Download Protein expression in plastids Peter B Heifetz* and Ann Marie Tuttle

157
Protein expression in plastids
Peter B Heifetz* and Ann Marie Tuttle†
Current Opinion in Plant Biology 2001, 4:157–161
including roots, contain leucoplasts. The different plastids
are further distinguished by their varying capacities for the
expression of organelle-encoded genes. Illuminated
chloroplasts, in particular, possess extraordinarily high
rates of transcription and translation. These facilitate the
accumulation of large amounts of soluble ribulose bisphosphate carboxylase (thus insuring that enzyme levels do not
limit photosynthetic carbon fixation) and allow the rapid
turnover of redox-sensitive electron transfer components,
such as the D1 protein of photosystem II [4]. This feature
makes leaf chloroplasts suitable for the production of large
amounts of recombinant proteins. In this review, we summarize recent progress in understanding plastid protein
expression and in transformation technology.
1369-5266/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Plastid transformation
The genome of the plastid has generated much interest as a
target for plant transformation. The characteristics of plastid
transgenes both reflect the prokaryotic origin of plastid
organelles and provide a unique set of features that are
currently lacking in genes introduced into the plant nucleus.
Recent progress has been made in understanding plastid
expression of recombinant proteins.
Addresses
*Torrey Mesa Research Institute, 3115 Merryfield Row,
San Diego, California 92121, USA; e-mail: [email protected]
† Syngenta Agribusiness Biotechnology Research Inc, 3054 Cornwallis
Road, Research Triangle Park, North Carolina 27709, USA
Abbreviations
aadA
adenosyl-3′-adenyltransferase
EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
GFP
green fluorescent protein
NEP
nuclear-encoded RNA polymerase
PEG
polyethylene glycol
PEP
plastid-encoded RNA polymerase
Introduction
One of the unique and defining characteristics of plants is
the presence of plastid organelles. These endosymbiotic
remnants of a once free-living cyanobacterial progenitor [1]
have, over evolutionary time, given up the vast majority of
their genes and cellular functions to become the energy
transduction and metabolic centers of the plant cell. Among
the primitive features of the cyanobacterial progenitor that
are retained in plastids is a circular and largely prokaryotic
chromosome. This genome of circa 50–290 kilobases is
remarkably similar across the algal and plant lineages with
regard to the complement of genes encoded within it, their
relative order and their sequence [2]. The plastid genome
is also distinguished by its copy number, which even in the
simplest of plants far exceeds the ploidy of the nuclear
chromosomes. In a mature leaf cell, in which there may be
as many as 100 plastids each containing upwards of 10–100
genomes, it is possible to find 10,000 or more identical
copies of each plastid gene [3].
Plastids are distributed ubiquitously throughout the differentiated cells of diverse plant organs and tissues. Although
each plant cell type contains identical copies of the plastid
genome because of their organelles’ shared biogenesis
from undifferentiated proplastids, the organelles themselves can vary tremendously in morphology and function.
Thus, leaves and green tissues contain photosynthetic
chloroplasts; mature fruits and flowers contain pigmented
chromoplasts; tubers and other storage organs contain amyloplasts or elaioplasts; and other non-green tissues,
Boynton and colleagues [5,6] demonstrated that all copies
of the plastid genome in the single large chloroplast of the
green alga Chlamydomonas reinhardtii could, following
delivery of cloned DNA by particle bombardment, integrate such DNA in a targeted manner via homologous
recombination. These pioneering studies made possible
subsequent efforts to transform the plastids of higher
plants [7]. They also laid out the unique ‘ground rules’ for
plastid transformation as compared to nuclear transformation: foreign DNA must be delivered across the double
membrane of the plastid envelope and across the cell
membrane (and across the cell wall, if present); homologous (legitimate) recombination is the normal mode of
DNA integration into the plastid genome. When suitable
regions of homology between the introduced DNA and
the target are provided, appropriate selectable markers
for plastid transformation must facilitate the segregation
of transformed genome copies at the expense of nontransformed ones; complete segregation of all the genome
copies within a plastid, and within all of the plastids of a
cell or tissue, must occur during clonal propagation in
order to confer a stably transformed phenotype; and
finally, bona fide plastid transformation events must be
readily distinguishable at the molecular level from spontaneous mutations or ectopic nuclear integrations of the
transforming DNA.
