411

The requirement for large quantities of therapeutic proteins has
fuelled interest in the production of recombinant proteins in
plants and animals. The first commercial products to be made
in this way have experienced much success, and it is predicted
that in the future a plethora of protein products will be made
using these ‘natural’ bioreactors.

Addresses
*

Planet Biotechnology, Inc., 2438 Wyandotte Street, Mountain View,

CA 94043, USA; e-mail: [e-mail]

†

Palo Alto Institute of Molecular Medicine, 2462 Wyandotte Street,

Mountain View, CA 94043, USA

Current Opinion in Biotechnology 2001, 12:411–418

0958-1669/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.

Abbreviations
AAT

α

1-antitrypsin

HC-Pro helper-component proteinase
PTGS

post-transcriptional gene silencing

TSP

total soluble protein

Introduction

The inexpensive production for large quantities (many
kilograms) of protein has led to a new industry to produce
recombinant proteins in transgenic plants and animals.
The potential of ‘molecular pharming’, using transgenic
plants or animals as ‘bioreactors’ to produce therapeutic
proteins, has been apparent for over a decade and several
proteins produced in these systems are now in clinical tri-
als. Depending on the production system used, there are
several issues of concern. Animal systems suffer from long
development timelines and possible contamination of
purified proteins with animal viruses and prions. Plant-
produced proteins have altered post-translational modifi-
cations that introduce novel carbohydrates. Here, we
review the most recent advances in these technologies and
discuss the methods that are being developed to
overcome these concerns.

Plant expression

Technological advances using plant bioreactors

Plants provide an attractive expression vehicle for numer-
ous proteins (see Table 1) [1–14]. Plant ‘bioreactors’ are
expected to yield over 10 kg of therapeutic protein per acre
in tobacco, maize, soybean and alfalfa [15,16

•

]. Compared

with the use of conventional steel tank bioreactors and
mammalian cells or microorganisms, the cost of producing
a protein under good manufacturing practice (GMP)
conditions is reduced to perhaps one-tenth.

Recent research demonstrates that plants will provide a
facile and economic bioreactor for the large-scale produc-
tion of industrial and pharmaceutical recombinant proteins
[15,17–23]. Genetically engineered, transgenic plants have

many advantages as sources of proteins compared with
human or animal fluids/tissues, recombinant microbes,
transfected animal cell lines or transgenic animals. First,
the cost of producing raw material on an agricultural scale
is low and there is the possibility, in some cases, of using
the edible plant material directly. Second, the use of plants
offers reduced capitalization costs relative to fermentation
methods. Third, production can be rapidly up-scaled.
Fourth, unlike bacteria, plants can produce multimeric
proteins, such as antibodies, in the correct assembly. Fifth,
plant proteins are considered to be safer, as plants do not
serve as hosts for human pathogens such as human
immunodeficiency virus (HIV), prions, hepatitis viruses
and so on. This final point is noteworthy, given the spread
of foot-and-mouth disease and bovine spongiform
encephalopathy from the United Kingdom into Europe.

Depending upon the promoters used, transgenic proteins
can be deposited throughout the plant, in specific parts of
the plant (e.g. in seeds) or in specific plant cell organelles
(e.g. chloroplasts) within a given plant cell. Numerous lab-
oratories have shown transgenic protein accumulation in
seed of corn [24,25], soybean [3], tobacco [26] or barley
[27

•

]. Proteins produced in seed exhibit remarkable stabil-

ity; for example, enzymes and antibodies expressed in
seed and stored for more than three years at refrigerator
temperature retain full enzymatic or binding activity.
Recently, success was reported using tropical plants, such
as cassava (Manihot esculenta Crantz) [28

•

] and banana [29].

This opens up the possibility of delivering oral vaccines
and recombinant pharmaceuticals directly to consumers in
less developed equatorial countries.

