with DNA and shot into young plant cells (leaf or callus tissue), is now the most widely used and effective method for transforming plastids. The first successful chloroplast transformation using this method was reported in Chlamydomonas by complementation of a native gene fragment in a deletion mutant33.
An alternative approach is PEG-mediated transformation34,35. After protoplast isolation, PEG incubation with foreign DNA facilitates entry of DNA through cell and chloroplast membranes. However, this technique has a lower success rate than biolistic transformation and is not often reproducible.
After transformation, transplastomic plants are regenerated either by direct organogenesis or somatic embryogenesis. Regeneration by direct organogenesis is relatively straightforward and was first achieved in tobacco36, followed by several other crops (Table 2). Regeneration in the first, second or third round of selection via direct organogenesis follows the same time frame in lettuce and tobacco16. However, regeneration of homoplasmic transplastomic plants through somatic embryogenesis continues to remain a major challenge because homoplasmy must be achieved before initiation of differentiation in embryogenic cells.
The first stable plastid transformation of nongreen embryogenic cell cultures and regeneration via somatic embryogenesis was established in carrot37 and then extended to soybean, cotton and rice (Table 2). Foreign gene expression has been observed in nongreen tissues such as microtuber23 (potato), fruit24 (tomato) and root37 (carrot). Nongreen plastids (amyloplasts and chromoplasts) are generally known to be less active in gene expression than chloroplasts in photosynthetically active leaves. However, there are currently no well-characterized chloroplast regulatory elements that express in nongreen plastids that can be used to achieve high levels of transgene expression. Recently, the levels of transcription, mRNA accumulation and formation of polysomes were analyzed in amyloplasts of potato tuber. The transcriptional activity was reduced in amyloplasts when compared with chloroplasts, whereas there were small differences in the specific transcript levels. Also, the polysomes associated with the specific transcripts could not be detected38. Therefore, further study of gene expression in nongreen plastids is required.
Applications of protein expression in chloroplasts
Engineering plants with improved agronomic traits Several foreign genes have been used to engineer agronomic traits via the chloroplast genome, including insect and pathogen resistance, drought and salt tolerance, phytoremediation and cytoplasmic male sterility (Table 3). Insecticidal proteins expressed via the chloroplast genome showed the highest levels of expression reported in transgenic plants (up to 46.1% wt/wt of total leaf protein), formed cuboidal crystals7 and showed 100% mortality when transplastomic leaves were fed to susceptible or resistant insects (up to 40,000-fold resistance was created by breeding insects resistant to the insecticidal protein). Similarly, transplastomic plants conferred the highest levels of protection against biotic or abiotic stress reported in the literature.
Expression of therapeutic proteins and vaccines Several vaccine antigens have been expressed in tobacco chloroplasts, including the cholera toxin B (CTB) subunit of Vibrio cholerae17, the anthrax protective antigen1,39, the C terminus of Clostridium tetani40,41, the 2L21 peptide from the canine parvovirus42,43 and the LecA from Entamoeba histolytica18 (Table 4). Tobacco chloroplasts have also been used for the production of valuable therapeutic proteins, such as human somatotropin14, human serum albumin44, a broad-spectrum topical agent, systemic antibiotic, wound-healing stimulant, and a potential anticancer agent45, type I interferons6,15 and human proinsulin16 (Table 5). Most of these chloroplast-derived proteins have proven to be functional by appropriate tests in vitro or in animal studies4,46.
Soybean, lettuce and carrot plastid transformation have been accomplished using species-specific vectors37,47,48. A human therapeutic protein has already been stably expressed in lettuce chloroplasts16. Oral administration of transplastomic leaves expressing a CTB–proinsulin fusion protein conferred protection against the development of insulitis in nonobese diabetic mice16. These studies open the door for oral therapeutic delivery through plant cells, significantly reducing the cost of purification, processing, cold storage, transportation and sterile delivery. Engineering of the chloroplast genome for expression of vaccine antigens or biopharmaceuticals has ushered in a new era in biotechnology46,49,50.
Expression of industrially valuable biomaterials In addition to therapeutic proteins and vaccines, plastid genetic engineering offers an ideal opportunity for the cost-effective production of industrially valuable biomaterials (Table 6). The successful engineering of several pathways and enzymes has resulted in the effective production of many important biomaterials. For example the xynA gene, which encodes xylanase, was transformed into the tobacco chloroplast genome; the resulting protein was as biologically active as the bacterial-derived enzyme and retained its substrate specificity51. Liquid crystal polymer can also be produced to very high levels—up to 25% wt/wt of plant dry weight52.