In addition to particle bombardment, polyethylene glycol
(PEG) treatment of protoplasts [8,9] has been found to
be suitable for the delivery of DNA to plastids for stable
transformation. This method appears more prone to creating unexpected nuclear mutations than is particle
bombardment, however, as demonstrated by the relatively high level of selection escapes [10] and
physiological artifacts [11] observed in PEG-transformed
tobacco. Recently, an alternative means of directly delivering DNA to plastids was developed by Knoblauch et al.
[12]. These workers used a sub-micron-diameter syringe
158 Plant biotechnology
driven by the controlled thermal expansion of a liquid
metal alloy to microinject DNA into chloroplasts.
Although this method has not yet been shown to yield
regenerated and fertile transformed plants, transient
expression of the green fluorescent protein (GFP) transgene was achieved in tobacco without apparent harm to
the injected cells.
The ease with which plastid DNA can undergo homologous recombination between even short regions of
sequence identity [13] has significant implications for the
engineering of gene expression within plastids. First,
targeted integration is the norm rather than the exception.
This means that the location of transgenes can be predicted
with precision so as to minimize interference with endogenous plastid genes and to prevent regulatory sequences
near the integration site from influencing transgene
expression. An added benefit is that comparatively few
transgenic events need to be characterized as, in the
absence of illegitimate recombination, only single-locus
insertion events occur. (An exception to this norm results
when the transforming DNA has been targeted to the
inverted-repeat region of the plastid genome. Dual integration then occurs due to homologous copy correction
between the two repeat regions.) Second, homologous
recombination occurring spontaneously between directly
repeated sequences flanking an integrated selectable
marker gene can result in the excision of the marker gene
from the genome when selective conditions are relaxed
[14]. This approach has recently been shown to be feasible
in plastids of higher plants [15••], raising the possibilities
of sequential transformation by marker recycling and of
elimination of bacterial antibiotic resistance genes from
the final transgenic plants.
A recent advance in selectable marker technology for plastids was the discovery that GFP can be stably transformed
and expressed in the leaf chloroplasts of tobacco [16,17••]
and potato [18••]. In potato, GFP was also expressed in
tuber amyloplasts, albeit at a 100-fold lower level than in
chloroplasts. Transient expression of GFP has been
achieved in carrot, marigold and pepper [12], demonstrating
that DNA can be delivered to plastids in these plant
species. An even more versatile use of GFP was developed
by Khan and Maliga [17••] who fused its coding sequence
to that of the aadA gene, which encodes adenosyl-3′adenyltransferase. The resulting fusion molecule was
functional both as an antibiotic-resistance gene and as a
fluorescent protein in multiple types of plastids in tobacco.
Moreover, following its introduction by bombardment and
selection using streptomycin, activity of this fusion molecule was detectable at low levels in the plastids of rice
suspension cells. These rice cells proved to be highly heteroplasmic, however, with only a small fraction of the
plastid genomes transformed and no plants regenerated.
Nonetheless, this remains the only successful demonstration of the stable delivery of foreign DNA to the plastids
of a cereal species.
Controlling plastid gene expression
The plastid rpoA, rpoB, rpoC1 and rpoC2 genes encode the
catalytic subunits of a eubacterial-type RNA polymerase
that recognizes upstream sequences that have high homology to the consensus –10 and –35 regions typically found
in eubacterial promoters. Promoter recognition by this
plastid-encoded RNA polymerase (PEP) is mediated by
sigma-like factors that are encoded in the plant nucleus.
These factors are expressed in a regulated manner in
response to environmental or developmental cues and are
imported post-translationally into plastids via transit peptides [19]. Interestingly, plastids contain a second (and in
spinach, likely a third) complete transcriptional apparatus
that is entirely nuclear encoded. This apparatus contains a
single-subunit that is phylogenetically related to the RNA
polymerases of fungal and plant mitochondria, and of bacteriophages such as T7 and T3 [20–22]. The promoter
elements recognized by the nuclear-encoded RNA polymerase (NEP) bear little similarity to eubacterial or PEP
promoters [23] and require one or more specificity factors
for their correct interaction with the polymerase [24].
Plastid genes can have only PEP promoters, only NEP
promoters, or hybrid promoter regions that contain both
PEP and NEP elements. The significance for message
accumulation of these multiple transcription initiation
sites is unclear. A recent study suggests, however, that the
NEP may recognize DNA promiscuously and, thus, could
be capable of transcribing any plastid gene [25].
Nonetheless, PEP and NEP elements are each capable
of directing the expression of foreign genes in
plastids [26,27].