In the past year, notable advances have been made using
chloroplast expression of recombinant proteins. A foreign
gene is introduced into a spacer region between the
functional genes of the chloroplast using homologous
recombination. This eliminates position effects of nuclear
transformation, and gene silencing has not been a problem.
Staub et al., [30

••

] demonstrated that chloroplasts can

process and fully assemble a disulfide-bonded form of
human somatotropin (expressed as 7% of the total soluble
protein [TSP]) and an oligomeric form of cholera toxin B
(4–5% TSP). Henriques and Daniell [31] expressed
foreign genes (up to 10,000 copies/cell) in tobacco chloro-
plasts, resulting in the accumulation of foreign proteins
that comprise up to 47% of the TSP [32].

A novel Tobravirus-based system [33] for root expression
was reported last year, and new vector technologies contin-
ue to be developed. For example, internal ribosome entry
sites (IRES) have been used to direct the expression of
bicistronic mRNA [34] and Dow AgroSciences described an
adenosine deaminase selection system for maize [35].

Producing proteins in transgenic plants and animals
James W Larrick*

†

and David W Thomas

†

Issues regarding protein production in plants
Glycosylation

Differences in the glycosylation patterns of proteins
produced in plants and humans give perhaps most cause for
concern regarding therapeutic protein production.
Cabanes-Macheteau et al. [36

••

] reported the first detailed

analysis of the glycosylation of a plant-produced monoclon-
al antibody (plantibody). Both N-glycosylation sites located
on the heavy-chain of the plantibody are N-glycosylated;
however, the number of glycoforms is higher in the plant
than in mammalian-expressed antibodies. In addition to
high-mannose-type N-glycans, 60% of the oligosaccharides
N-linked to the plantibody have

β

(1,2)-xylose and

α

(1,3)-fucose residues linked to the core Man3GlcNAc2.

These oligosaccharides linkages, not found on mammalian
N-linked glycans, are potentially immunogenic and raise
the possibility that plantibodies containing N-linked
glycans might have limited scope as parenterals or even
when applied topically or orally, particularly in patients with
severe food allergies. Food allergens bearing

β

(1,2)-xylose

and

α

(1,3)-fucose have been linked to specific IgE in

serum and to biological activity (i.e. histamine release) in
allergic patients [37,38]. In contrast, however, the mere
presence of serum IgE against cross-reactive carbohydrate
determinants, which include the

β

(1,2)-xylose and

α

(1,3)-fucose linkages to the core Man3GlcNAc2 of plant

glycans, has been shown to be a poor predictor of clinical
allergy [39–41]. These results preclude generalities about
the potential toxicity of plantibody glycans in humans.

When a mouse plantibody (mouse amino acid sequence
and plant glycans) was used to immunize mice there was
no, or only a minimally detectable, serum immune
response. Thus, in the mouse at least, a ‘self’ primary
protein structure decorated with plant N-linked glycans
can be non-immunogenic. This plantibody has been
applied topically in the mouth of humans with no
detection of human antimouse antibodies [2].

Recent efforts have focused on the humanization of plant
glycans to reduce their immunogenic potential. Comparison
of plant and mammalian N-glycan biosynthesis indicates
that

β

1,4-galactosyltransferase is the most important

enzyme that is missing for conversion of typical plant
N-glycans into mammalian-like N-glycans. Bakker et al. [42]
reported the stable expression of human

β

1,4-galactosyl-

transferase in tobacco plants. Crossing a tobacco plant
expressing human

β

1,4-galactosyltransferase with a plant

expressing the heavy- and light-chains of a mouse antibody
resulted in the expression of a plantibody that exhibits
partially galactosylated N-glycans (30%). This level of
carbohydrate incorporation is approximately as abundant as
when the same antibody is produced by hybridoma cells.
These results represent a major step forward in the engi-
neering of the N-glycosylation of recombinant proteins.
Aglycosylated antibodies lacking carbohydrates altogether
can also be created by altering the peptide recognition
sequence for N-linked glycosylation (Asn-X-Ser/Thr). High-
mannose-type glycosylation, which does not contain the
core-linked xylose and fucose residues, may be favored by
the addition of a C-terminal Lys-Asp-Glu-Leu sequence
[43] and the subsequent targeting of proteins to the proxi-
mal endoplasmic reticulum. Alternatively, the plant-specific
fucosyl transferase [44] or xylosyl transferase, which are
active in the transgolgi, may be targeted for silencing.

