Gene Therapy and Molecular Biology Vol 15, page 14

14

Gene Ther Mol Biol Vol 15, 14-29 2013


Green factories: plastids for the production of
foreign proteins at high levels
Original Research Article
Niaz Ahmad
*
and Zahid Mukhtar
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan

*Correspondence: Dr. Niaz Ahmad, Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic
Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan
Postal code: 38000, Phone: +92(0)412 651 475 Ext 309, Fax: +92(0)412 651 472, Email: niazbloch@yahoo.com
Keywords: Chloroplast transformation, recombinant proteins, biopharming
Abbreviations: TSP=Total soluble proteins, ptDNA= Plastid DNA, LSC=Large single copy, SSC=Small single copy, IR=Inverted
repeat, UTRs =Untranslated regions


Received: 10 September 2013; Revised: 20 September 2013
Accepted: 2 October 2013; electronically published: 5 October 2013


Summary
The soaring costs along with the increasing demand of recombinant proteins in different walks of life have fuelled
the quest to find out alternate modes for their production, which are cheaper, safer and can deliver the modern
safety standards. In this context, plants are emerging as an alternative production platform for foreign proteins.
Chloroplasts the green plastids with an in-built remarkable capacity to express foreign proteins at high levels
offer several attractive features, which make plants an exceptionally useful system for low-cost production of high-
value targets at large scale. Transformation of chloroplasts, therefore, holds a great potential to meet the challenges
in the areas of food, feed and medicine posed by a population on the rise. Numerous developments have been made
in the field, all of which set plastids to become a centrepoint of future plant engineering efforts. The present review
briefly describes the state of the art of the technology along with its salient features whilst highlighting the latest
trends in the area of chloroplast transformation.


I. Introduction
The use of recombinant proteins in different spheres of
life is creating a huge demand for their large-scale
production. For example, the antibodies used as topical
medicines are required in large doses due to their
stoichiometric mechanism of action and therefore their
production is also needed in huge quantities, sometimes
ranging from 100-1,000 kg to cope with the demand
(Twyman et al., 2012). Our current capacity to meet this
challenge with the established techniques is lacking
(Bosch et al., 2013). The award of US$ 100 million for
research into plant-based expression systems by the US
Government agency, Defense Advanced Research
Projects Agency (DARPA), is the recognition of the fact
that making antibodies and vaccines using plants as
expression vehicles is the technology of choice for
low-cost production at a large scale (Bosch et al., 2013).
Expressing proteins in plants offers a unique expression
platform with low capital costs, excellent scalability,
post-translational modifications and the ability to
properly fold and process the recombinant proteins (see
Rybicki, 2010; Lssl and Waheed, 2011 for reviews). It
has been estimated that Sanofi Pasteur, a conventional
vaccine production plant in Pennsylvania (USA) would
cost 150 million US$ for the production of 100 million
doses of influenza vaccine in a year. Similarly Novartis,
in North Carolina, USA, will cost one billion US$ to
produce 300 million doses of the same vaccine in a year.
On the other hand, tobacco (Nicotiana benthamiana)
would produce one billion doses for 15 million US$ in
one year (Penney et al., 2011). Even the costly
purification requirements can be eliminated where plant
tissue containing the recombinant protein/antigen is
consumed directly as food material (Gunn et al., 2012).
In terms of SWOT (strengths, weaknesses, opportunities
and threats) the advantage of transgenic plants and their
potential to mass-produce recombinant proteins becomes
very distinctive over the conventional approaches
(Raskin et al., 2002).
The plant-based expression system for cost-
effective production of recombinant proteins is therefore
more affordable as compared to conventional systems
(Table 1).
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Parameter
Transgenic
plants
Bacteria Yeast Insect cells
Mammalian
cells
Capital cost Low Medium Medium High Very High
Operating cost Low Low Medium High Very high
Speed Slow Fast Fast Medium Slow
Multigene
engineering
Yes Yes No No No
Glycosylation Yes but absent
in chloroplasts
Absent Incorrect Yes Yes
Multimeric
assembly
Yes No No No No
Protein
folding
High Low Medium High High
Protein yield Low-High High Medium-high Medium Medium
Scale up cost Very low High High Very high Very high
Safety High Low Unknown Medium Low
Storage Very cheap Costly Costly Expensive Very
expensive
Distribution Easy Feasible Feasible Difficult Difficult