Here, we provide a protocol to construct transformation vectors, transform chloroplasts, regenerate transgenic plants using tissue culture, select putative transformants, characterize transgenic plants, extract total proteins, evaluate transgene expression, quantitate foreign proteins and purify foreign proteins using tags. These protocols have been used, and their efficacy and reproducibility have been evaluated, and published in peer-reviewed scientific literature, as mentioned earlier.
Chloroplast targeting vector, containing flanking sequences specific to the genome to be targeted (Fig. 2)
Chloroplast expression cassette, containing the necessary genome-specific regulatory sequences and the GOI (Fig. 3a), each fragment is verified by DNA sequencing. All published chloroplast vectors from the Daniell Laboratory listed in Tables 2, 3, 4, 5, 6 are available to investigators involved in basic research upon signing of a Material Transfer Agreement
Young, green, healthy tobacco leaves
DNeasy Plant Mini Kit (Qiagen, cat. no. 69106)
dNTPs (Invitrogen, cat. no. 10297018)
Pfu DNA polymerase (Promega, cat. no. M7748)
Set of primers for evaluation of site specific transgene integration (Table 7)
Sterile molecular biology-grade water (Eppendorf, cat. no. 955155033)
PCR cloning vector (Promega pGEM-T Easy, cat. no. A1360)
Restriction endonucleases (New England Biolabs)
T4 DNA polymerase (Invitrogen, cat. no. 18005017)
T4 DNA ligase (Invitrogen, cat. no. 15224017)
Calf intestinal alkaline phosphatase (CIAP; Promega, cat. no. M1821)
QIAquick Gel Extraction Kit (Qiagen, cat. no. 28704)
QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)
QIAprep Spin Miniprep Kit (Qiagen, cat. no. 27104)
Antibiotics (see REAGENT SETUP)
Plasmid Midiprep Kit (Bio-Rad, cat. no. 732-6120)
Ethanol 190 proof for molecular biology (Sigma, cat. no. E7148-1GA)
Murashige and Skoog salts 4.33 g for 1 l (MS; Caisson, cat. no. MSP 001)
6-Benzylamino purine (BAP; Sigma, cat. no. B3408)
1-Naphthaleneacetic acid (NAA; Sigma, cat. no. N0640)
Sucrose (Sigma, cat. no. S0389-5KG)
Phytoblend (Caisson, cat. no. PTC 001)
Soil (Miracle Gro—Pot-mix)
Water-soluble all purpose plant food (Miracle Gro)
1 M phenyl methyl sulfonyl fluoride (PMSF; Sigma; cat. no. P7626; see REAGENT SETUP)
Plant extraction buffer (PEB; see REAGENT SETUP)
Coating buffer (CB; see REAGENT SETUP)
Tween-20 (Sigma, cat. no. P1379)
Nonfat powdered milk (e.g., Carnation)
3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for ELISA (American Qualex, cat. no. K3620)
Electrochemiluminescent (ECL) substrate for western blot (Pierce, cat. no. 32109)
Sulfuric acid (dilute to 2M; Sigma, cat. no. 320501)
BSA (Sigma, cat. no. A7030)
Bradford reagent (Bio-Rad, cat. no. 500-0006)
Sample buffer (SDS reducing buffer; see REAGENT SETUP)
10 × Electrode buffer (EB; see REAGENT SETUP)
Transfer buffer (see REAGENT SETUP)
MSO (see REAGENT SETUP)
RMOP (see REAGENT SETUP)
Binding buffer (see REAGENT SETUP)
Wash buffer (see REAGENT SETUP)
Elution buffer (see REAGENT SETUP)
Nitrocellulose membrane (Bio-Rad, cat. no. 162-0146)
PVDF membrane (Bio-Rad, cat. no. 162-0174)
Whatman filter paper, 70 mm (Whatman; cat. no. 1001-070)
Sterile 100 × 25 mm2 Petri dishes (Midwest Scientific, cat. no. TPP 93100)
6-Well and 96-well tissue culture plates (Corning, cat. no. 3506 and Corning, cat. no. 3585)