Post-transcriptional gene silencing

Silencing of introduced transgenes has frequently been
observed in plants, constituting a major commercial prob-
lem [45

•

]. Post-transcriptional gene silencing (PTGS) is a

sequence-specific RNA degradation mechanism that is
widespread in eukaryotic organisms. It is often associated
with methylation of the transcribed region of the silenced
gene and with the accumulation of small RNA molecules
(21 to 25 nucleotides) homologous to the silenced gene. In
plants, PTGS can be triggered locally and then spread
throughout the organism via a mobile signal that can cross
a graft junction. PTGS has been shown to occur in many

412

Protein technologies and commercial enzymes

Table 1

Representative therapeutic proteins produced in transgenic
plants.

Recombinant proteins

Plants

Reference/group

Antibodies
SIgA anti-S. mutans

Tobacco

[1,2]

Planet Biotechnology

Hayward, CA

IgG anti-herpes simplex

Soybean

[3]

virus

Monsanto,

St Louis, MO

Various antibodies

Maize/rice

EPIcyte,

San Diego, CA

Anti-carcinoembryonic

Rice and wheat

[88]

antigen

Lymphoma idiotypes

Tobacco

[89]

Vaccines
Hepatitis B surface antigen

Tobacco

[4–6]

Rabies vaccine

Tobacco

[90]

Norwalk capsid protein

Tobacco, potato

[7]

Porcine transmissible

Tobacco

[63]

gastroenteritis virus

E. coli toxin (LT-B)

Potato

[8–10]

Cholera toxin (CT-B)

Potato

[11,12]

Mouse GAD67

Potato

[13]

VP2 capsid protein of mink

Black-eyed bean,

[14]

enteritis virus inserted into

Vigna unguiculata

cowpea mosaic virus

Other proteins
Collagen

Tobacco

[56

•

]

Hirudin

Canola, Sembiosys

Calgary,

(Brassica napus)

Alberta, Canada

Lactoferrin

Potato

[55]

Lipase

Tobacco, maize

Meristem Therapeutics

Clermont Ferrand,

France

Growth hormone

Tobacco

[30

••

]

Erythropoietin

Tobacco

[17]

transgenic dicotyledonous plants and more recently in
monocotyledonous plants [46].

Until now, the most efficient strategy to avoid PTGS was to
carefully design transgene constructs (e.g. eliminating
repeat sequences) and by thorough analysis of transfor-
mants at the molecular level [47]. When matrix attachment
regions (MARs) are positioned on either side of a transgene
their presence usually results in higher and more stable
expression in transgenic plants or cell lines, most likely by
minimizing gene silencing [48]. The helper-component
proteinase (HC-Pro) of plant potyviruses suppresses
PTGS. Introduction of HC-Pro into plants results in loss of
PTGS, loss of small RNAs, and partial loss of methylation.
Grafting experiments indicate that HC-Pro prevents the
plant from responding to the mobile silencing signal, but
does not eliminate its ability to produce or send the signal
[49

•

]. HC-Pro targets a PTGS maintenance component (as

opposed to an initiation or signaling component) at a point
that affects accumulation of small RNAs and methylation of
genomic DNA [50

••

]. Recent mutagenesis studies are

beginning to define the plant genes involved in this inter-
esting and important process [51].

Therapeutic advances using plant-produced proteins

The most widely studied therapeutic proteins produced in
plants have been monoclonal antibodies for passive
immunotherapy and antigens for use in oral vaccines.
Although a large number of useful proteins have been suc-
cessfully expressed in transgenic plants (Table 1) [52

•

], a

limited number of therapeutic products have entered the
clinic. Products that have entered clinical trials include two
antibodies, an oral vaccine and pancreatic lipase. The most
advanced product is an anti-Streptococcus mutans secretory
IgA (SIgA) plantibody currently in phase II clinical trials for
the prevention of dental caries. At the present time, plants
offer the only large-scale, commercially viable system for
production of this unique form of antibody [1]. SIgA is the
most abundant antibody class produced by the body (