Table 1. Comparison of different protein expression systems

Transgenic plants are now being developed to
produce better food and nutrients. For example, golden
rice genetically engineered rice fortified with vitamin
A would help eliminate the effects of vitamin A
deficiency, which results in the blindness of 124 million
children around the world (Ye et al., 2000). Similarly,
the development of transgenic plants with improved
performance against biotic and abiotic stresses has
resulted in significant increase in world food production
(Herrera-Estrella et al., 2005).
Plants are unique in a sense that they possess
three distinct genomes in different cellular
compartments: nucleus, plastids and mitochondria; each
one equipped with its own genetic system of DNA
replication, repair, recombination, transcription,
processing of RNA and translation (Maliga and Bock,
2011). Over the past few years, significant developments
have been made to use a diverse range of plant species
for the expression of recombinant proteins (Demain and
Vaishnav, 2009) using different parts such as leaves,
seeds, cell suspension cultures, and more recently
temporary immersion bioreactors. The technical aspects
of all these plant-based approaches have been discussed
elsewhere (Chichester et al., 2009; Orzaez et al., 2009;
Shih and Doran, 2009; Boothe et al., 2010; Komarova et
al., 2010; Pogue et al., 2010; Hensel et al., 2011;
Michoux et al., 2013). It is noteworthy that the majority
of recombinant proteins expressed in plants have been
produced via nucleus (Paul and Ma, 2011; Bosch et al.,
2013; Okuzaki et al., 2013).
Expressing proteins through plant nucleus,
however, has met several challenges such as poor and
variable protein expression levels, unwanted DNA re-
arrangements, gene silencing and the risk of out-crossing
of the transgenes to the weedy relatives (Daniell, 2002).
By delivering inexpensive, extraordinary expression
levels, tight natural gene containment (mostly in
angiosperms) and site-specific integration of the
transgenes, plastid transformation has emerged as a
serious competitor of the conventional protein
production platforms. This review argues that the
chloroplast transformation technology has been shown as
a successful alternative to conventional approaches used
to engineer plants and discusses its potential for
becoming a method of choice for the production of
recombinant proteins at large scale.

II. Chloroplast: a bona fide
organelle
The characteristic feature of plant cells is the
presence of specialized organelles called plastids, which
evolved from free-living prokaryotes through the process
of endosymbiosis and became permanent cellular
inclusions of exogenous origin (Theissen and Martin,
2006; Tveitaskog et al., 2007). Chloroplasts are
observable as flat discs; usually 5-10 !m in length and 2-
5 !m in width with an average surface area of 50 !m
2

