Download Green Factory: Recombinant Protein Production in Chloroplasts

Green Factory: Recombinant Protein
Production in Chloroplasts
R
ecombinant proteins are an increas-
ingly important component of medicine and applied chemistry. They are
needed for a vast range of applications including therapeutics, vaccines, diagnostics and enzymes. Exciting new technologies now exist for
the large-scale production of these
bio-molecules in transgenic plants,
often termed molecular farming. An
innovative approach exploits plant
organelles as a production platform
by genetic manipulation of the
chloroplast genome.
Uses of Recombinant Proteins
With the understanding of the molecular
basis of many diseases it now becomes
possible to treat these with customized
biomolecules, mostly proteins. These
include enzymes (e.g. treatment of
phenylketonuria), growth or regulatory
factors (e.g. insulin), vaccines (e.g. hepatitis antigen), antibodies (e.g. antiHer2) or structural substances (e.g. collagen). Recombinant proteins are
preferable to the extraction from natural
sources due to risks of contamination
with pathogenic agents such as prions or
viruses.
Industrial and food chemistry (e.g. cellulases for paper production, proteinases
for detergents or chymosin for cheese)
also benefit from the availability of synthetic enzymes.
Currently, most recombinant proteins
originate from genetically engineered
bacteria. Other sources are eukaryotes
like yeast, human or animal cell lines or
even transgenic animals. Compared to
these systems the production costs in
plants are low and an almost unlimited
scale-up is rapidly possible.
Chloroplast Transformation
The genetic information in plants is distributed between three cellular compartments, the nucleus, the mitochondria
and the plastids (Fig. 1). Until recently
transgenic plants were exclusively generated by introducing foreign genes into
the nuclear genome. Here we focus on
plastid transformation as an alternative.
Plastids have their own rudimentary
genome, the plastome, which encodes
around 120 genes [1]. Plastids in green
tissues are “chloroplasts”, whose primary function is photosynthesis.
In contrast to the nuclear genome with
typically two copies of each gene per cell,
the plastome is present in over 10,000
copies (a leaf cell has up
to 100 chloroplasts each
containing about 100
plastome molecules). Due
to this high gene dosage
the plastid represents a
very attractive compartment for high-level expression of recombinant
proteins.
Site-directed
insertion of the transgenes into the plastid
avoids ‘position effects’
and leads to uniform and
reproducible expression.
Silencing of transgenes,
also often observed in
the nucleus, is not
known for plastids. It is
possible to co-express Fig. 1: Genetic compartments of the plant cell
several genes by creating artificial operons.
Furthermore, since plastid traits in most crop
plants are not inherited
through
the
pollen,
spread of novel genes to
other crops or closely
related wild species is
prevented [2]. This is a
major contribution to
bio-safety and should
reduce
environmental
concerns relating to GM
plants.
Plastid
transformation
vectors carry a gene of
interest together with a
selection marker between
flanking sequences, which Fig. 2: Vector systems for plastid transformation. a. Standard plastid
direct site-specific inser- transformation vector with marker gene cloned between flanking setion into the plastome quences. The marker gene (M) and gene of interest (GOI) are both sta(Fig. 2a). DNA can be de- bly integrated into the transformed plastome. b. Plastid transformation
vector used for the generation of marker-free plants. Here only the GOI
livered to the chloroplast is stably integrated in the plastome. c. Plastid transformation using vivia particle bombardment sual selection.
BIOforum Europe 04/2004, p 32–34, GIT VERLAG GmbH & Co. KG, Darmstadt, Germany, www.gitverlag.com/go/bioint
[3], where microscopic gold particles
coated with DNA are propelled at high
velocity directly into intact tissues or
cells, or using polyethylene glycol (PEG),
where DNA uptake into protoplasts
(plant cells without the protective cell
wall) is induced chemically [4]. Treated
tissues or cells are then cultured in vitro
under antibiotic selection until green
resistant clones appear (Fig. 3a, b, c).
Improvements in Transplastomic
Technology
Although an established technique, applied plastid transformation still needs
improvement in several key areas: The
approach needs to be extended from the
model plant tobacco to other important
crops. Other challenges include optimisation of gene expression and protein
stability. In addition, transplastomic
plants containing antibiotic resistance
markers are undesirable. Furthermore,
inducible gene expression would allow
the production of toxic proteins, since
the vegetative and production phases
could be separated. A suite of novel technologies has been developed targeted
towards solving these problems.