PTC-100 Peltier thermal cycler (Bio-Rad, cat. no. PTC-1196)
PDS-1000/He Biolistic particle delivery system (Bio-Rad, cat. no. 165-2257)
0.6-μm Gold microcarriers (Bio-Rad, cat. no. 165-2262)
Macrocarrier holder (Bio-Rad, cat. no. 165-2322)
Macrocarrier (Bio-Rad, cat. no. 165-2335)
Stopping screen (Bio-Rad, cat. no. 165-2336)
1,100 psi Rupture disks (Bio-Rad, cat. no. 165-2329)
Laminar air flow (model no. NU-201-630; Nuaire)
Vacuum pump (Fisher, cat. no. 01-257-8c)
Growth chamber fitted with fluorescent lights on a controlled timer (16 h light/8 h dark photoperiod)
5-Gallon pots (12 in diameter and 11 in height)
Commercial mason jars
Surgical blade #21 (Henry Schein, cat. no. 100-3535)
Porcelain mortars and pestles (50 ml capacity; e.g., Coors, cat. no. Z247464)
1.7-ml Microcentrifuge tubes (Midwest Scientific, cat. no. AVSS1700)
Thin wall 0.2-ml PCR tubes (Midwest Scientific, cat. no. AVTW2)
Hand-operated homogenizer (Sigma, cat. no. Z359971) with sterile polypropylene pestle adapters (Sigma, cat. no. Z359947)
Refrigerated microcentrifuge (Eppendorf 5415R; Fisher, cat. no. 05-401-05)
Costar 96-well enzymatic immunoassay (EIA) plates for ELISA and protein determination (Corning, cat. no. 3590)
Syringe-operated filter units, 0.22-μm pore size (Sigma, cat. no. Z359904)
Mini-PROTEAN 3 (Bio-Rad, cat. no. 165-3323)
Film developer mini-medical series (AFP Imaging Corp, cat. no. 9992305300)
Autoradiography cassette (Fisher, cat. no. FBXC810)
Microtiter plate reader (BioTek Instruments, EL403), equipped with 450-, 570- and 595-nm filters
1-ml Nickel chelate-charged columns (Amersham Biosciences, cat. no. 17-1880-01)
Antibiotics Prepare stock solutions as indicated in the following table.
1 M PMSF Dissolve 17.4 mg PMSF in 1 ml methanol. Vortex solution and store at −20 °C for up to 1 month.
PEB 100 mM sodium chloride (NaCl), 10 mM EDTA pH 8 (Sigma, cat. no. 46081), 200 mM Tris–HCl,pH 8 (Fisher, cat. no. BP152-5), 0.05% vol/vol Tween-20 (Fisher, cat. no. BP337-100), 0.1% wt/vol SDS (Fisher, cat. no. BP166-500), 14 mM β-mercaptoethanol (β-ME; Sigma, cat. no. M 3148), 200 mM sucrose and 2 mM PMSF.
Modified PEB 15 mM sodium carbonate (Na2CO3; Sigma, cat. no. S7795), 35 mM sodium bicarbonate (NaHCO3; Sigma, cat. no. S6297), 3 mM sodium azide (NaN3; Sigma, cat. no. S8032), adjusted to pH 9.6 and Tween 0.05% vol/vol and 1 M PMSF.
Sample buffer (SDS reducing buffer), for 10 ml 3.55 ml H2O, 1.25 ml Tris–HCl (0.5 M, pH 6.8), 2.5 ml glycerol (Shelton Scientific, cat. no. IB15762), 2 ml 10% wt/vol SDS and 0.2 ml 0.5% wt/vol bromophenol blue (Sigma, cat. no. B8026). Add 50 μl β-ME to 950 μl sample buffer before use.
10 × EB, for 1 l Dissolve 30.3 g Tris base, 144 g Gly (Bio-Rad, cat. no. 161-0724) and 10 g SDS in dH2O.
Transfer buffer, for 1.5 l 300 ml 10 × EB, 300 ml methanol (Fisher, cat. no. A411), 900-ml deionized water and 0.15 g SDS.
MSO, for 1 l MS salts, 30 g sucrose, pH 5.8 and 6 g phytoblend.
RMOP36, for 1l MS salts, 100 mg myo-inositol, 1 mg thiamine HCl, 1 mg BAP, 0.1 mg NAA, 30 g sucrose, pH 5.8 and 6 g phytoblend (with or without spectinomycin).
Binding buffer 20 mM Sodium phosphate, 0.5 M NaCl, 30 mM imidazole, (pH 7.4).
Wash buffer 20 mM Sodium phosphate, 0.5 M NaCl, 5 mM imidazole, (pH 7.4).
Elution buffer 20 mM Sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, (pH 7.4).