(Pyke, 1999). Chloroplasts are surrounded by a double-
membrane envelope, which is made up of galactolipids
in contrast to the rest of the cell membrane, which is
phospholipid derived. Therefore, deficiency of
galactolipids severely impairs chloroplast development
(Jarvis et al., 2000). Chloroplasts contain a fluid-like
material called stroma, which is the location of the
carbon fixation reactions (Benson et al., 1950).
Immersed in the stroma is an extensive network of
thylakoid membranes, forming stacks known as grana
(Mustardy and Garab, 2003). Thylakoids possess the
necessary machinery to carry out light capture and
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energy transduction; the process of oxygenic
photosynthesis in which oxygen is evolved as a by-
product (Anderson and Aro, 1994). A notable feature of
plastids is that they are capable of dividing within plant
cells by the process of binary fission (Pyke, 1999).
Chloroplasts are green photosynthetic plastids,
which are primarily responsible for the conversion of
sunlight into chemical energy (Waters and Pyke, 2004).
Apart from housing photosynthetic machinery, they also
have got their own genome the plastome and the
related genetic machinery, which resembles to that of the
prokaryotes (Harris et al., 1994). However, the majority
of the genetic material was either lost or transferred to
the host-cell nucleus during the course of evolution
(Martin and Herrmann, 1998; Martin et al., 1998;
Bhattacharya et al., 2007; Chan et al., 2011). The plastid
DNA (ptDNA) is found in the form of DNA-protein
complexes called plastid nucleoids, which are attached to
the inner envelope membrane of the plastids (Kuroiwa,
1991). Each nucleoid contains 10-100 copies of ptDNA
(Sugiura, 1992).
It was not until early 1970s when it was
demonstrated that the chloroplasts have their own
genome with a size ranging from 120-160 kbp except for
Epifagus virginiana (size 70 kbp) and other parasitic,
non-photosynthetic plants (Bungard, 2004). The
chloroplast genome exhibits a high copy number ranging
from 1,000-10,000 per plant cell (Sugiura, 1992). This
high copy number coupled with homologous
recombination is thought to preserve the integrity of
chloroplast genome by slowing down the mutation rates.
The ptDNA is rich in adenine (A) and thymine (T) (~60-
70% in coding region and up to 80% in non-coding
regions) as compared to cytosine (C) and guanine (G).
Therefore a high degree of propensity towards a codon
having A or T at the third position exists in the
plastid genetic machinery. One of the unique features of
the chloroplast genome in most of the crop plants is the
presence of a large inverted repeat (IR), which ranges
from 6-76 kbp in length (Palmer, 1985). The size of the
repeat is variable and accounts for most of the variations
in the plastome (Sugiura, 1992). Any transgene inserted
into IR is copied into the other by a mechanism called
copy correction thereby doubling the transgene number
(Khan et al., 2007).
The genes in the repeats are therefore present
twice in the genome. The regions outside the repeats are
called large single-copy (LSC) and small single-copy
(SSC) regions. The chloroplast genome consists of 120
to 135 genes, of which 76 encode different proteins,
whereas, the rest encode RNAs (Rivas et al., 2002).
Genes present on the ptDNA can be divided into three
groups: a) photosynthetic (~46 in number), b) involved
in in-house genetic system (~21 genes plus four rRNAs)
and c) involved in miscellaneous functions. The
miscellaneous group represents a mixed bag including
some open reading frames known as hypothetical
chloroplast reading frames (ycf). Some of which are
conserved across different species, while some are not.
All of the proteins encoded by the ptDNA remains
within the chloroplast and are not exported. Majority of
the genes are organized into operons and therefore
transcribed in a polycistronic fashion (Sugiura, 1992).
The genome organization of the tobacco plastome is
shown in Figure 1.
Although plastids have retained many of the
prokaryotic features, the gene expression mechanism in
plastids is undauntedly complex. Transcription is carried
out by the concerted action of two different types of
RNA polymerase complexes. One of them, plastid-
encoded polymerase (PEP) is encoded by four plastid
genes: rpoA, rpoB, rpoC1, and rpoC2. This PEP is
similar to that of E. coli polymerase and initiates
transcription by recognizing -35 and -10-like conserved
recognition sequences. However, it is much more
specific than its bacterial counterpart. Its specificity is
determined by the nuclear-encoded sigma factor.
The expression of such sigma factors is
controlled by the light, and therefore PEP activity is
dependent on the light-controlled sigma factors present
in the nucleus. The second polymerase is a nucleus-
encoded polymerase (NEP), a single component
molecule that resembles to the polymerases found in
bacteriophage and yeast mitochondria.
Initially, the NEP is expressed in nucleus and is
imported to proplastids, where it transcribes genes
essential for plastid genetic system such as components
of PEP, for example. Once the PEP is assembled and
other necessary components of transcription such as
sigma factors and ribosomes are recruited, it starts
transcribing photosynthetic genes. As soon as transcripts
of PEP become available, the activity of NEP is
repressed, for example, by binding of glutamyl t-RNA to
NEP, so as PEP becomes the dominant RNA polymerase
in mature green chloroplasts. In non-green plastids such
as those present in roots where sigma factors are not
available due to the light, the NEP continues to remain
the dominant enzyme for transcription of photosynthetic
genes.
Before the translation of messages occurs,
newly synthesized transcripts undergo a series of
refinement processes such as cutting of polycistrons into
monocistrons, RNA editing and removal of introns to
produce mature transcripts.
A plastid mRNA molecule ready for translation
exhibits several features required for efficient translation
such as 5' untranslated regions required for suitable
context, translation initiation codon, translation
termination codon, and the 3' untranslated regions
needed for transcript stability. All these features,
therefore, have a strong influence on the yield of final
product (Hartz et al., 1991). It has been shown that
protein expression levels can be significantly increased
or decreased by the judicious use of such elements. For
example, the levels of 5-enolpyruvylshikimate-3-
phosphate synthase (EPSPS) accumulation in tobacco
plastids were increased by 10,000 fold by simply using a
different 5' UTRs (Ye et al., 2001).
Initiation of translation in plastids begins by
stepwise binding of 30S and 50S ribosomes with the
help of several initiation factors imported from the
nucleus into plastids at a location on the chloroplast
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17
genome known as ribosomal binding sites (RBS).
Elongation of the polypeptide takes place with the help
of t-RNA molecules, all of which are transcribed by the
plastid genome. The elongation factors involved in the
process are all nuclear encoded, and therefore are
imported from the cytoplasm. After reaching the
termination codon, the ribosomal complex disassociates
itself from the mRNA molecule and newly translated
polypeptide is released.


Figure 1: Structural map of tobacco chloroplast genome. Physical map of the tobacco chloroplast genome showing
the organization of different plastid genes. Genes on the inside are transcribed clockwise, whereas, genes on the
outside are transcribed anticlockwise. The open reading frames of unknown functions are shown by ycf (hypothetical
chloroplast reading frames) plus designation number. The map was drawn by a web-based tool, OGDRAWV1.1
(http://ogdraw.mpimp-golm.mpg.de/ssi_faqs.shtml) using the sequence information of tobacco chloroplast from
GenBank under the accession number Z00044. Abbreviations: LSC = Large single-copy region, SSC= Small single-
copy region, IR = Inverted repeat




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Figure 2: Schematic representation of the assembly of chloroplast transformation vectors. Chloroplast transformation vectors consist of
a selection cassette and an expression cassette, both of which are placed within chloroplast sequences called flanking regions. Flanking
regions are essentially plastome regions where insertion of the transgene is made. Commonly used regulatory elements for driving
expression of transgene as well as selectable markers and the exploited insertion sites are shown. Abbreviations used: GOI = Gene of
interest; UTR = Un-translated regions