Trait / Application
Herbicide resistance
resistance to PPT, e.g. Basta‚
resistance to glyphosate, e.g. Roundup‚
Pest resistance
insect resistance
Pathogen resistance
antimicrobial peptide
Pharmaceuticals
artificial blood serum
treatment of growth defects
modulation of immune response
modulation of immune response
Vaccines
tetanus immunisation
protection against gastroenteritis
cholera immunisation
protection against diarrhoea
protection against parvovirus
Miscellaneous
phytoremediation of mercury contaminated soils
polyhydroxybutyrate (PHB) synthesis
protein-based biopolymer
xylanase for paper production
tryptophan biosynthesis
trehalose synthesis for drought tolerance
Protein
phosphinothricin acetyltransferase
mutant enol-pyruvyl-shikimate-phosphate synthase
Bt (cry2Aa2) toxin
MSI-99 (magainin analogue)
human serum albumin
human growth hormone
human interferon-alpha
human interferon-gamma
tetanus toxin fragment
subunit from enterotoxigenic E. coli toxin
subunit from cholera toxin
rotavirus capsid protein
parvovirus capsid protein
mercuric ion reductase/organomercurial lyase
acetyl-CoA reductase, α-keto thiolase, phb-synthase
EG121 polymeric protein
xylanase
anthranilate synthase
trehalose phosphate synthase
Tab. 1: Recombinant proteins expressed in tobacco plastids
Recombinant Protein Expression in
Tobacco
To-date efficient and reproducible production of transplastomic plants is
mainly restricted to tobacco. Tobacco
has several features that make it an
attractive production platform for molecular farming. It produces a relatively
large amount of biomass (Fig. 3d) from
which proteins can be easily extracted
and purified. Furthermore, since it is not
used for food or feed, segregation from
materials intended for human or animal
consumption is not a problem. A number
of biotechnologically and agriculturally
interesting genes have been expressed in
tobacco chloroplasts in various laboratories (Tab. 1).
Transplastomic Crop Plants
So far other transplastomic crop plants
have only been reproducibly recovered
from potato [5] and tomato [6]. The challenge still is to extend the initial success
with plants of the nightshade family to
important crops such as wheat, rice,
maize and rapeseed.
Expression Levels
Very high yields of recombinant protein
can be achieved in plastids: Depending on
the stability of the protein these are in the
Fig. 3: Tobacco chloroplast transformation a. Antibiotic resistant shoot developing from a bombarded leaf. b. Transformed cell lines selected from a PEG-transformation experiment. c. In vitro
grown transplastomic tobacco plant. d. Glasshouse grown tobacco plants expressing high levels
of recombinant protein.
range of 1–10 % TSP (total soluble protein). High production levels depend on
the availability of suitable regulatory elements. Generally plastid transformation
vectors use separate promoters, it is however also possible to use promoter-less
vectors, in which transcription is coupled
to strongly expressed plastid genes. This
approach increases transgene stability
since inadvertent recombination between
duplicated sequences is avoided.
However, high expression levels can also
cause toxic side effects for the plant. Using an inducible expression system such
as activating plastid genes by external
application of chemicals can circumvent
this and will provide additional safety
and alleviate concerns about the production of pharmaceuticals in plants.
Transformants Without Antibiotic Resistance Markers
For the genetic stability of the transformed lines it is necessary to eliminate
all wild type plastome molecules. In this
respect antibiotic resistance markers
have two functions: firstly for the initial
selection of cells containing transformed
plastids and then for promoting loss of
wild-type copies. The presence of antibi-
otic resistance genes in transgenic plants
is of major concern to the public. In order
to address this problem, a novel visual
selection system was developed [7]. Here
an antibiotic resistance marker is still
used for the initial selection, but the
marker is automatically excluded later.
New vectors were designed, that carry
the marker gene in the vector backbone
rather than between the flanking sequences (Fig. 2b). In this configuration a
gene of interest (GOI) can be stably introduced, whereas the marker gene gets
lost via recombination once the selection
pressure is removed [8]. Visual selection
is achieved using albino mutants as an
alternative to wild-type. Such mutant
lines were created by inactivation of pigmentation-related genes (Fig. 2c). Secondary transformation of mutant tissues
with a functional copy of the pigmentation gene linked to the GOI allows the
visual recognition of transplastomic areas
through re-greening. Plants regenerated
from green sectors are marker-free.
Conclusions
High yields, unlimited scalability, stable
and predictable expression, biological
safety, inducible expression and marker
removal make chloroplasts a highly attractive platform for expression of recombinant proteins. Plastids are a truly
impressive green factory.
References
A list of references can be obtained from the
authors.
Authors picture left to right: Timothy James
Golds, Hans-Ulrich Koop, Christian Eibl
Dr. Timothy James Golds
Dr. Christian Eibl
Prof. Hans-Ulrich Koop
Icon Genetics
Research Centre Freising
Lise-Meitner-Str. 30
85354 Freising, Germany
[email protected]
www.icongenetics.com