>

60%

of total immunoglobulin) and is secreted onto mucosal sur-
faces to provide local protection from toxins and pathogens.
SIgA is comprised of four different protein chains: heavy
and light immunoglobulin chains that form the antigen-
binding hypervariable region; the J chain that dimerizes two
IgA molecules (SIgA has four antigen-binding sites); and the
secretory component that is derived from the polyim-
munoglobulin receptor of mucosal epithelial cells [53].
Previously, it was not possible to obtain therapeutic quanti-
ties of this class of immunoglobulin. The initial clinical
studies demonstrate that topically applied anti-S. mutans
SIgA plantibody is safe (i.e. no human antimouse antibody
response [HAMA] and no local or systemic toxicity) and pre-
vents colonization by S. mutans, the major cause of human
dental caries [2]. Planet Biotechnology Inc. completed
United States Food and Drug Administration (FDA)-
approved phase I/II confirmatory clinical trials at the School
of Dentistry at the University of California in San Francisco
in late 2000. Details will be published elsewhere; however,

initial analysis of the data indicates that the plantibody
significantly reduces the levels of S. mutans. Further
confirmatory trials have been designed to optimize the
treatment regimen.

The recent availability of large amounts of sIgA plantibod-
ies opens up a number of novel therapeutic opportunities
for disorders of the mucosal immune system. These
include therapies for intestinal pathogens such as hepatitis
viruses, Helicobacter pylori, enterotoxigenic Escherichia coli
and cholera, respiratory pathogens (e.g. rhinovirus and
influenza), and genitourinary sexually transmitted diseases
(e.g. herpes simplex virus [3]). SIgA might also be devel-
oped as a method of contraception. A number of other
plantibodies, usually of the IgG class, are currently under
development. The most advanced of these, an anti-
EPCAM plantibody (co-developed by NeoRx and
Monsanto) was recently discontinued after phase II trials
demonstrated significant gastrointestinal side-effects.

Recombinant bioactive avidin and

β

-glucuronidase [22,54]

are the first recombinant plant-derived proteins to be pro-
duced commercially (made by Prodigene Inc., College
Station, TX) and sold by Sigma Chemical company
(St Louis, MO). Two other proteins that will be required in
large quantity for nutriceutical use (lactoferrin [55]) or cos-
meceutical use (collagen [56

•

]) were recently produced in

transgenic plants. Hirudin, a therapeutic anticoagulant, has
been produced by Sembiosys (Calgary, Canada) using
oleosin-fusion technology [57], which allows extraction
using canola oil bodies.

Edible vaccines [58] produced in transgenic plants have
the potential to revolutionize vaccination, particularly in
less-developed countries. Numerous vaccine proteins have
been produced in plants and demonstrated to be both
immunogenic and protective against pathogen challenge
(see Table 1). Recently, the first human trials were report-
ed of plant-produced bacterial [8] and viral [59] oral
vaccines. These demonstrated modest levels of protective
antibodies in serum and stool.

Recently, respiratory syncytial virus vaccine [60], canine par-
vovirus [61], measles [62], transmissible gastroenteritis virus
(TGEV) [63], and improved hepatitis B virus vaccines have
been prepared in plants [6,64]. Wigdorovitz et al., [65] devel-
oped transgenic alfalfa expressing the structural protein VP1
of foot-and-mouth disease virus. Mice, parenterally immu-
nized using leaf extracts or fed freshly harvested leaves from
the transgenic plants, developed a virus-specific protective
immune response. These studies demonstrate the feasibili-
ty of this ‘appropriate technology’ for developing countries
where vaccines are urgently needed.