Preparation of the gold particle stock for coating DNA Take 50 mg of gold particles (0.6 μm) in a siliconized 1.5-ml Eppendorf tube and add 1 ml molecular grade 100% ethanol. Vortex for 2 min and centrifuge at 10,000g for 3 min. Carefully discard the supernatant and resuspend the gold particles in 1 ml of 70% vol/vol ethanol by vortexing for 1 min. Incubate at room temperature (24 ± 2 °C) for 15 min and mix intermittently by gentle shaking. Pellet the gold particles by centrifuging at 5,000g for 2 min. Discard the supernatant. Wash a total of four times by resuspending the gold particles in 1 ml sterile dH2O, incubating at room temperature for 1 min followed by centrifugation at 5,000g for 2 min. After the final centrifugation step, resuspend the gold particles in 1 ml of sterile 50% vol/vol glycerol and store at −20 °C until ready to use.
Timing: 1 h
Coating gold particles with DNA Transfer 50 μl gold particles (prepared for coating) from a resuspended stock to a 1.5-ml microcentrifuge tube. While vortexing, add 5 μg of plasmid DNA followed by 50 μl of 2.5 M CaCl2 and 20 μl of 0.1 M spermidine. Continue vortexing for 20 min at 4 °C. Centrifuge the DNA-coated gold particles at 10,000g for 1 min. Remove the supernatant. Wash the pellet with 200 μl of 70% vol/vol ethanol followed by 100% ethanol. Resuspend the DNA-coated pellet in 50 μl of 100% ethanol. The DNA-coated gold particles can be stored on ice for 2–3 h and should be used as soon as possible.
Timing: 40 min (five shots)
15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, adjusted to pH 9.6.
Preparation of the chloroplast targeting vector for ligation
Digest the chloroplast targeting vector with the appropriate restriction enzyme(s). As an example we provide details for the vector shown in Figure 2, which should be digested with PvuII (Fig. 3b). Assemble the following components in a microfuge tube and incubate at 37 °C for 1 h.
Component Amount (μl) Final 10 × NEBuffer 2 5 1 × Chloroplast targeting vector (1 μg μl−1) 1 1 μg PvuII (10,000 U ml−1) 0.2 2 U Nuclease-free water 43.8
Dephosphorylate the vector using CIAP, as instructed by the supplier. This treatment minimizes self-ligation of the vector during later ligation steps (Steps 8–10).
Run the entire CIAP-treated sample on a 0.8% wt/vol agarose gel, excise the required band with a clean blade and purify the DNA from the gel using the QIAquick Gel Extraction Kit as instructed by manufacturer.
Preparation of the chloroplast expression cassette for ligation
Digest the chloroplast expression cassette with the appropriate restriction enzyme(s). The expression cassette might be a linear DNA fragment assembled by a series of ligated PCR products or part of a previously constructed plasmid (see INTRODUCTION). For illustrative purposes, we will use the example shown in Figure 3, in which sequential digestion with SalI and SacI is required. Assemble the following components in a microfuge tube and incubate at 37 °C for 2 h.
After digestion, run the entire sample on a 0.8% wt/vol agarose gel, excise the required band with a clean blade and purify the DNA from the gel using the QIAquick Gel Extraction Kit as instructed by manufacturer. If a second restriction step is required, repeat Steps 4 and 5 using the purified DNA and the second enzyme.
Add the following components to the completely purified restriction digest in a 0.2-ml PCR tube kept on ice; T4 DNA polymerase will 'polish' the ends of each fragment, making them blunt and enabling them to ligate to the blunt-ended PvuII site in the targeting vector (from Step 3). Incubate at 11 °C for 15 min.
Component Amount Final 5 × T4 DNA polymerase buffer 20 μl 1 × 10 mM dNTP mix 1 μl 0.1 mM Restricted chloroplast-expression cassette 0.5–2.5 μg 0.5–2.5 μg T4 DNA polymerase (5 U μl−1) 2 μl 10 U Nuclease-free water Up to 100 μl
Run the entire T4 DNA polymerase-treated sample on a 0.8% wt/vol agarose gel, excise the required band with a clean blade and purify the DNA from the gel using the QIAquick Gel Extraction Kit as instructed by manufacturer.
Ligation of the expression cassette and targeting vector to make the transformation vector
Set up 20-μl ligation reactions containing the chloroplast expression cassette (insert, from Step 7) and targeting vector (vector, from Step 3), as detailed below following manufacturer instructions. Set up 1:1 and 3:1 insert:vector ratios, not exceeding a total of 1.0 μg DNA. Also use vector alone as a control to test the efficiency of dephosphorylation. The number of colonies should be minimal in self-ligated vector when compared with the vector and insert ligation. Incubate the reactions at 14 °C for 16–24 h.
Component Amount Final 5 × Ligase reaction buffer 4 μl 1 × Vector ends 15–60 fmol 15–60 fmol Insert ends 45–180 fmol 45–180 fmol T4 DNA ligase (1 U μl−1) 1 μl 1 U Nuclease-free water Up to 20 μl
Transform competent Escherichia coli cells with 2–4 μl of the ligation mix using standard methods (CaCl2 method)53 and plate on selective plates. Grow overnight at 37 °C.
Screen for recombinant clones by colony PCR54 and confirm the presence of the appropriate insert by restriction analysis. Each clone should be verified by sequencing to rule out any errors introduced by PCR.
Set up an overnight culture to grow the positive clone and isolate the plasmid using a Plasmid Midiprep Kit, as instructed by the manufacturer. The chloroplast transformation vector is now ready for delivery to plants.
DNA delivery into leaf explants and selection of transplastomic shoots
Take 100–200 tobacco seeds in a 1.7-ml Eppendorf tube. Wash with 1 ml of 70% (vol/vol) ethanol for 30 s to remove any greasy material.
Add 1 ml diluted commercial bleach, for example, chlorox [1.5% (vol/vol) sodium hypochlorite in water] containing 0.1% (vol/vol) Tween 20. Incubate for 10 min, gently mixing by inverting the microfuge tube.
Remove the bleach and wash the seeds five times with 1 ml sterile deionized water.
Inoculate ∼40 seeds per Petri dish in MSO medium for germination. Keep in culture room under white fluorescent lamps (1,900 lux) with 16 h light/8 h dark cycle at 26 °C for 7–10 d.
Transfer individual germinated seedlings to magenta boxes containing MSO medium and keep in culture room for 4–7 weeks. Alternatively, transfer nodal segments of aseptically grown plants to magenta boxes containing MSO medium. This latter method decreases the time needed to obtain leaves of an adequate size for particle bombardment.
Harvest leaves at five- to seven-leaf stage of plant growth. Place an autoclaved Whatman 70-mm circle filter disk on RMOP medium in a deep Petri dish. Place leaf on filter disk with its adaxial side facing the medium.
Load 10 μl DNA-coated gold particles, prepared as outlined in REAGENT SETUP, onto the sterile macrocarrier placed in its holder. Allow it to dry in the laminar flow hood. Proceed with DNA delivery using a standard particle bombardment method55,56; our method is outlined in Box 1.
After 2 d in dark, cut 5-mm2 pieces of bombarded leaves and place on RMOP selection medium (bombarded side in contact with medium) for the first round of selection. Seal the Petri dish with parafilm.
Keep Petri dishes in culture room under white fluorescent lamps (1,900 lux) with 16 h light/8 h dark cycle at 26 °C. After 4–8 weeks, putative transgenic shoots appear (Fig. 4, panel a). Screen the putative transplastomic shoots for transgene integration by PCR (Fig. 5a,b) as described later.
Confirmation of transgene integration in putative transplastomic plants by PCR
Before the second round of selection, harvest 100 mg of leaf material from the putative transplastomic shoots. Isolate DNA using the DNeasy Plant Mini Kit, following the manufacturer's protocol. This procedure yields ∼20–30 μg of DNA.
Set up two separate 50 μl PCRs in 0.2-ml PCR tubes as detailed in the table below. One reaction (3P and 3M primers) will check integration of selectable marker gene into the chloroplast genome, the second (5P and 2M primers) will check integration of the transgene expression cassette. Primer sequences are given in Table 7. Also amplify untransformed leaf DNA in a separate PCR tube for use as control.
Component Amount per reaction (μl) Final concentration 10 × PCR buffer 5 1 × 50 mM MgCl2 2 2 mM dNTP mix, 10 mM each 1.5 300 μM each Template DNA (1 μg μl−1) 0.5 0.5 μg 3P/5P (10 pmol μl−1) 1 0.2 pmol 3M/2M (10 pmol μl−1) 1 0.2 pmol Taq DNA polymerase 1 1 U 50 μl−1 Nuclease-free water 38
Amplify using the following PCR program. Maintain the reaction at 4 °C after cycling.
Cycle number Denature Anneal Extend 1 5 min at 94 °C 2–31 1 min at 94 °C 1 min at 60 °C 1 min kb−1 at 72 °C 32 10 min at 72 °C
Examine 5 μl PCR product by agarose gel electrophoresis. Visualize amplified PCR products by staining with ethidium bromide (Fig. 5b). Once the plants have been confirmed for transgene integration by PCR, they are subjected to two further rounds of selection.