Figure 3: Schematic representation of the events involved in chloroplast transformation by biolistic delivery system. (A)
Transformation. 4 to 6-week-old leaves are selected for transformation; DNA is coated on the surface of microparticles of gold or
tungsten and then shot onto the leaf surface with a great force using a helium-driven particle delivery device (gene gun). (B)
Regeneration. Leaves are then cut into small leaf discs after 48 hours of incubation in dark and put on selection media. Primary
shoots generally arise within 4-6 weeks. (C) Homoplastomy. Initially few copies of plastid genome are transformed and leaf cells
are called heteroplastomic. Homoplastomy, a state where all copies of chloroplast genome are transformed, is achieved by
performing few rounds of regeneration under selection. The putative transgenic shoots are then further characterized for
transgene integration and protein expression accordingly.
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III. Transforming chloroplasts:
the state of the art
Transformation of chloroplasts consists of
series of events, which include construction of vectors
tailored for high-level expression in chloroplasts (Figure
2), delivery of transgenes into chloroplast genome
(Figure 3A) and then recovery of the transplastomic
plants (Figure 3B, C). Since the procedural details of all
these steps are beyond the scope of this article, the
reader is therefore referred to the detailed protocol
published on transformation of the chloroplasts by the
Daniell Laboratory (Verma et al., 2008). The foremost
requirement of transforming chloroplasts is the
construction of expression vectors, which serves as a
vehicle to integrate the transgene(s) into the plastome.
A schematic design of chloroplast
transformation/expression vector is shown in Figure 2.
Typically, it contains a selection cassette, an expression
cassette and two flanking regions taken from the
plastome where insertion of the transgenes is intended.
The incorporation of ptDNA fragments in the final
vector is to allow the delivery of the transgenes at the
chosen site in the plastome using homologous
recombination.
Once the vector has been constructed, providing
all the necessary regulatory elements required for the
integration of transgenes into ptDNA as well as to
achieve an efficient transcription and translation, the
next step is to deliver the expression cassettes to the
chloroplasts. The classical approach to deliver the
transgene into chloroplast is the biolistic process
(Boynton et al., 1988; Svab et al., 1990) in which DNA
is coated on the surface of nanoscale particles, for
example, tungsten or gold and then bombarded onto the
leaves with a great force (Figure 3A). Other methods
include PEG-mediated transformation (Tyagi et al.,
1989; Sporlein et al., 1991) and microinjection
(Knoblauch et al., 1999). The PEG-mediated
transformation of the chloroplasts is not very practical in
terms of transformation efficiency and regeneration,
which remains very poor (Koop and Schweiger, 1985).
In case of microinjection, no successful regeneration has
been reported yet.
After transformation of the transgenes into
chloroplasts, the explant is then tissue cultured in the
presence of a selection marker to recover the
transplastomic plant lines. Initially, few copies of the
plastome are transformed; the transformants are
therefore a mixture of both transformed and wild-type
ptDNA (Figure 3B), a state known as heteroplastomy.
However, with repeated regeneration cycles under
constant selection pressure, these non-transformed or
wild-type copies are gradually bleached out, resulting in
a homoplastomic (with all copies of plastome
transformed) transplastomic plant line (Figure 3C)
(Maliga, 2004). The aminoglycoside antibiotics such as
spectinomycin, streptomycin or kanamycin are
frequently used for recovering stable homoplastomic
plants. These antibiotics block protein synthesis by
binding to a 30S, the smaller subunit, of a prokaryotic-
type 70S ribosome or by inhibiting ribosomal
translocation during translation, and therefore provide
selection by preventing cell division, greening and shoot
formation (Maliga, 2004). The aadA selection cassette
provided in the expression vector encodes an enzyme
called aminoglycoside 3''-adenylyltransferase type A. It
confers resistance to spectinomycin and streptomycin by
adding an ATP moiety, which results in the destruction
of inhibitory activities of these antibiotics to the
ribosomes.
Although organogenesis through repeated
cycles of regeneration under selection pressure is the
preferred strategy to recover homoplastomic lines
(Ahmad et al., 2012a; Ahmad et al., 2012b; Scotti et al.,
2012; Lu et al., 2013), some studies have also used
somatic embryogenesis to recover homoplastomic plants
(Kumar et al., 2004a; b). Use of embryogenesis offers an
opportunity to extend technology to non-green plastids
as well, which is a bottleneck in making technology
available to other plant species.
The integration of transgenes at a pre-
determined location in the plastid genome using
homologous recombination is the hallmark of chloroplast
transformation technology. Therefore, all types of
vectors used in transformation of the chloroplasts
include two fragments from the chloroplast DNA around
a region, where insertion of the gene(s) is desired
(Figure 4).
This homologous recombination, however,
requires a prior knowledge of the sequence of the
plastome region where insertion is to be made. It is
indeed a barrier in the expansion of chloroplast
transformation technology to those crops whose plastid
genome has not been sequenced yet. Although attempts
have been made to develop universal transformation
vectors to maximize the potential of chloroplast
transformation technology (Daniell et al., 1998; Sidorov
et al., 1999; Ruf et al., 2001), the transformation
efficiency, however, was compromised. For example,
when flanking regions of petunia were used for
chloroplast transformation in tobacco, the transformation
efficiency was significantly reduced (DeGray et al.,
2001).
Recently, a study reported a drastic reduction,
80% and 97% respectively, in expression levels of
anthrax protective antigen (PA) and human proinsulin
(Pins) fused with the cholera toxin B subunit (CTB)
(CTB-Pins) in comparison to endogenous regulatory
elements (Ruhlman et al., 2010).