Trans-activation expression systems using completely heterologous RNA polymerases, such as those from T7 phage,
have also been developed for plastids [28,29•]. This
approach allows the efficient and controlled transcription
of target plastid transgenes as long as appropriate promoters,
which are recognized by the T7 polymerase but not by
NEP or PEP, are located upstream. The primary advantage
of trans-activation is that it allows the imposition of developmental, tissue-specific, or chemically-inducible regulation
upon the expression of plastid transgenes through control
of the polymerase by nuclear promoters of the desired
specificity. Using the tobacco PR-1a promoter [30] to
direct the expression of a plastid-targeted T7 polymerase,
β-glucuronidase (GUS) and a cellulose-degrading enzyme
were expressed at high levels in tobacco chloroplasts in
response to foliar application of an inexpensive fieldregistered compound [29•].
The production of primary transcripts is not typically the
limiting step for the expression of plastid genes, although
this step does limit plant nuclear expression and, to some
degree, eubacterial expression. Instead, the rate of protein
synthesis in plastids is much more dependent on posttranscriptional processes, which include transcript
processing and stability, the conversion (by post-transcription
RNA-editing mechanisms) of cytidines to uridines in certain
Protein expression in plastids Heifetz and Tuttle
editing-dependent plastid messages, and translational initiation or elongation on polyribosomes. These events are
mediated at the RNA level by the binding of nuclearencoded factors to the 5′ untranslated leader and 3′
untranslated trailer regions of the plastid messages [31]
and, in the case of RNA editing, by the site-specific interaction of an editing complex with RNA segments flanking
the targeted nucleotides [32,33]. The efficient expression
of transgenes in plastids thus requires not only the use of
appropriate promoters but also the presence of the correct
sequences in the 5′ and 3′ untranslated regions. The
occurrence of RNA editing also necessitates care in the
choice of the cloned sequences flanking a plastid expression cassette that are designed to direct homologous
integration into the plastid genome. Editing sites, but not
editing itself, may be conserved among related plant
species [34]. Hence, it is important to prevent the inadvertent introduction of editing-dependent sequences into
a genetic background that lacks the capacity for correct
processing when flanking sequences from one plant
species are used to direct integration into the plastids of a
second plant species [34].
Uniparental inheritance of plastid genes
Another unusual feature of plastid genes is their nonMendelian mode of inheritance. In gymnosperm plants,
plastid DNA is maintained in sperm cells but not egg cells
and is therefore transmitted uniparentally by the male. In
the majority of angiosperms (including most of the important crops with the exception of alfalfa and to a limited
extent potato), however, it is the pollen that loses the plastid
DNA after mitosis I. Consequently, pollen cannot transmit
the contents of the plastid genome to the zygote [35]. This
could, in theory, prevent the spread of plastid transgenes
through pollen to neighboring crops or related wild
species [36•].
Recombinant protein expression in plastids
The plastid genome is an attractive location for the engineering of pest-resistance and herbicide-tolerance traits,
which typically benefit from high and stable levels of gene
expression. Insecticidal Bt proteins derived from Bacillus
thuringienesis cry genes are particularly amenable to plastid
expression in tobacco. McBride et al. [37] and, more
recently, Kota et al. [38•] demonstrated that plastid transformation with native bacterial cry sequences, produces
extraordinarily high levels of protein accumulation as compared to nuclear transformation with codon-optimized
transgenes. The resulting leaf tissue is extremely toxic to
target insect larvae. In a recent follow-up study [39••], it
was found that the introduction of the complete Bt cry2Aa2
operon into tobacco plastids caused even higher levels of
expression than did the single genes. This was accompanied by the formation of cuboidal Bt protein crystals.
This work is the first to demonstrate the expression of a
natural bacterial operon in plastids and follows on from the
initial report by Staub and Maliga [40] of chimeric operon
expression from the tobacco plastid genome. Daniell et al.
159
[41] demonstrated that expression of a wild-type
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
gene and its complete transit-peptide sequence from petunia
in the plastids of tobacco resulted in a ten-fold-greater
level of tolerance to the herbicide glyphosate. The expression of additional herbicide-tolerance genes in plastids,
including mutant EPSPS, protoporphyrinogen IX oxidase,
and several bacterial genes, has been described recently in
the patent literature [29•].