Transgenic animals

Technological advances using transgenic animals

One of the more promising approaches to the large-scale
production of recombinant proteins has been the secretion

Producing proteins in transgenic plants and animals Larrick and Thomas 413

of proteins into the milk of transgenic mammals. This has
been successfully demonstrated for over two dozen differ-
ent proteins in either cows, goats, sheep, pigs, rabbits or
mice and appears particularly promising for the production
of large amounts of monoclonal antibodies [66]. Indeed,
the commercial promise for using animals as bioreactors for
protein production is being realized through several com-
panies, including Genzyme Transgenics, Pharming Group,
PPL Therapeutics and Infigen Inc. Although recombinant
proteins can be expressed in other tissues, milk has
remained the major mode of production due to the large
volume produced by lactating female animals and the ease
of collection and purification of the protein product. In
goats, for example, a lactating female can produce up to
800 L of milk a year, which can contain around 5 g recom-
binant protein/L to yield around 4 kg of protein per year.
Thus, even a modest herd of transgenic goats can produce
several hundred kilograms of protein product each year at
a low production cost. In addition to milk, urine has been
suggested as another feasible route for the production of
recombinant proteins [67,P1]. Another more recent
approach is to produce recombinant proteins in transgenic
chicken eggs; high-yield protein can be expressed in eggs,
harvesting is straightforward and production is easy to
scale-up. Studies into this method of protein production
have been undertaken by the Avian Initiative, a collabora-
tion between Viragen and the Roslin Institute.

To date, most recombinant proteins produced in transgenic
animals have involved the microinjection of a genetic
construct the expression of which is driven by a mammary-
gland-specific promoter (e.g. for the production of caseins,
whey acidic protein, lactalbumins and lactoglobulin).
Although successful, this approach is inefficient and time-
consuming. The challenge is to improve the efficiency and
speed of producing high-yield protein-expressing founder
animals. One way to quickly determine the activity and sta-
bility of a recombinant protein in milk is to transfect
mammary tissue in vivo using an expression plasmid encod-
ing the protein, as shown with human growth hormone
[68

••

] which was infused through the nipple canal. A

similar strategy could also be used to potentially transfect
bladder epithelial cells for expression of proteins in urine.
However, neither of these approaches would result in the
production of large amounts of protein.

One very promising approach to increasing the efficiency of
creating a herd of animal bioreactors with improved recom-
binant protein expression is through nuclear transfer
technology. Transgenic mice have been produced for some
years using genetically modified pluripotential embryonic
stem cells that were introduced into the early embryo to
create a chimeric mouse. Although transfer of nuclei from
embryonic cells into enucleated oocytes was used success-
fully to create cloned animals in larger mammalian species,
these embryonic cells were difficult to genetically modify
and there was an absence of a cloned embryonic stem cell
line in which transfectants could be more readily identified

and propagated. Recently, however, there have been
reports of established embryonic stem cells from cattle [69]
and goats [70]. A surprising breakthrough was made sever-
al years ago by Wilmut et al. [71] who showed that a sheep,
Dolly, could be created from the transfer of a nucleus from
an adult somatic cell as opposed to an embryonic cell. Since
that time a number of different adult cell types have been
used as nuclear donors for the cloning of sheep, cattle,
goats, pigs and mice. These include tissue fibroblast cells
from ears and tails, as well as fibroblast cell lines, tissue
cumulus cells, mammary gland epithelial cells, oviduct,
uterine, skin, muscle, and liver cells) [69,72–75]. The opti-
mal technology in each case is still being developed and
includes variables on the timing and method for activation
of the recipient enucleated oocyte, the cell cycle of the
donor nucleus, and the fusion method. Polejaeva et al.
[76

••

] developed a new double nuclear transfer procedure

in pigs using granulosa cells as the nuclear donor; this
approach minimizes in vitro activation and increases the
efficiency of producing viable births. The next step is to
produce animals that have been genetically modified by
using nuclei from engineered cell lines. Ideally, this would
include targeted gene insertion for optimal expression, con-
trolled copy number, and inducible expression of the
introduced gene. McCreath et al. [77

••

] used gene targeting

in ovine fibroblast cells to introduce the gene encoding
human

α

1-antitrypsin (AAT) and these cells were then

used as nuclear donors. Several sheep were born that
expressed AAT and the milk of one of them expressed AAT
at 650

µ

g/ml following induced lactation. Unfortunately,

the majority of the lambs died and showed evidence of a
spectrum of genetic abnormalities, as has been observed
with cloned calves [78]. This raises the possibility that
manipulation of the nucleus may introduce alterations to
reprogramming that impair full development, as found with
nuclei that were repeatedly recycled [79

••

].

In addition to unknown effects of nuclear transfer on
embryonic development, there is also the question about
long-term effects on cloned animals after birth. It was sur-
prising to discover that Dolly had shortened telomeres of a
length similar to that of the donor nuclei [80]. As shortened
telomeres are a process of aging and are thought to be asso-
ciated with decreased replicative ability and lifespan, this
observation raised questions about the feasibility of pro-
ducing cloned animals by nuclear transfer without causing
major problems, including a shortened life. By contrast,
additional studies have found that telomerase activity,
which maintains telomere length, is reprogrammed in
nuclear transferred embryos and is similar to that obtained
from conventional embryos [81]. Furthermore, the telom-
ere length in cloned fetuses and calves was the same as in
normal animals even when the donor nuclei from adult
fibroblasts had shortened telomeres [82

••

]. In a similar

fashion, Lanza et al. [83] found that nuclei from senescent
cells could be rejuvenated upon transfer and were able to
generate cloned calves with extended telomeres. These
latter studies would suggest that normal cloned animals

414

Protein technologies and commercial enzymes

can be produced by nuclear transfer from aged adult donor
cells and that that the age-limiting telomere shortening can
be reversed. The differences between these observations
and those reported by Shiels et al. [80] remain to be
resolved but could relate to the species and/or donor cells
or the technology used to optimize each system.

Therapeutic advances using proteins produced in
animal bioreactors

The expression of proteins in milk offers several uses: to
produce large amounts of therapeutics for human or veteri-
nary use; to improve the health of dairy livestock; and to
produce large amounts of proteins used in commercial
processes or in drug formulation, such as human serum
albumin. Although an increasing number of proteins are
being produced in milk, most are in preclinical develop-
ment and few are yet in clinical testing. Pharming Group
and Genzyme are developing recombinant human

α

-glucosidase to treat infants with Pompe’s disease, which

results from a genetic deficiency in this enzyme. Enzyme
produced in rabbits milk was well-tolerated and showed
clinical benefit in treated patients [84]. Pharming Group
and Baxter Healthcare Corporation have also recently initi-
ated clinical testing with a rabbit-milk-derived recombinant
human C1 inhibitor to treat patients with hereditary
angioedema who exhibit C1 inhibitor deficiency. Pharming
Group has also just completed a phase I clinical study with
recombinant human lactoferrin — the protein was found to
be safe and well tolerated. Lactoferrin will be tested initial-
ly for heparin neutralization in coronary artery angioplasty
or bypass surgery. Recently, human lactoferrin was also
found to have potent antibacterial activity in animal models
using antibiotic-resistant Staphylococci, which could broad-
en its clinical use [85]. Genzyme Transgenics and Genzyme
General have produced antithrombin III in goats milk; the
protein is currently in phase III clinical trials to prevent
blood clotting during cardiac surgery in heparin-resistant
patients [86]. Goat herds are also being developed by
Genzyme Transgenics for the large-scale production of the
tumor necrosis inhibitory monoclonal antibody, Remicade.
Remicade is marketed by Centocor for the treatment of
inflammatory conditions, including Crohn’s disease and
rheumatoid arthritis.

Another use for the expression of recombinant proteins in
milk is to prevent mastitis caused by Staphylococcus in
dairy cattle. For example, Kerr and colleagues [87

••

]

showed that expression of recombinant lysostaphin in the
milk of mice prevented Staphylococcus aureus infection.

Issues regarding the production of proteins in animals

The technologies discussed above go some way towards
addressing the two big issues facing animal production of
recombinant proteins: efficiency and the speed with which
a commercial product can be produced. The current meth-
ods for producing transgenic animals lead to live births at a
low rate and the success rate is generally below 10%. Even
with nuclear transfer, the success rate is low and many

embryos suffer abnormalities and are not viable. When live
births do occur, the next challenge is to identify animals that
produce high levels of protein and can serve as founders for
a herd. This selection procedure has a relatively low success
rate, particularly with animals produced using microinjec-
tion technology. Theoretically, the use of nuclear transfer
could improve this situation as all animals are derived from
the same nucleus and the animals are not chimeric. This
approach should also permit greater stability and retention
of the introduced gene in subsequent generations. Using
gene targeting it should be possible to design optimal
insertion points in the genome for expression of the intro-
duced gene, which could remove some of the expression
variability that results from more random insertion.

The second issue facing protein production in recombi-
nant animals is the speed to commercial product. With
microinjection technology, to produce large amounts of
product the founders must first be identified and then
used to breed and establish the herd. With cows, for exam-
ple, the age to sexual maturity is around 15 months and
each cow has a single offspring. In females natural lactation
also occurs at around three years of age. It is clear that to
produce a sufficient herd for commercial production takes
a number of years. Again, this could be greatly expedited
using nuclear transfer; once a female founder is identified,
cells from that animal can donate nuclei to produce a herd
in one generation.

Another issue that needs to be considered with animal
production of recombinant proteins is that of infectious
diseases. The potential for products to contain prions has
been a concern for some time, and most companies have
designed a process and testing to assure that their products
are prion-free. A current problem is the occurrence of
sudden epidemics that can devastate livestock herds, such
as foot-and-mouth disease. There is a necessity to place
herds in different regions of the world to avoid such
catastrophes, but even this cannot avoid potential loss of a
manufacturing herd. For example, the foot-and-mouth
disease outbreak in England quickly spread to other parts
of Europe. Foot and mouth is also prevalent in South
America and the Middle East. One can control as much as
possible to ensure a safe and healthy herd of transgenic
animals, but there are unknown risks that can be
catastrophic. This concern may stimulate even greater
efforts to develop animals resistant to disease, as pointed
out above for lysostaphin, and to produce new effective
vaccines to ward off known pathogens.

Finally, the therapeutic proteins produced in transgenic
animals undergo post-translational processing characteris-
tic of that animal, although the protein itself is of human
sequence. Because carbohydrates in recombinant glyco-
proteins, in particular, are somewhat different from those
of human origin, there is still a question about whether or
not this will have adverse effects, such as eliciting an
immune response or conferring altered biodistribution or

Producing proteins in transgenic plants and animals Larrick and Thomas 415

retention. Although only few animal-produced protein
drugs have yet been administered to humans, there does
not appear to be any issue with respect to the circulating
half-life or production of antibodies.

Conclusions

In summary, the future production of recombinant proteins
in plants or in the milk of animals looks promising. The
number of applications will increase as more therapeutic
proteins are required in quantities that cannot be attained
economically in cell culture. With the advent of nuclear
transfer technology, optimized production of recombinant
proteins in milk should become even more efficient and
inexpensive. In plants, efforts to modify glycosylation and
control post-transcriptional gene silencing will enhance
the value of this important technology. An increasing
number of transgenic plant- and animal-derived proteins
are entering clinical testing. The initial success of these
development programs suggests an important role for cost-
effective and large-scale production technologies.

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

1.

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van Dolleweerd C, Mostov K, Lehner T: Generation and assembly of
secretory antibodies in plants. Science 1995, 268:716-719.

2.

Ma J, Hikmat B, Wycoff K, Vine N, Chargelegue D, Yu L, Hein M,
Lehner T: Characterization of a recombinant plant monoclonal
secretory antibody and preventive immunotherapy in humans. Nat
Med 1998, 4:601-606.

3.

Zeitlin L, Olmsted SS, Moench TR, Co MS, Martinell BJ, Paradkar VM,
Russell DR, Queen C, Cone RA, Whaley KJ: A humanized monoclonal
antibody produced in transgenic plants for immunoprotection of the
vagina against genital herpes. Nat Biotechnol 1998, 16:1361-1364.

4.

Mason HS, Lam DM, Arntzen CJ: Expression of hepatitis B surface
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5.

Thanavala Y, Wang Y-F, Lyons P, Mason HS, Arntzen C:
Immunogenicity of transgenic plant-derived hepatitis B surface
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6.

Richter LJ, Thanavala Y, Arntzen CJ, Mason HS: Production of
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Haq TA, Mason HS, