Jim M. Dunwell and Andy C. Wetten (eds.), Transgenic Plants: Methods and Protocols, Methods in Molecular Biology, vol. 847,
DOI 10.1007/978-1-61779-558-9_35, © Springer Science+Business Media, LLC 2012
Plastid Transformation as an Expression Tool
for Plant-Derived Biopharmaceuticals
Nunzia Scotti and Teodoro Cardi
The production of biopharmaceuticals in plants is currently one of the most attractive approaches to modern
medicine. Several effi cient plant-based expression systems have been developed so far. Among them, plastid
transformation has attracted biotechnologists because the plastid genome, unlike nuclear genome, bears a
number of unique advantages for plant genetic engineering. These include higher levels of protein produc-
tion, uniform gene expression of transformants due to the lack of epigenetic interference, and expression
of multiple genes (as in operons) from the same construct. Further, the plastid transformation technology
is an environmentally friendly method because plastid and their genetic information are maternally inherited
in many species with a consequent lack of transmission of plastid DNA by pollen. Recently, great progress
has been made with plastid-based production of biopharmaceuticals demonstrating that it is a promising
platform for such purposes. This chapter describes detailed protocols for plastid transformation including
the delivery of DNA by biolistic method, the selection/regeneration of transplastomic plants, and the
molecular analyses to select homoplasmic plants and confi rm transgene expression.
Key words: Plastid transformation , Tobacco , Biofactories , Biopharmaceuticals , Transgene containment
Plastids are a family of plant organelles derived from endosymbiosis
of a common ancestor of the present cyanobacteria. In plant cells,
they participate in a wide range of metabolic processes ( 1 ) . The
chloroplast, the best known among plastids, is the site of photosyn-
thesis and a number of biosynthetic pathways, including those for
fatty acids, amino acids, isoprenoids, etc. All plastids derive, directly
or indirectly, from a small and undifferentiated organelle, termed
the proplastid, and share a common small circular DNA molecule
(plastome) of about 120–160 kb present as numerous copies in
each plastid (up to 10,000 copies/cell).
452N. Scotti and T. Cardi
In recent years, plastids have long been the subject of biotech-
nological approaches based on plastid transformation opening new
opportunities in plant biotechnology. Plastid transformation has
many advantages, over conventional transgenic plants, linked to a
very high levels of gene expression (up to 70% of total soluble
proteins ( 2 ) ), ensured by the high copy number of plastomes per
plastid/cell, absence of gene silencing mechanisms, expression of
polycistronic mRNAs, and gene containment due to the maternal
inheritance of plastids in most crop species ( 3– 5 ) .
Recently, the expansion of the molecular farming concept
(production of biopharmaceuticals in plants) has given an exten-
sive boost to the exploration of the suitability of chloroplast
genetic engineering for such purposes. In fact, great and exciting
progress has been made with plastid-based production of
Several proteins belonging to the different subclasses of bio-
pharmaceuticals (vaccines, therapeutic proteins, autoantigens,
antibiotics, etc.) have been produced by plastid transformation
( 6– 8 ) , demonstrating that plastid transformation is a promising
tool for plant-derived pharmaceuticals.
To date, the species of choice for plastid transformation is
tobacco because of its large biomass and suitability for effi cient
transformation and regeneration. In fact, the main limitation of
this technology is currently its extension to plants other than
tobacco due to low transformation effi ciency. Generally, it has been
attributed to several factors such as the kind of explants, the regen-
eration protocol, the nonoptimal homology of fl anking regions
etc. ( 9– 14 ) . In some species, the use of species-specifi c transforma-
tion vectors and improved regeneration protocol resulted in an
improvement of the transformation effi ciency ( 15– 20 ) . Despite the
progress made for plants other than tobacco, only two crops
(tomato and lettuce) have been used successfully for the produc-
tion of biopharmaceuticals via plastid transformation ( 21– 23 ) .
Another limitation of plastid transformation is the absence of some
posttranslational modifi cations such as glycosylation important for
biological activity of some biopharmaceuticals.
In this chapter, we describe detailed procedures (delivery of
DNA by biolistic method, selection/regeneration of transplastomic
plants, molecular analyses to select homoplasmic plants, and confi rm
transgene expression) for chloroplast transformation of tobacco
used in our laboratory to produce plant-derived viral antigens
(HIV, HPV, Vaccinia virus) and other recombinant proteins
( 24– 26 ) , with particular emphasis on plant-derived HIV-1 Pr55 gag
45335 Plastid Transformation as an Expression Tool for Plant-Derived…
1. PDS-1000/He TM Gene Gun delivery system (Bio-Rad, Hercules,
CA, USA), gold particles (microcarrier, 0.6 m m), macrocarriers,
stopping screen, and rupture disks (1,100 psi).
2. Nicotiana tabacum (in vitro–grown plants).
3. 2.5 M CaCl 2 (sterilize by fi ltration, should always be prepared
4. 0.1 M spermidine (prepare a stock, aliquot into small volume,
and store at −20°C, see Note 1 ).
5. 70% and 100% ethanol (molecular grade).
6. Sterile water.
1. RMOP medium: Murashige and Skoog salts 4.30 g/L, thiamine
1 mg/L, myo -inositol 100 mg/L, sucrose 30 g/L, benzylam-
inopurine 1 mg/L, naphthylacetic acid 0.1 mg/L, and Micro
Agar 8 g/L. Adjust pH to 5.8 before autoclaving medium
(All chemicals from Duchefa, Haarlem, The Netherlands).
2. Sterile forceps and scalpel.
3. Sterile Petri dishes (100 × 20 mm and 60 × 15 mm).
4. Antibiotics: prepare stock solutions of spectinomycin
(100 mg/mL) and streptomycin (250 mg/mL).
5. MS30B5 medium: Murashige and Skoog salts 4.30 g/L, B5
vitamins 1 mL/L, sucrose 30 g/L, and Micro Agar 8 g/L.
Adjust pH to 5.8 before autoclaving medium (All chemicals
1. Total DNA from transplastomic and untransformed (wild-
2. PCR primers: primer 1 which anneals to the native integration
plastid sequence and primer 2 which anneals to the gene of
4. Taq DNA polymerase, MgCl 2 , and buffer (Invitrogen, Carlsvad,
5. DNA molecular weight marker (Fermentas, Ontario, Canada).
6. Agarose (Invitrogen).
1. Total DNA from transplastomic and untransformed (wild-
2. Restriction enzymes and buffer (New England Biolabs,
Ipswich, MA, USA, supplied by manufacturer).
3. DNA molecular weight marker (Fermentas).
2.2. Tobacco Plant
Tissue In Vitro Culture
2.3. Identifi cation
Plants by PCR
2.4. Identifi cation
Plants by Southern
454N. Scotti and T. Cardi
4. Agarose (Invitrogen).
5. 0.25 N HCl (depurination solution).
6. 0.4 N NaOH (transfer buffer).
7. Nylon membrane (Hybond-N+, GE Healthcare, Waukesha,
8. GS GeneLinker UV Chamber (Bio-Rad).
9. Flanking probe produced by PCR amplifi cation of the fl anking
sequences using digoxigenin-11 dUTP (Roche Applied
Science, Penzberg, Germany) and two primers which anneal to
the native plastid integration sequences ( see Note 2 ).
10. Hybridization oven (Thermo, Waltham, MA, USA) and roller
11. DIG Easy Hyb Granules, blocking solution, anti-digoxigenin-
AP conjugate, and CSPD (Roche Applied Science, DIG High
Prime DNA labeling and Detection Starter Kit II).
12. 2× SSC, 0.1% SDS and 0.5× SSC, 0.1% SDS.
13. Maleic acid buffer: 0.1 M maleic acid, 0.15 M NaCl, and adjust
pH to 7.5 with solid NaOH.
14. Washing buffer: 0.1 M maleic acid, 0.15 M NaCl, adjust pH to
7.5, and 0.3% (v/v) Tween 20.
15. Detection buffer: 0.1 M Tris–HCl, 0.1 M NaCl, and adjust
pH to 9.5.
16. Autoradiography cassette and fi lm.
1. Eppendorf tube (1.5 mL) and polypropylene pestle.
2. Liquid nitrogen.
3. 10× PBS: 80 g/L NaCl, 2 g/L KCl, 14.4 g/L Na 2 HPO 4 ,
2.4 g/L KH 2 PO 4 , and adjust pH to 7.5.
4. PBS HS: 1× PBS (1:10 diluted from 10× PBS), 0.5 M NaCl,
10 mM EDTA, 1 mM PMSF, and 5 mM DTT.
5. Bradford assay (Bio-Rad protein assay).
6. Loading sample buffer (6×): 50 mM Tris–HCl pH 6.8, 2%
SDS, 2 mM EDTA, 5 mM DTT, 10% glycerol, and 0.1% blue
7. 10% SDS and 100 mM PMSF ( see Note 3 ).
8. Acrylamide/bis (ready to use mixture from Bio-Rad).
9. TEMED (ready to use, Bio-Rad).
10. Separating buffer (4×): 1.5 M Tris–HCl pH 8.8 and 0.4% SDS.
11. Stacking buffer (4×): 1 M Tris–HCl pH 6.8 and 0.4% SDS.
12. 10% Ammonium persulfate (should always be prepared fresh).
13. Running buffer (5×): 25 mM Tris–HCl pH 8.8, 250 mM
Glycine, and 0.5% SDS.
2.5. Protein Extraction
and Western Blot
Analysis to Evaluate
the Expression of
45535 Plastid Transformation as an Expression Tool for Plant-Derived…
14. Prestained molecular weight marker (Fermentas).
15. Blotting buffer (10×): 48 mM Tris and 39 mM glycine.
16. 1× Blotting buffer (1:10 diluted from 10× blotting buffer) and
10% (v/v) methanol.
17. Nitrocellulose membrane (GE Healthcare).
18. 3MM chromatography paper (Whatman, Maidstone, UK).
19. Bovine serum albumin (BSA, Sigma, St. Louis, MO, USA).
20. Nonfat dry milk (Nestlé Carnation, Vevey, Switzerland).
21. Blocking buffer: 1× PBS and 3% BSA.
22. T-PBSW: 1× PBS containing 0.1% Tween 20.
23. Antibodies: antigen-specifi c primary antibody (ARP432 and
ARP431, Programme EVA Centre for AIDS Reagents, NIBSC,
UK); secondary antibody conjugated to horse radish peroxidase
(peroxidase-labeled anti-rabbit antibody, GE Healthcare).
24. Enhanced chemiluminescent reagents (ECL plus Western blotting
detection system, GE Healthcare).
25. ECL fi lm (GE Healthcare).
1. EIA-RIA 96-well plate.
2. 1× PBS.
3. T-PBSE: 1× PBS containing 0.05% Tween 20.
4. T-PBSEM5: T-PBS containing 5% nonfat dry milk.
5. T-PBSEM1: T-PBS containing 1% nonfat dry milk.
6. Antigen standard: HIV-1 p24 protein purifi ed from E. coli (EVA
673, Programme EVA Centre for AIDS Reagents, NIBSC, UK).
7. Antigen-specifi c primary antibody: mouse monoclonal antibody
anti-p24 (ARP 3243.4, Programme EVA Centre for AIDS
Reagents, NIBSC, UK); rabbit polyclonal antibody anti-p24
(ARP432, Programme EVA Centre for AIDS Reagents,
8. Secondary antibody: horseradish peroxidase (HRP)—
conjugated anti-rabbit antibody (GE Healthcare).
9. TMB substrate (Bio-Rad).
10. 2 N Sulfuric acid.
11. Microtiter plate reader (Victor, Perkin Elmer, Waltham, MA,
1. Homogenization buffer: 50 mM Tris–HCl pH 8.0, 1.3 M
NaCl, 25 mM EDTA pH 8.0, 0.2% BSA, 0.05% cysteine, and
56 mM b -mercaptoethanol.
2. Lysis buffer: 10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0,
5 mM DTT, 1 mM PMSF, and 0.5% Triton X-100.
to Quantify the
Expression Level of
456N. Scotti and T. Cardi
3. Miracloth Calbiochem (Merck, Darmstadt, Germany).
4. Bradford assay (Bio-Rad protein assay).
In this chapter, we describe the biolistic DNA delivery method to
transform plant plastids. It is a system of choice for most laborato-
ries and requires a PDS-1000/He TM Gene Gun delivery apparatus.
DNA from plastid transformation vector is coated on gold parti-
cles, delivered at high velocity to go through the cell wall and
membranes (both cellular and plastidial), then integrated into
the plastome by a double recombination event. Further, detailed
protocols for in vitro culture to select and regenerate transfor-
mants, and to confi rm transformation and transgene expression are
1. Take 35 mg of gold microcarrier (0.6 m m) in an Eppendorf
tube, add 1 mL of 100% ethanol, and vortex for 5 min.
2. Spin for 2 s at 4,300 × g .
3. Carefully remove the supernatant, add 1 mL of 100% ethanol,
and vortex for 5 min (repeat steps 2 and 3 three times).
4. To perform fi ve shots, transfer 50 m L of microcarrier stock in a
new Eppendorf tube and spin for 2 s at 4,300 × g , remove the
supernatant, and wash with 50 m L of cold sterile water (repeat
5. Add sequentially 10 m L of plasmid DNA (1 m g/ m L), 50 m L
2.5 M CaCl 2 , and 20 m L 0.1 M spermidine and vortex the
mixture for 20 min at 4°C.
6. Precipitate the plasmid DNA on microcarrier gold, add 200 m L
of cold ethanol to the mixture, and spin for 2 s at 4,300 × g .
7. Remove carefully the supernatant, wash the pellet by adding
200 m L of cold ethanol, and spin for 2 s at 4,300 × g (repeat
8. Resuspend the DNA-coated gold particles in 30 m L of ethanol
and store on ice.
9. Before shooting, spread 5 m L of particles over the center of the
macrocarrier, and as soon as they are dry, they are ready for use
in biolistic transformation.
1. Harvest leaves from 20 to 30-day-old in vitro–grown tobacco
2. Place the leaf on RMOP medium (without antibiotics) in Petri
dish (60 × 15 mm) with the abaxial side uppermost.
3. Switch on the vacuum pump.
of Gold Particle
Stock and Coating
3.2. Particle Delivery
into Tobacco Leaf
45735 Plastid Transformation as an Expression Tool for Plant-Derived…
4. Open the knob of the helium gas and set up the helium pressure
200 psi higher than desired pressure of the rupture disk.
5. Switch on the PDS system.
6. Place the rupture disk in its holder ring and screw the holder
into the PDS chamber.
7. Place the stopping screen in the retaining assembly. On top of
this place, upside down the macrocarrier with DNA on it and
screw the metal ring.
8. Place the retaining assembly in the fi fth slot from the bottom
in the PDS chamber.
9. Place the Petri dish containing the tobacco leaf into the target
plate holder, then into the third slot (6 cm) from the bottom
of the PDS chamber, and close the chamber door.
10. Turn the vacuum button on VAC position. Allow the vacuum
pressure to reach 27–28 in. of Hg, turn the vacuum button on
HOLD position, and press continuously the FIRE (shooting)
button until the rupture disk bursts.
11. Release the FIRE button and turn the vacuum button to
VENT position. Remove the bombarded sample when the
vacuum pressure reaches 0 in. of Hg.
12. Repeat steps 6 – 11 for additional samples.
13. Shut down the system by rotating the knob of the gas cylinder
clockwise. Create vacuum as in step 10 to release the gas pressure
remaining inside the helium tube.
14. Press and release the FIRE button until the pressure falls to
zero on the meter gauge of the gas cylinder.
15. Switch off the vacuum pump and PDS system.
16. Place the Petri dishes containing the bombarded leaves in the
culture room for 2 days.
1. Cut each bombarded leaf into small pieces (3 × 3 mm) and
place them ( see Note 4 ) with their abaxial side uppermost on
RMOP selection medium containing 500 mg/L of spectino-
mycin for the fi rst round of selection in the growth chamber
under dim light (about 10 m E/m 2 /s), in 16-h light, and 8-h
dark regime. Within 4–6 weeks, putative transformed shoots
should appear (Fig. 1 ).
2. Cut each primary shoot into small pieces (3 × 3 mm) and place
two-third of them on RMOP selection medium containing
500 mg/L of spectinomycin (second round of regeneration)
and one-third of them on RMOP selection medium containing
500 mg/L of both spectinomycin and streptomycin. If the
primary shoot is a true transformant, the tissue will form a
green callus on Spec-Strep plate (Fig. 2 ) and secondary green
shoots on Spec plate.
3.3. In Vitro
Selection of Shoots
458N. Scotti and T. Cardi
3. Transfer the secondary green shoots on MS30B5 medium
containing 500 mg/L of spectinomycin in Magenta box for
rooting under controlled condition (16-h light 40 m E/m 2 /s
and 8-h dark at 24°C). Within 3–4 weeks, rooted plants
with three to four leaves will be obtained (Fig. 3 ). At this stage,
leaf DNA will be extracted and used to select transplastomic
homoplasmic plants by PCR and Southern blot analyses.
Fig. 1. Regeneration of a putative transformed shoot after 4–6 weeks of bombardment on RMOP selection medium (fi rst
round of regeneration).
Fig. 2. Selection of primary regenerants on RMOP selection medium containing 500 mg/L of both spectinomycin and
streptomycin. ( a ) positive explants, ( b ) negative explants.
459 35 Plastid Transformation as an Expression Tool for Plant-Derived…
4. If the plants are homoplasmic, transfer to soil for seed
5. If the plants are not homoplasmic, cut into small pieces and
transfer onto RMOP selection medium for the third round of
1. Extract total leaf DNA from wild-type (control) plants and
putative transformed plants ( see Note 5 ).
2. Perform PCR reaction (25 m L) for each sample as follow:
50 ng of total DNA from putative transformants and control
plant, 2.5 m L of 10× PCR buffer, 0.75 m L of 50 mM MgCl 2 ,
0.5 m L (20 m M) of each primer, 0.5 m L of 10 mM dNTPs,
1.25 units of Taq DNA polymerase, and adjust the fi nal volume
with sterile water.
3. Set the PCR reaction as follows: initial denaturation at 94°C
for 3 min; 30 cycles of denaturation at 94°C for 1 min, annealing
at 55–60°C for 1 min, elongation at 72°C for 1 min /kb; and
fi nal elongation at 72°C for 5 min.
4. Analyze the PCR products by agarose gel (0.8% w/v) electro-
phoresis/ethidium bromide staining. PCR products from
putative transplastomic plants show a variable size depending
on the size of the foreign gene inserted and from primers
selected for the analysis. The wild-type plants show no amplifi -
3.4. Identifi cation
Plants by PCR
Fig. 3. Rooted secondary shoots (positive to both spectinomycin and spectinomycin/
streptomycin selections) on MS30B5 medium 500 mg/L of spectinomycin ready for leaf
460N. Scotti and T. Cardi
1. Extract total leaf DNA from wild-type (control) plants and
transformed plants ( see Note 5 ).
2. Digest 1.5–2.5 m g of total DNA from transformed and control
plants with suitable restriction enzymes and incubate at suggested
3. Run digested DNA and DNA molecular weight marker on
0.8% agarose gel, take a picture of your gel, and nick a corner
to defi ne the orientation of the gel.
4. Depurinate DNA in 0.25 N HCl for 10 min by shaking gently
and rinse twice with distilled water.
5. Denature DNA in transfer buffer (0.4 N NaOH) for 10–15 min
by shaking gently.
6. Turn the gel upside down. Prewet the nylon membrane (nick
a corner to defi ne the orientation of the membrane) in transfer
solution, place it on top of the gel, and smooth out bubbles.
Put on the top of the membrane six pieces of Whatman paper
and blotting paper. Apply a weight of 500 g on top and allow
the DNA to transfer overnight.