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Figure 4: Illustration of homologous recombination in chloroplasts. Chloroplast transformation vectors are
constructed with two flanking regions of the plastome where gene(s) integration is required. DNA sequences/gene of interest and the
selection marker are inserted in between these fragments. Homologous recombination between flanking fragments of vector and ptDNA
then transfers transgene(s) from the vector to the plastome.


IV. Chloroplast transformation:
the salient features
Chloroplast transformation offers a number of
attractive advantages over conventional genetic
engineering approaches such as nuclear transformation.
For example, several studies have shown that
chloroplasts are able to express proteins at extra ordinary
levels (De Cosa et al., 2001; Oey et al., 2009a; Ruhlman
et al., 2010; Michoux et al., 2011; Ahmad et al., 2012a).
This high expression level could be attributed to
the high copy number, which ptDNA exhibits. For
example, each chloroplast depending upon the tissue it
resides in contains up to 100 ptDNA copies, giving rise
to 10,000 copies per cell. Further duplication of the
transgene if inserted into the inverted repeat region
would double the copy number of the transgene per plant
cell. This feature of chloroplast genome makes it highly
beneficial for the expression of recombinant proteins in
large quantities.
The other attractive feature of transforming
chloroplasts is in the fact that chloroplasts are not
normally transmitted through pollen and exhibit, in most
of the crop plants, maternal inheritance (Bogorad, 2000).
Though, very low frequency (0.01%) of leakage of
plastids with pollen has been reported in tobacco plants
(Svab and Maliga, 2007), this mode of inheritance
provides a natural means of preventing unwanted gene
flow from transgenic plants to non-transformed plants
and weedy relatives.
Due to a prokaryotic origin, genes in
chloroplasts are arranged in operons and therefore are
transcribed in a polycistronic manner; thus providing an
opportunity of engineering a whole metabolic pathway.
Taking the advantage of this feature, several studies have
reported successful engineering of various metabolic
pathways in higher plant chloroplasts such as enhancing
!-carotene and vitamin E conent (Vidi et al., 2006;
Wurbs et al., 2007; Lu et al., 2013; Yabuta et al., 2013).
Transformation of chloroplasts allows multi-gene
genetic engineering, which means several genes can be
transformed in a single transformation event. Genetic
studies have shown that several traits are controlled by a
number of genes. For example, nitrogen-fixation-enzyme
complex from Klebsiella pneumoniae is encoded by
twenty different genes (Arnold et al., 1988), whereas,
polysaccharide formation gene cluster in Streptococcus
is made up of sixteen genes (Cieslewicz et al., 2001).
Engineering such traits via nucleus would require several
transformation events and several backcrosses, which is
time consuming. The situation is further aggravated by
the fact that gene expression levels remain low and
variable. For example, it took seven years to engineer
the !-carotene biosynthetic pathway in rice to fortify
vitamin A (Ye et al., 2000).
On the other hand, transformation of a complete
Cry2Aa2 pathway into tobacco chloroplasts could be
accomplished in a single transformation event, resulting
in an expression as high as 45.1% of total soluble
proteins (TSP) (De Cosa et al., 2001).

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21
Similarly, a recent study demonstrated a
successful transformation of a 50-kbp DNA fragment
into tobacco chloroplasts (Adachi et al., 2007). The
introduced DNA was not only stable in chloroplasts but
was successfully passed on to the next generation,
demonstrating the remarkable capacity of chloroplasts to
accept foreign DNA, and the extent to which metabolic
pathways can be engineered in plastids.
Chloroplasts are packed into a double
membrane layer, and with no protein export mechanism
installed, therefore, their transformation offers a natural
compartmentalization of recombinant proteins within the
plant cell. This means that even toxic proteins can be
expressed in chloroplasts without having lethal effects
on plant phenotype. For example, plants expressing even
low-levels of cholera toxin B subunit (CTB) via nucleus
showed a significant growth inhibition. When the same
protein was expressed in tobacco chloroplasts, the host
plants did not show any symptoms of growth retardation
even with 410-3,300-fold higher CTB accumulation in
transplastomic plant leaves (Daniell et al., 2001).
However, the downside of this technology is
that chloroplasts do not glycosylate proteins.
Glycosylation the enzymatic attachment of
polysaccharide units to the proteins is one of the
common post-translational modifications, and is often
considered necessary for the protein stability. However,
experiments in our lab as well as others have shown it is
not always a stringent requirement for proper folding of
the proteins (Kim and Langridge, 2004; Huang et al.,
2006; Arlen et al., 2007). For example, xylanases are
single chain glycoproteins and when expressed in
tobacco chloroplasts were observed to be as active as its
bacterially-produced version (Leelavathi et al., 2003). In
another study, Arlen et al. (2007) expressed Type I
Interferon "2b (IFN-"2b), a member of human
glycoprotein family (cytokines), in tobacco chloroplasts
and reported that the chloroplast-made INF-"2b induced
up-regulation of MHCI molecules at the level
comparable to its commercially available counterpart
(Arlen et al., 2007). However, more studies are needed
to determine the level of effect the glycosylation may
have on protein stability by taking a range of
glycoproteins and then comparing them with their
unglycosylated versions, for instance, by expressing
them in chloroplasts.
Similarly, the stroma of chloroplasts contains
many proteases (Kamiya and Shen, 2003) and some
degradation of the recombinant proteins has been
observed in chloroplasts (Birch-Machin et al., 2004).
For example, human insulin was unstable in tobacco
chloroplasts unless fused with cholera toxin B subunit
(CTB) (Ruhlman et al., 2007). Similarly, VP6, the most
immunogenic rotavirus subunit and a potential target for
an oral subunit vaccine was observed to be rapidly lost in
mature leaves and could only be stabilized after the
addition of the 5!-UTRs from the gene 10 of
bacteriophage T7 (T7g10) and the addition of 15
nucleotides at the 5!-end of the coding region (Inka
Borchers et al., 2012). Chloroplast transformation
technology is a tissue-culture-dependent technology, and
is therefore not available yet in those crops, which do not
respond to current tissue culture protocols such as
monocots (Cui et al., 2011), which make most of the
food crops in the form of cereals (Li et al., 2010). Table
2 gives an updated list of crop plants in which successful
transformation of chloroplasts has been reported.