For the second round of selection, cut 2-mm2 pieces of leaves from PCR-positive plants and place them on RMOP selection medium. Grow in culture room under white fluorescent lamps (1,900 lux) with 16 h light/8 h dark cycle at 26 °C. These leaf sections produce transgenic shoots in 3–4 weeks (Fig. 4b).
Excise the regenerated shoots and transfer to MSO medium containing the appropriate antibiotic. Grow in culture room under white fluorescent lamps (1,900 lux) with 16 h light/8 h dark cycle at 26 °C. This step is termed as the third round of selection, where rooting occurs in 3–4 weeks (Fig. 4c). Southern blot analysis (Fig. 5a,c) can be carried out to confirm integration and determine homoplasmy, as described previously53,56.
Growth of plants containing integrated transgenes
Take homoplasmic plants with roots from Step 26 and wash thoroughly with water to remove the phytoblend or agar.
Soak jiffy pellet in water for 20 min. Transfer the plant to jiffy pellet in a small container with enough water so that it covers the surface. Cover it with plastic bag to maintain humidity. Keep in growth chamber maintained at 26 °C and 16-h photoperiod of 1,900 lux.
After 4 d, make a small hole in plastic bag to facilitate exchange of air. Remove the bag after a total of 7 d.
After removing the bag, grow the plant for a week in growth chamber and water the plant every 2 d.
Transfer the jiffy pellet with plant to a pot containing autoclaved soil in a greenhouse. Water plants every 2 d and add water-soluble all-purpose plant food every week, according to manufacturer's instructions.
After 5 weeks, collect the healthy leaves for characterization of transgenic protein (TP) (Steps 33–59). When the flower heads appear, either check for maternal inheritance, as outlined in Box 2 (Fig. 6), or collect the seeds. To do this, cover the flower heads with a waterproof paper bag (moisture in pods increases fungal infection) until the pods mature. Fasten the mouth of paper bag securely to the stalk below the flower branches using a string or rubber band. When the seed pods have matured, remove the bag, collect the pods and dry them in a desiccator. These seeds can further be used to grow transplastomic plants.
Extraction of total soluble protein
Timing: 1 h
Collect green and healthy leaves from transformed and untransformed plants growing in the greenhouse. Wash soil and debris from leaves and chop off the midrib portion.
Grind the leaf material in liquid nitrogen to a fine powder. Add 200 μl freshly prepared PEB to each pulverized 100-mg plant sample on ice.
Homogenize the leaf tissue using a hand-held homogenizer for 5 min, keeping the samples on ice.
Spin the homogenized samples at 15,000g for 10 min at 4 °C.
Save the supernatant and quantify the total soluble protein (TSP) as outlined in Box 3.
Confirmation of transgene expression by western blot analysis
Boil various quantities (e.g., 100, 10 and 1 μg) of crude extracts (from Step 37) diluted in an equal amount (by volume) of sample buffer for 4–20 min. Some proteins may not be detected after boiling because of destruction of epitopes (e.g., GFP). Note that western analysis may also be performed on purified protein (from Step 72) as described.
Load samples (including unboiled control samples) into wells of a 12% wt/vol SDS-polyacrylamide gel53.
Separate the proteins by electrophoresis. Set the initial current at 85 V in 1 × electrode buffer until proteins migrate into the resolving gel, then increase the current to 110 V and electrophorese until dye reaches the bottom of the gel.
Using electroblotting apparatus, transfer the separated proteins to a nitrocellulose or PVDF membrane at 15 V overnight.
After transfer, soak the membrane in a sufficient volume of PBS-T (PBS supplemented with 0.1% vol/vol Tween-20) to fully cover the membrane for 5 min at room temperature (25 °C). Pour off the PBS-T.
To block nonspecific binding, incubate the membrane at room temperature for 1 h with gentle rocking in PTM (PBS-T supplemented with 3% wt/vol nonfat milk) so that it fully covers the membrane. Pour off the PTM.
To detect the TP, fully cover the membrane with primary Ab diluted in PTM (dilution ratio depends on the Ab titer). Incubate the membrane and primary Ab solution at room temperature for 2 h (or overnight at 4 °C) with gentle rocking.
Wash the membrane one time with 1 × PBS-T for 5 min at room temperature and then add secondary Ab (conjugated to horseradish peroxidase (HRP)) at an appropriate dilution in PTM. Incubate for 1.5 h with gentle shaking.
Wash the membrane three times with PBS-T for 15 min each and one time with 1 × PBS for 10 min.
Add ECL substrate and incubate at room temperature for 5 min with gentle shaking.