7. Remove the membrane with transferred DNA (use a pencil to
mark the wells) and cross-link it by using GS GeneLinker UV
Chamber (C3 program).
8. Prepare fl anking probe by PCR amplifi cation of the fl anking
sequences using digoxigenin-11 dUTP. Perform PCR reaction
(50 m L) as follows: 1–50 ng of total DNA from wild-type plant,
5 m L of 10× PCR buffer, 1.5 m L of 50 mM MgCl 2 , 1 m L
(20 m M) of each primer, 5 m L of DIG dNTPs (fi nal concentra-
tion 200 m M dATP, dCTP, and dGTP, 130–185 m M dTTP,
and 15–70 m M DIG-dUTP, see Note 2 ), 2.5 units of Taq DNA
polymerase, and adjust the fi nal volume with sterile water.
Prepare a tube for unlabeled control probe using a standard
9. Set the PCR reaction as follows: initial denaturation at 95°C
for 2 min; 40 cycles of denaturation at 95°C for 30 s, annealing
at 55–60°C for 1 min, elongation at 72°C for 1 min /kb; and
fi nal elongation at 72°C for 7 min.
10. Analyze the PCR products by agarose gel (0.8% w/v) electro-
phoresis/ethidium bromide staining. The labeled probe will
migrate slower than unlabeled control probe due to the presence
of digoxigenin (DIG).
11. Preheat at 37–42°C an appropriate volume (10 mL/100 cm 2
membrane) of DIG Easy Hyb and prehybridize membrane for
at least 30 min by shaking gently ( see Note 6 to calculate
3.5. Identifi cation
Plants by Southern
461 35 Plastid Transformation as an Expression Tool for Plant-Derived…
12. Denature DIG-labeled probe (25 ng/mL DIG Easy Hyb) by
boiling for 5 min and cool in ice. Add denatured DIG-labeled
probe to preheated DIG Easy Hyb (3.5 mL/100 cm 2 mem-
brane) and mix.
13. Pour off prehybridization solution, add probe/hybridization
mixture to membrane, and incubate overnight by shaking
14. Wash the membrane twice with 2× SSC, 0.1% SDS at room
temperature for 5 min by shaking.
15. Wash the membrane twice with preheated 0.5× SSC, 0.1% SDS
at 65–68°C for 15 min by shaking.
16. Rinse the membrane briefl y in washing buffer.
17. Incubate the membrane for 30 min in 100 mL blocking
solution at room temperature by shaking.
18. Incubate the membrane for 30 min in 20 mL antibody
solution at room temperature by shaking.
19. Wash the membrane twice in 100 mL washing buffer for
15 min at room temperature by shaking.
20. Equilibrate the membrane in 20 mL detection buffer for
15 min at room temperature.
21. Place membrane with DNA side facing up on a plastic wrap
and apply 1 mL CSPD ready to use. Immediately cover the
membrane with plastic wrap, spread the substrate over the
membrane, and smooth out bubbles. Incubate at room tem-
perature for 5 min.
22. Squeeze out excess liquid and incubate the damp membrane
for 10 min at 37°C to enhance the luminescent reaction.
23. Expose to X-ray fi lm for 15–30 min.
1. Grind 50–100 mg leaf and add 200–400 m L of freshly prepared
PBS HS on ice.
2. Homogenize for 5 min the tissue by using polypropylene
pestle and keeping the sample in ice to prevent protein degra-
dation. Vortex briefl y.
3. Centrifuge at 11,200 × g for 10 min at 4°C.
4. Aliquot the supernatant (soluble proteins) and proceed to pro-
tein quantitation by Bradford assay.
1. Prepare a 12% separating gel (1.5-mm thick) using a Mini
PROTEAN 3 system (Bio-Rad) by mixing 3.375 mL water,
1.875 mL separating buffer (4×), 2.25 mL acrylamide/bis
solution, 37.5 m L 10% ammonium persulfate solution, and
7.5 m L TEMED. Pour the solution smoothly, leaving space for
the stacking gel, and overlay with isopropanol. Gel polymeriza-
tion should take 45–60 min.
3.6. Extraction of Total
3.7. Western Blot
Analysis to Evaluate
the Expression of
462N. Scotti and T. Cardi
2. Pour off the isopropanol, rinse the gel surface with water, and dry
the area between glass plates with a piece of Whatman paper.
3. Prepare the stacking gel by mixing 3.075 mL water, 1.25 mL
stacking buffer (4×), 625 m L acrylamide/bis solution, 50 m L
10% ammonium persulfate solution, and 10 m L TEMED. Pour
the solution until the top of the short place is reached, and
then insert the desired comb between the spacers. Gel polym-
erization should take 30–40 min.
4. Prepare 1 L of running buffer 1× (1:5 diluted from 5× solution).
5. Remove the comb and wash the wells with water and running
buffer using a syringe.
6. Prepare the samples (plant extracts and standard protein) diluting
them in loading sample buffer and boil for 5–10 min.
7. Put immediately the samples on ice and load them and protein
marker into wells of 12% SDS-polyacrylamide gel.
8. Run the gel with initial current at 80 V until proteins migrate
into separating gel, then increase the current at 150 V. Stop the
run when the dye reaches the bottom of the separating gel.
9. Transfer the separated proteins to nitrocellulose membrane in
blotting buffer 1× for 1 h at 100 V.
10. Remove the membrane and rinse with 1× PBS.
11. Incubate the membrane in blocking buffer for 2 h at room
temperature or overnight at 4°C by shaking.
12. Pour off the blocking buffer and wash with T-PBSW for 10 min
13. Add primary antibody diluted in blocking buffer ( see Note 7 )
and incubate at room temperature for 2 h by shaking.
14. Wash the membrane with T-PBSW for 10 min by shaking
(repeat three times).
15. Add secondary antibody (anti-rabbit) diluted (1:60,000) in 1×
PBS containing 5% nonfat dry milk and incubate at room tem-
perature for 1 h by shaking.
16. Wash the membrane with T-PBSW for 10 min by shaking
(repeat three times).
17. Add ECL substrate, spread it on the membrane, and incubate
for 5 min at room temperature.
18. Squeeze out excess liquid and expose the membrane to ECL
fi lm for about 5 min (Fig. 4 ).
The procedure described is referred to a sandwich (or capture)
ELISA that measures the amount of antigen between two layers
of antibodies. This procedure is particularly indicated when the
concentration of antigen is contained in high concentrations of
contaminating proteins (e.g., crude plant extracts). The crude plant
to Quantify the
Expression Level of
46335 Plastid Transformation as an Expression Tool for Plant-Derived…
proteins used for this procedure were extracted using 1× PBS HS
without DTT (native condition).
1. Coat the plate with 50 m L/well of monoclonal antibody anti-
Pr55/p24 (ARP 3243.4) diluted in 1× PBS (2.5 mg/mL) and
incubate the plate overnight at 4°C.
2. Pour off the solution and wash the plate twice with T-PBSE.
3. Block the plate with 150 m L/well of T-PBSEM5 for 1.5–2 h
4. Pour off the solution and wash the plate twice with T-PBSE.
5. Add 50 m L/well of T-PBSEM1, then sample crude extract or
antigen standard, and incubate for 1 h at 37°C.
6. Pour off the samples and wash the plate three times with
7. Add 50 m L/well of polyclonal anti-p24 antibody (ARP432)
diluted in T-PBSEM1 (1:5,000) and incubate for 1 h at 37°C.
8. Pour off the solution and wash the plate three times with T-PBSE.
9. Add 50 m L/well of polyclonal anti-rabbit antibody conjugated
to horse radish peroxidase diluted in T-PBSEM1 (1:5,000)
and incubate for 1 h at 37°C.
10. Pour off the solution and wash the plate four times with T-PBSE.
11. Develop with TMB substrate (50 m L/well) for 5–10 min.
12. Stop the reaction by adding (50 m L/well) of 2 N sulfuric acid.
13. Read the plate on a microtiter plate reader using 450-nm
Fig. 4. Western blot analysis of total protein extracts (TE) and chloroplast protein extracts
(CpE) of transplastomic NS40 plant overexpressing the HIV-1 Pr55 gag polyprotein using a
rabbit polyclonal antibody against p24. p24 = recombinant p24 protein produced in E. coli
(0.1 and 0.05 m g); NS40-TE = total soluble proteins from N. tabacum transformed with
pNS40 vector (0.5, 1, and 2 m g); NS40-CpE = chloroplast proteins from N. tabacum trans-
formed with pNS40 vector (1 and 2 m g).
464 N. Scotti and T. Cardi
This protocol describes a rapid procedure to isolate chloroplasts
(based on differential centrifugations) and extract total proteins
from enriched chloroplast fraction. It generally ensures a higher
yield than methods based on the isolation of purifi ed chloroplasts
on percoll or sucrose gradients, although more contaminations are
present. All operations are carried out at 4°C.
1. Keep plants in the dark overnight before harvesting ( see
Note 8 ).
2. Homogenize 4–20 g of leaves with 16–80 mL of homogeni-
zation buffer two times for 5 s in a Waring blender at 4°C (see
Note 9 ).
3. Filter the homogenate rapidly trough two layers of Miracloth.
4. Centrifuge at 500 × g for 5 min at 4°C.
5. Recover the supernatant and centrifuge it twice at 2,600 × g for
15 min and 10 min (the second time) at 4°C.
6. Resuspend the pellets, carefully, with 10 mL of homogeniza-
7. Centrifuge the chloroplast solution at 2,600 × g for 15 min
8. Pour off the supernatant and resuspend the pellet with
1.2–6 mL of lysis buffer on ice.
9. Leave on ice for 10 min and then vortex vigorously.
10. Aliquot the crude chloroplast proteins and proceed to protein
quantitation by Bradford assay.
1. Spermidine is very hygroscopic and air sensitive. Prepare stock
solution and aliquot into small volumes to avoid freeze and
thaw. After use, discard the remaining volume.
2. The production of the fl anking probe by PCR amplifi cation
using DIG-dUTP can require an adjustment of the DIG-dUT
concentration, and generally, 70 m M as fi nal concentration of
DIG-dUTP works well for labeling probes up to 1 kb long;
reduce the fi nal concentration to 35 m M for probes 1–3 kb
long. The labeling of probes >3 kb can require the use of a
Long Taq DNA polymerase and a gradual reduction of the
fi nal concentration of DIG-dUTP up to 7 m M (depending on
the specifi c-target sequences).
3. SDS and PMSF are harmful by inhalation or in contact with skin
and can cause irritation to the eyes, skin, and respiratory system.
4. Place no more than nine pieces per Petri dish because leaf
segments expand during in vitro culture.
46535 Plastid Transformation as an Expression Tool for Plant-Derived…
5. Total DNA can be isolated by both kit and standard procedure
6. The appropriate hybridization temperature is calculated accor-
ding to manufacturer’s instructions and should be 20–25°C
below the calculated Tm. Tm is calculated according the
( ) (
Tm49.82 0.41 %GC 600 / l
[ l = length of hybrid in base pairs].
7. The appropriate dilution of the primary antibody are the fol-
lowing: 1:10,000 for anti-p24 antibody (ARP432, NIBSC)
and 1:15,000 for anti-p17 antibody (ARP431, NIBSC).
8. Starch granules present in chloroplasts interfere with the isola-
tion of intact organelle. In fact, chloroplasts containing large
starch grains are generally broken during centrifugation.
Therefore, prior to proceeding with the experiment, the plants
should be kept in the dark to reduce the amount of starch.
9. The homogenization of plant material with the Waring blender
must be as short as possible to reduce the portion of broken
The authors would like to thank Ms. Lorenza Sannino and Ms.
Silvia Silletti for the technical assistance and photography. HIV-1
antisera and recombinant proteins were provided by the Programme
EVA Centre for AIDS Reagents, NIBSC, UK, supported by the
EC FP6/7 Europrise Network of Excellence, AVIP and NGIN
consortia and the Bill and Melinda Gates GHRC-CAVD Project
IGV publication no. 368.
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