V. Latest trends in chloroplast
transformation
Since the machinery to carry out photosynthesis
is present in the chloroplasts, transformation of this
organelle therefore offers an invaluable tool to
understand how photosynthesis works at molecular
levels. Employing homologous recombination feature
naturally available in plastids, an insertion or deletion
mutation can be introduced to disrupt the function of an
important gene and then study its effect on plant
phenotype. Using this approach, the functions of a
number of genes have been elucidated. However, study
of essential genes for plant survival was no more
successful using this approach. More recently, the
development of inducible gene expression system allows
suspending the function of essential genes, which are
critical to plant survival, and therefore cannot be studied
by conventional approaches (Verhounig et al., 2010;
Ramundo et al., 2013).
Transformation of chloroplasts has become a necessary
tool for plant scientists to directly test mathematical
modelling for enhancing photosynthesis (Sharwood et
al., 2008; Whitney et al., 2011a; Whitney et al., 2011b;
Hanson et al., 2013; Parry et al., 2013; Price et al.,
2013). The development of the
cm
trL tobacco master line,
for example, has emerged as a useful tool to rapidly
analyse recombinant RuBisCOs constructed from
diverse sources (Whitney et al., 2011a; Parry et al.,
2013). Adaptation of high throughput cloning techniques
such as Gateway

technology for the construction of

expression vectors for higher plant chloroplasts would
allow a speedy and restriction-ligation independent
assembly of multiple gene constructs in one simple
reaction (Gottschamel et al., 2013).
Higher plant chloroplasts have been used to
express difficult proteins whose expression was not
otherwise possible such as cell-wall degrading enzymes
(Leelavathi et al., 2003; Yu et al., 2007; Gray et al.,
2009; Petersen and Bock, 2011), next-generation
antibiotics (Oey et al., 2009a; b) and membrane proteins
(Singh et al., 2008; Ahmad et al., 2012b). Several recent
advances such as engineering of metabolic pathways in
higher plant chloroplasts such as tobacco, tomato and
lettuce (Vidi et al., 2006; Lu et al., 2013; Yabuta et al.,
2013) and the ability to engineer the plastome of non-
green plastids (Zhang et al., 2012; Lu et al., 2013) has
opened the doors for engineering novel metabolic
pathways in plants using plastid transformation.




Gene Therapy and Molecular Biology Vol 15, page 14

22
Similarly, the use of new selection markers
would help to expand the technology to other crops such
as cereals (Li et al., 2010). These developments set
chloroplasts to become even more attractive platforms
for the low cost production of high-value targets such as
therapeutics, vaccine antigens and commercial enzymes
at large scale. Table 3 gives a comprehensive list of
different foreign proteins expressed in chloroplasts along
with their expression levels and various traits engineered
using plastid transformation technology.





Crop Protein/trait Gene Reference
Alfalfa Aminoglycoside adenylyl transferase,
Green fluorescent protein
aadA, gfp Wei et al. (2011)
Arabidopsis Aminoglycoside adenylyl transferase aadA Sikdar et al. (1998)
Cabbage Aminoglycoside adenylyl transferase aadA, uidA Liu et al. (2007)
Carrot Aminoglycoside adenylyl transferase,
Betaine aldehyde
aadA, badh Kumar et al. (2004a)
Cauliflower Aminoglycoside adenylyl transferase aadA Nugent et al. (2006)
Cotton Aminoglycoside transferase,
Neomycin phosphotransferase II
aphA6, nptII Kumar et al. (2004b)
Eggplant Aminoglycoside adenylyl transferase aadA Singh et al. (2010a)
Lettuce Aminoglycoside adenylyl transferase,
Green fluorescent protein
aadA, gfp Lelivelt et al. (2005)
Oilseed
rape
Aminoglycoside adenylyl transferase,
Crystal protein insecticidal
aadA,
cry1Aa10
Hou et al. (2003)
Petunia Aminoglycoside adenylyl transferase,
#-Glucuronidase
aadA, uidA Zubko et al. (2004)
Poplar Fusion protein aadA/gfp Okumura et al. (2006)
Potato Aminoglycoside adenylyl transferase,
Green fluorescent portein
aadA, gfp Sidorov et al. (1999)
Rice Aminoglycoside adenylyl transferase,
Green fluorescent portein
aadA, gfp Lee et al. (2006)
Soybean Aminoglycoside adenylyl transferase aadA Dufourmantel et al. (2004)
Sugarbeet Aminoglycoside adenylyl transferase,
Green fluorescent portein
aadA, gfp De Marchis et al. (2008)
Tobacco Aminoglycoside adenylyl transferase aadA Svab et al. (1990a)
Tomato Aminoglycoside adenylyl transferase aadA Ruf et al. (2001)
Wheat Neomycin phosphotransferase II nptII, gfp Cui et al. (2011)

Table 2. Crops in which plastid transformation has been achieved
Note: Only first report is included in the table.

Ahmad & Mukhtar: plastids for the production of foreign proteins at high levels

23




Enzymes/protein/trait Gene Host plant
Expression
observed
Reference
Biopharmaceuticals
Porcine post-weaning
diarrhoea (PWD)
FaeG Tobacco 1% DW Kolotilin et al.
(2012)
Human granulocyte
colony-stimulating factor
hG-CSF Lettuce ND Sharifi Tabar et al.
(2013)
Bacterial phage lytic
protein
plyGBS Tobacco >70% TSP Oey et al. (2009a)
Human proinsulin CTB-Pins Tobacco
Lettuce
16% TSP
2.5% TSP
Ruhlman et al.
(2007)
Human serum albumin hsa Tobacco 11% TSP Fernandez-San
Millan et al.
(2003)
Human Somatotropin hST Tobacco 7% TSP Staub et al. (2000)
IFN-" GUS-IFN-" Tobacco 6% TSP Leelavathi and
Reddy (2003)
Insulin-like growth factor IGF-1n
IGF-1s
Tobacco 32% TSP Daniell et al.
(2005)
Interferon-"2b (IFN-"2b) IFN-#2b Tobacco 21% TSP Arlen et al. (2007)
Monoclonal Ab Guy's 13 Tobacco ND Daniell et al.
(2005)
Cardiotrophin-1 rhct1 Tobacco 5% TSP Farran et al.
(2008)
"1-antitrypsin SERPINA1 Tobacco 2% TSP Nadai et al. (2009)
CTB-F.IX (Coagulation
factor IX)
CTB-FIX Tobacco 3.8% TSP Verma et al.
(2010)
Retrocyclin-101-GFP RC101-
GFP
Tobacco 38% TSP Lee et al. (2011)
Protegrin-1-GFP PG1-GFP Tobacco 26%TSP Lee et al. (2011)
Endolysin Cpl-1 cpl-1 Tobacco 10%TSP Oey et al. (2009b)
Endolysin Pal pal Tobacco 20% TSP Oey et al. (2009b)
Thioredoxin 1 hTrx1 Lettuce 1% TSP Lim et al. (2011)
Aprotinin APR Tobacco 0.5%TSP Tissot et al. (2008)
Enzymes/biomaterials
Cellulases bgl1C,
cel6B,
cel9A,
xeg74
Tobacco 5-40% TSP Petersen and Bock
(2011)
Elastin-derived polymer eg121 Tobacco ND Guda et al. (2000)
Endo-1,4-Beta-glucanase celA Tobacco 10.7% TSP Gray et al. (2009)
Exo-cellobiohydrolase celB Tobacco 3 % TSP Yu et al. (2007)
Fibronectin extra domain
A
EDA Tobacco 2 % TCP Farran et al.
(2010)
Monellin monellin Tobacco 2.5% TSP Roh et al. (2006)
p-Hydroxybenzoic acid ubiC Tobacco 13-18% TSP Viitanen (2004)
Polyhydroxybutyrate phb operon Tobacco 18.8% DW Maclean et al.
(2007) Bohmert-
Tatarev et al. (
2011)
Trp asa2 Tobacco ND Zhang et al. (2001)
Xylanase xynA Tobacco 6% TSP Leelavathi et al.
(2003)
!-glucosidase Bgl1 Tobacco ND Jin et al. (2011)
Gene Therapy and Molecular Biology Vol 15, page 14

24
Vaccine Antigens
Anthrax protective antigen pagA Tobacco 29% TSP Ruhlman et al.
(2010)
Cholera toxin B-
Proinsulin fusion protein
CTB-pins Tobacco 72% TSP Ruhlman et al.
(2010)
Cholera Toxin B Subunit CTB-AMA1
CTB-MSP1
Tobacco 13% TSP
10% TSP
Davoodi-
Semiromi et al.
(2010)
CTB-fibronectin binding
domain
CTB-D2 Chlamydomonas 23% TSP Dreesen et al.
(2010)
HIV-1 Gag structural
Poly-protein
Pr55
gag
Tobacco 8% TSP Scotti et al. (2009)
HPV Hpv16 L1 Tobacco 26%TSP Fernandez-San
Millan et al.
(2008)
HPV16-E7 Chlamydomonas 0.12% TSP Demurtas et al.
(2013)
Human b-site APP
cleaving enzyme
hBACE Tobacco 2% TSP Youm et al. (2010)
LTB fused with
Hemagglutinin
Neuraminidase
neutralizing
Epitope
LTB-HNE Tobacco 0.5% TSP Sim et al. (2009)
Heat labile toxin B subunit
fused with the heat stable
toxin
LTB-ST Tobacco 2.3% TSP Rosales-Mendoza
et al. (2009)
Vaccinia virus envelope A27L Tobacco 18% TSP Rigano et al.
(2009)

Amoebiasis LecA Tobacco 6.3% TSP Chebolu and
Daniell (2007)
Anthrax protective antigen pag Tobacco 18% TSP Koya et al. (2005)
Canine parvovirus (CPV) CTB-2L21
GFP-2L21
Tobacco 22.6-31%
TSP
Molina et al.
(2004; 2005)
Cholera toxin CtxB Tobacco 4.1% TSP Daniell et al.
(2001a)
Lyme disease OspAOspA-
T
1-10% TSP Glenz (2006)
Rotavirus vp6 Tobacco 0.3-3% TSP Birch-Machin et
al. (2004)
Malaria vaccine antigens CTB-AMA1 Tobacco
Lettuce
13.2% TSP
7.3% TSP
Davoodi-
Semiromi et al.
(2010)
CTB-MSP1 Tobacco
Lettuce
10.1% TSP
6.1% TSP
Davoodi-
Semiromi et al. (
2010)
Dengue virus DENV-
1,2,3,4
Lettuce ND Maldaner et al.
(2013)
Anti-Toxoplasma GRA4 Tobacco 6 !g/g FW Ycono et al.
(2012)
Tuberculosis antigens CTB-ESAT6
CTB-
Mtb72F
Tobacco 7.5% TSP
1.2% TSP

Lakshmi et al.
(2013)
CTB-ESAT6 Lettuce 0.75% TSP Lakshmi et al.
(2013)
Tetanus toxin TetC Tobacco 10-25% TSP Tregoning et al.
(2003)
Hemorrhagic colitis EIT Tobacco 1.4% TSP Karimi et al. 2013
Ahmad & Mukhtar: plastids for the production of foreign proteins at high levels

25
Agronomic Traits
Cytoplasmic male sterility phaA Tobacco ND Ruiz and Daniell
(2005)
Disease resistance msi-99 Tobacco ND DeGray et al.
(2001)
Drought tolerance tps1 (yeast) Tobacco ND Lee et al. (2003)
Herbicide resistance aroA Tobacco ND Daniell et al.
(1998)
Herbicide resistance bar Tobacco ND Iamtham and Day
(2000)
Insect resistance cry1A(c) Tobacco 5% TSP McBride et al.
(1995)
cry2Aa2 Tobacco 3% TSP Kota (1999)
cry2Aa2
Operon
Tobacco 45.3% TSP De Cosa et al.
(2001)
cry1Aa10 Oilseed rape ND Hou et al. (2003)
cry1Ab Soyabean ND Dufourmantel et
al. (2005)
cry9Aa2 Tobacco 10% TSP Chakrabarti et al.
(2006)
Phytoremediation merA/merB Tobacco ND Ruiz et al. (2003)
Hussein et al.
(2007)
Salt tolerance badh Carrot ND Kumar et al.
(2004a)

Table 3: Heterologous expression of recombinant proteins and vaccine antigens in higher plant chloroplasts
Abbreviations: TSP: Total soluble proteins, TCP: Total cellular proteins, ND: Not determined, FW: Fresh weight,
DW: Dry weight
VI. Conclusion and future
perspectives
Although a large number of proteins have been
expressed in plants via chloroplast transformation
technology, no chloroplast-made product has reached to
the market yet. One of the major reasons for this vacuum
is that the technology is heavily patented and therefore is
not yet open to commercial entrepreneurs. However, the
expiry of patents in the near future should result in the
removal of such barriers. Other factors include time
course involved in the development of transplastomic
plants, costs associated with the purification of
recombinant proteins, and lack of well-characterized
good manufacturing practices (GMPs) and regulations
for plant-made pharmaceuticals. Successful
commercialization of plant-made pharmaceuticals would
require the establishment of rapid and non-
chromatographic techniques for the purification of
recombinant proteins, facile transformation and

regeneration methodologies including new class of
selection markers to be used in the recovery of
transplastomic plants, and the availability of GMPs used
to regulate the production of pharmaceuticals in plants.
Similarly, the future attempts must focus on the
transformation of non-green plastids such as roots, tubers
and other storage organs, which are directly consumed
up by mammals and humans.

VII. Acknowledgments
The work in authors lab is supported by Higher
Education Commission (HEC) and Punjab Agricultural
Research Board (PARB). We sincerely apologise to
those colleagues whose work could not be discussed here
due to space limitation.






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