Develop the chemiluminescent signal by exposing the membrane to x-ray film (Fig. 7a). The initial exposure should be for 1 min; depending on the signal obtained, subsequent exposure times can be extended up to 30 min.
Quantitation of TP
Prepare various dilutions of crude extract ranging from 1:5 to 1:5,000 in CB. A suitable purified standard for the TP must be selected and an appropriate dilution range must be empirically determined. The standard is also diluted in CB.
Aliquot 100 μl of the diluted plant protein extract and standards into separate wells of a 96-well microtiter EIA plate, in duplicate. Cover the plate with parafilm and incubate either at room temperature for 4 h or at 4 °C overnight. Wash with PSB-T and block with 100 μl of PTM at 37 °C for 1 h.
Wash wells three times with PBS-T, followed by three washes with dH2O. Pat the plate on paper towels to remove excess solution but do not let the wells dry completely.
Add 100 μl primary Ab diluted in PTM (appropriate dilution must be empirically determined). Incubate the plate at 37 °C for 2 h.
Wash and dry wells as in Step 51.
Add 100 μl secondary Ab (conjugated to HRP) diluted in PTM (appropriate dilution must be empirically determined). Incubate the plate at 37 °C for 1 h.
Wash and dry wells as in Step 51.
Add 100 μl TMB substrate to the wells. When the color begins to change, add 50 μl of 2 M sulfuric acid to stop the reaction.
Read the plate immediately on a microtiter plate reader using a 450-nm filter.
Use formula 1 to calculate the amount of TP as a percentage of the TSP of transformed leaf material (see Fig. 7b for an example). This provides the level of expression and facilitates comparison with expression levels reported in the literature.
[TP] = Concentration of transgenic protein in ng ml−1 (from the ELISA results of Step 57) and [TSP] = concentration of total soluble protein in ng ml−1 (from the results of the protein assay of Step 37 and Box 3).
Use formula 2 to calculate the amount of TP relative to the frozen weight of transformed leaf material (in milligrams of TP per 100 mg of freeze-dried leaf material, see Fig. 7c for an example). This information is useful for oral delivery of known amount of antigens or other therapeutic proteins.
[TP] = Concentration of transgenic protein in nanograms per milliliter (from the ELISA results of Step 57), VPEB = volume of PEB in milliliters used in Step 34 (normally, this value is 0.2 ml) and WTLM = weight of transformed leaf material in grams used in Step 34 (normally, this value is 0.1 g).
Purification of tagged protein (optional)
If the GOI was designed to include an affinity tag, the tag can be used to purify the TP, which may be subsequently used for functional assays. Purified proteins are essential for subcutaneous injections or other forms of delivery into the circulatory system or for in vitro studies in cultured cells. As an example, we describe affinity purification of a His-tagged TP. Grind 6 g of leaf material in liquid nitrogen to a fine powder. Add 15 ml of modified PEB in a 50-ml Falcon tube to each pulverized plant sample on ice.
Homogenize the leaf tissue using a hand-held homogenizer for 5 min, keeping the samples on ice.
Centrifuge the homogenized plant samples at 15,000g for 10 min at 4 °C. Save the supernatant and proceed directly to protein purification.
Dilute the supernatant with binding buffer in a ratio of 1:1. This step ensures the binding of His-tagged protein in the extract to the column.
We use an Akta prime machine (Amersham Biosciences) for purification of His-tagged protein as per manufacturer's instructions. In brief, insert inlet tubing A1, B and A3 in the binding buffer, elution buffer and wash buffer, respectively.
Connect the column between port 1 on the injection valve and the upper port of the UV flow cell and fill the fraction collector rack with 18-mm tubes.
Carefully inject the sample into the sample loop through injection valve.
Set up the following purification steps: equilibrate the column with binding buffer, apply the sample, wash the column with wash buffer and elute using elution buffer by gradient elution with a linear gradient of 0–100%.
Collect the flow-through from wash step and analyze for any loss of transplastomic protein during washing using ELISA. Use binding buffer and elution buffer as negative control.
Identify all the fractions containing purified protein collected in elution step by PrimeView evaluation module showing the corresponding peaks of the protein.
Pool together all the fractions containing purified protein and dialyze overnight in the dialysis cassette against cold PBS (pH 7.4) to remove imidazole from collected fractions.
Load the dialyzed protein onto the Amicon Ultra-4 centrifugal filter device and concentrate protein according to manufacturer's guidelines and centrifuge at 2,500g at 4 °C until the required concentration is obtained.
Run the concentrated protein on SDS-PAGE gel to check the purity of protein and western blot (Steps 38–48) to check for any degradation of purified protein.
Determine the protein concentration by ELISA as described in (Steps 49–59). Aliquot concentrated protein and store at −80 °C for further studies or characterization.
Troubleshooting advice can be found in Table 8.
Steps 1–3, preparation of the chloroplast targeting vector for ligation: 3–4 h
Steps 4–7, preparation of the chloroplast expression cassette for ligation: 1 d
Steps 8–11, ligation of the expression cassette and targeting vector to make the transformation vector: 5–7 d
Steps 12–20, DNA delivery into leaf explants and selection of transplastomic shoots: 3–5 months
Steps 21–24, confirmation of transgene integration in putative transplastomic plants by PCR: 6–7 h
Steps 25 and 26, second and third round of selection: 6–8 weeks
Steps 27–32, growth of plants containing integrated transgenes: 8–16 weeks
Steps 33–37, extraction of TSP: 1 h
Steps 38–48, confirmation of transgene expression by western blot analysis: 2 d
Steps 49–59, quantitation of TP: 6 h to 2 d
Steps 60–73, purification of His-tagged protein by affinity chromatography: 3–7 d
Successful construction of a chloroplast-transformation vector should result in a fully functional vector (Fig. 1). Determination of complete sequence of the integration cassette should reveal proper alignment of the promoter and UTRs with the coding sequence of the GOI and the selectable marker gene. Before determining DNA sequence, restriction enzyme analysis should provide predicted sizes of restriction fragments. E. coli cells harboring the chloroplast vector are expected to grow in Luria–Bertani medium supplemented with 100 mg l−1 spectinomycin (or other appropriate selection agent). The protein extract from overnight grown cultures of E.coli is expected to show protein of correct molecular weight or their multimers in western blots. Putative transplastomic shoots are likely to appear on RMOP selection medium in 4–8 weeks after bombardment in tobacco (Fig. 4a). Efficiency of plastid transformation is highly predictable in tobacco. One set of bombardment with five young leaves should yield at least five independent transformation events. Under optimal conditions, as many as 10–50 transformation events have been obtained. However, the number of transformants may vary depending on the efficiency of DNA delivery, choice of competent leaves and toxicity of the foreign protein to plant cells. Plastid transformation efficiency via direct organogenesis is predictable. For example, under optimized conditions plastid transformation and regeneration via organogenesis in lettuce is as efficient as in tobacco. However, plastid transformation efficiency is not highly predictable in plant species where regeneration requires somatic embryogenesis.
Several shoots are expected within 3–4 weeks of the second round of selection (Fig. 4b) from leaves of putative transplastomic shoots, confirmed by PCR for transgene integration (Fig. 5b). Rooting of shoots on MSO selection medium occurs within 3–4 weeks (Fig. 4c). Southern blot analysis of plants after rooting under selection are likely to show the presence of only transformed chloroplast genomes (homoplasmy; Fig. 5c, lane 2), however, a few plants may also show both transformed and untransformed chloroplast genomes (heteroplasmy; Fig. 5c, lane 3). Untransformed plants are anticipated to show a fragment of the native chloroplast genome (Fig. 5c, lane 1).
Seeds obtained from self pollination of transplastomic plants and F1 hybrids with pollen from wild type are expected to germinate and grow into uniformly green plants (Fig. 6), whereas untransformed plants and F1 hybrid with pollen from transplastomic plants should not grow on spectinomycin containing media. The absence of Mendelian segregation of transgenes should indicate that they are maternally inherited. Western blot analysis of transplastomic plants should show the expected size protein (Fig. 7a) and ELISA should quantify the amount of foreign protein. In the case of tobacco, we also determined that the age of the leaf significantly impacts the yield of foreign protein, with mature leaves showing the highest levels of expression. Older leaves show the lowest level of expression, which is correlated with senescence and high proteolytic activity. In the example of interferon expressed in transgenic chloroplasts, both young and mature leaves showed high levels of expression, ranging from 1.5 to 2 mg g−1 fresh weight (Fig. 7b), or ∼15% TSP (Fig. 7c), in T1 transgenic lines. The purification of His-tagged protein from transplastomic plant is essential for functional evaluation of chloroplast-derived proteins by in vitro cell culture assays or in vivo studies using suitable animal models.
The results reported in this article were supported in part by grants from United States Department of Agriculture 3611-21000-017-00D and National Institutes of Health R01 GM 63879 to H.D. The authors are grateful to Drs. Philip Arlen and Dolendro Singh for critically reading this article and Dr. Arlen for redrawing Figure 7.