One of the most exciting potential applications of plastid
protein expression is in the production of recombinant
proteins for industrial, pharmaceutical or other valueadded uses. The biological containment afforded by
plastid localization is attractive from the standpoint of
reducing the risks of the pollen transmission of genes
that may encode metabolically active proteins.
Moreover, the high levels of expression that have been
reported for plastid-encoded proteins could effect substantial cost savings in cases in which large amounts of
tissue must be processed in order to yield purified molecules. Plastids are also capable of disulfide-bond
formation and hydrolysis via their endogenous thioredoxin and protein disulfide isomerase enzyme systems.
Mammalian proteins, such as the growth hormone somatotropin, normally require passage through the
endoplasmic reticulum in order to attain their mature
disulfide-bonded conformation. Recently, Staub et al.
[42••] demonstrated that metabolically active human
somatotropin could be expressed and correctly processed
in the plastids of tobacco where it accumulated to 7% of
total soluble protein. In contrast to this successful
expression of a mammalian protein, attempts by Guda et
al. [43] to express a protein biopolymer derived from
mammalian elastin were disappointing. These authors
found that although the synthetic biopolymer gene consisting of multiple repeating units of the amino-acid
sequence GVGVP was transcribed effectively in tobacco
plastids, the accumulated message was either not readily
translatable by the plastid ribosomes or was rapidly
turned over. Consequently, the levels of polymer product
detected in plastids were low and thus highly inefficient
compared to expression of the same recombinant protein in the plant nucleus.
Conclusions and future prospects
Plastid biotechnology has now progressed to the point at
which a range of proteins from prokaryotic and eukaryotic
sources have been expressed effectively in leaf chloroplasts
and other plastids of tobacco. Progress is still needed in the
improvement of transformation technology (to expand the
range of plant species that can be efficiently transformed)
and in the control of heterologous gene expression in
plastids at the post-transcriptional level. Overcoming these
challenges will result in a means of producing high levels
of recombinant proteins or pathway-derived metabolites in
a stable and predictable manner with the potential for
robust physical and genetic containment.
160 Plant biotechnology
Acknowledgements
The authors thank Eric Boudreau for a critical reading of
the manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
resistant to high levels of this antibiotic because of naturally occurring mutations in their 16S ribosomal RNA.
18. Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM,
•• Nehra NS: Stable chloroplast transformation in potato: use of
green fluorescent protein as a plastid marker. Plant J 1999,
19:209-216.
The authors describe both the first published use of GFP as a plastid marker
and stable plastid transformation in a food crop. Their results show a greater
than 100-fold difference in the expression of protein in chloroplasts and
amyloplasts.
19. Allison LA: The role of sigma factors in plastid transcription.
Biochimie 2000, 82:537-548.
1.
Moreira D, Le Guyader H, Philippe H: The origin of red algae and
the evolution of chloroplasts. Nature 2000, 405:69-72.
2.
Gray MW: Evolution of organellar genomes. Curr Opin Genet Dev
1999, 9:678-687.
3.
Bendich AJ: Why do chloroplasts and mitochondria contain so
many copies of their genome? BioEssays 1987, 6:279-282.
4.
Heifetz PB, Lers A, Turpin DH, Gillham NW, Boynton JE, Osmond CB:
dr and spr/sr mutants of Chlamydomonas reinhardtii affecting D1
protein function and synthesis define two independent steps
leading to chronic photoinhibition and confer differential fitness.
Plant Cell Environ 1997, 20:1145-1157.
23. Hajdukiewicz PTJ, Allison LA, Maliga P: The two RNA polymerases
encoded by the nuclear and the plastid compartments transcribe
distinct groups of genes in tobacco plastids. EMBO J 1997,
16:4041-4048.
5.
Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM,
Jones AR, Randolph-Anderson BL, Robertson D, Klein TM,
Shark KB et al.: Chloroplast transformation in Chlamydomonas
with high velocity microprojectiles. Science 1988, 240:1534-1538.
24. Bligny M, Courtois F, Thaminy S, Chang C-C, Lagrange T,
Baruah-Wolff J, Stern D, Lerbs-Mache S: Regulation of plastid rDNA
transcription by interaction of CDF2 with two different RNA
polymerases. EMBO J 2000, 19:1851-1860.
6.
Newman SM, Boynton JE, Gillham NW, Randolph-Anderson BL,
Johnson AM, Harris EH: Transformation of chloroplast ribosomal
RNA genes in Chlamydomonas: molecular and genetic
characterization of integration events. Genetics 1990, 126:875-888.
7.
Svab Z, Hajdukiewicz P, Maliga P: Stable transformation of plastids
in higher plants. Proc Natl Acad Sci USA 1990, 87:8526-8530.
25. Krause K, Maier RM, Kofer W, Krupkinska K, Herrmann RG:
Disruption of plastid-encoded RNA polymerase genes in tobacco:
expression of only a distinct set of genes is not based on
selective transcription of the plastid chromosome. Mol Gen
Genet 2000, 263:1022-1030.
8.
Golds T, Maliga P, Koop H-U: Stable plastid transformation in
PEG-treated protoplasts of Nicotiana tabacum. Biotechnology
1993, 11:95-97.
9.
O’Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P: Chloroplast
transformation in plants: polyethylene glycol (PEG) treatment
of protoplasts is an alternative to biolistic delivery systems.
Plant J 1993, 3:729-738.
20. Gray MW, Lang BF: Transcription in chloroplasts and mitochondria:
a tale of two polymerases. Trends Microbiol 1998, 6:1-3.
21. Hess WR, Borner T: Organellar RNA polymerases of higher plants.
Int Rev Cytol 1999, 190:1-59.
22. Lerbs-Mache S: Regulation of rDNA transcription in plastids of
higher plants. Biochimie 2000, 82:525-535.
26. Sriraman P, Silhavy D, Maliga P: The phage-type PclpP-53 plastid
promoter comprises sequences downstream of the transcription
initiation site. Nucleic Acids Res 1998, 26:4874-4879.
27.
Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU: In vivo analysis
of plastid psbA, rbcL and rpl32 UTR elements by chloroplast
transformation: tobacco plastid gene expression is controlled
by modulation of transcript levels and translation efficiency.
Plant J 1999, 19:333-345.
10. Koop HU, Steinmuller K, Wagner H, Rossler C, Eibl C, Sacher L:
Integration of foreign sequences into the tobacco plastome via
polyethylene glycol-mediated protoplast transformation. Planta
1996, 199:193-201.
28. McBride KE, Schaaf DJ, Daley M, Stalker DM: Controlled expression
of plastid transgenes in plants based on a nuclear DNA-encoded
and plastid-targeted T7 RNA polymerase. Proc Natl Acad Sci USA
1994, 91:7301-7305.
… chlororespiration: only half a story.
11. Nixon PJ, Maliga P: Reply…
Trends Plant Sci 1999, 4:51.
29. Heifetz PB: Genetic engineering of the chloroplast. Biochimie
•
2000, 82:655-666.
This paper includes a description of a chemically inducible transactivation
system for the expression of genes from the plastid genome. Such an
approach is important when heterologous gene products are deleterious to
the plant because of either their high expression levels or their biological
activity. The use of induction in mature plants allows the expression of otherwise toxic molecules as transformation is performed in the absence of
expression, which is only enabled once the nuclear transactivator and
plastid target are brought together in a sexual cross.
12. Knoblauch M, Hibberd JM, Gray JC, van Bel AJ: A galinstan
expansion femtosyringe for microinjection of eukaryotic
organelles and prokaryotes. Nat Biotechnol 1999, 17:906-909.
13. Kavanagh TA, Thanh ND, Lao NT, McGrath N, Peter SO, Horvath EM,
Dix PJ, Medgyesy P: Homeologous plastid DNA transformation in
tobacco is mediated by multiple recombination events. Genetics
1999, 152:1111-1122.
14. Fischer N, Stampacchia O, Redding K, Rochaix JD: Selectable
marker recycling in the chloroplast. Mol Gen Genet 1996,
251:373-380.
15. Lamtham S, Day A: Removal of antibiotic resistance genes from
•• transgenic tobacco plastids. Nat Biotechnol 2000, 18:1172-1176.
The work described in this paper and [14] demonstrates the feasibility of
excising selectable marker genes or other sequences from the plastid
genomes of stably transformed plants.
16. Gray JC, Hibberd JM, Linley PJ, Uijtewaal B: GFP movement
between chloroplasts. Nat Biotechnol 1999, 17:1146.
17.
••
Khan MS, Maliga P: Fluorescent antibiotic resistance marker for
tracking plastid transformation in higher plants. Nat Biotechnol
1999, 17:910-915.
A technical advance resulting in the first stable integration of foreign DNA
into the plastid genome of a monocot plant. The work uses an approach in
which the initial selection of cultured cells on streptomycin is followed by
screening for green fluorescence. The aadA gene product catalyzes the
covalent modification of both streptomycin and spectinomycin. Although the
latter is a more effective plastid-selectable marker, most cereals are naturally
30. Uknes S, Dincher S, Friedrich L, Negrotto D, Williams S,
Thompson-Taylor H, Potter S, Ward E, Ryals J: Regulation of
pathogenesis-related protein-1a gene expression in tobacco.
Plant Cell 1993, 5:159-169.
31. Barkan A, Goldschmidt-Clermont M: Participation of nuclear genes
in chloroplast gene expression. Biochimie 2000, 82:559-572.
32. Chaudhuri S, Carrer H, Maliga P: Site-specific factor involved in the
editing of the psbL mRNA in tobacco plastids. EMBO J 1995,
14:2951-2957.
33. Bock R, Koop U: Extraplastidic site-specific factors mediate RNA
editing in chloroplasts. EMBO J 1997, 16:3282-3288.
34. Corneille S, Lutz K, Maliga P: Conservation of RNA editing between
rice and maize plastids: are most editing events dispensable? Mol
Gen Genet 2000, 264:419-424.
35. Nagata N, Saito C, Sakai A, Kuroiwa H, Kuroiwa T: The selective
increase or decrease of organellar DNA in generative cells just
after pollen mitosis one controls cytoplasmic inheritance. Planta
1999, 209:53-65.
Protein expression in plastids Heifetz and Tuttle
36. Scott SE, Wilkinson MJ: Low probability of chloroplast movement
•
from oilseed rape (Brassica napus) into wild Brassica rapa. Nat
Biotechnol 1999, 17:390-392.
A theoretical study comparing the effect of nuclear and plastid transformation on transgene flow in a readily out-crossed plant species. Plastid
expression blocks pollen transmission but still allows the production of
weedy hybrids if the female parent is pollinated by a related species.
37.
McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P:
Amplification of a chimeric Bacillus gene in chloroplasts leads to
an extraordinary level of an insecticidal protein in tobacco.
Biotechnology 1995, 13:362-365.
38. Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ:
•
Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2
protein in chloroplasts confers resistance to plants against
susceptible and Bt-resistant insects. Proc Natl Acad Sci USA
1999, 96:1840-1845.
In a similar study to the pioneering work described in [37], a single bacterial
gene encoding a Bt toxin was expressed in tobacco plastids and found to
express at high levels, causing considerable mortality to target insect
species. The use of highly insect-resistant plants as part of a ‘high dose
resistance management strategy’ is discussed.
39. De Cosa B, Moar W, Lee S-B, Miller M, Daniell H: Overexpression of
•• the Bt cry2Aa2 operon in chloroplasts leads to formation of
insecticidal crystals. Nat Biotechnol 2001, 19:71-74.
Insecticidal proteins from Bacillus thuringiensis are normally expressed in
the insect as protoxins from operons, which can include a putative chaperonin.
161
These protoxins form crystalline inclusion bodies in the natural host, which
increase their stability. The authors achieved the expression of one such
complete operon, which was encoded by three genes, in tobacco plastids.
This expression resulted in the production of cuboidal crystals within leaf
chloroplasts. The resulting proteins were immensely toxic even to recalcitrant
pests such as beet armyworm and cotton bollworm.
40. Staub JM, Maliga P: Expression of a chimeric uidA gene indicates
that polycistronic mRNAs are efficiently translated in tobacco
plastids. Plant J 1995, 7:845-848.
41. Daniell H, Datta R, Varma S, Gray S, Lee SB: Containment of
herbicide resistance through genetic engineering of the
chloroplast genome. Nat Biotechnol 1998, 16:345-348.
42. Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N,
•• Paradkar V, Schlittler M, Carroll JA, Spatola L et al.: High-yield
production of a human therapeutic protein in tobacco
chloroplasts. Nat Biotechnol 2000, 18:333-338.
Proteins from eukaryotic sources, which require complex folding or disulfide
bond formation, are generally not easily expressed in prokaryotic systems.
The authors demonstrate that the tobacco chloroplast is capable of correctly
processing fusions of human somatotropin and ubiquitin expressed from
the plastid genome. Moreover, these transformed chloroplasts accumulate
metabolically active protein to high levels.
43. Guda C, Lee SB, Daniell H: Stable expression of a biodegradable
protein-based polymer in tobacco chloroplasts. Plant Cell Rep
2000, 19:257-262.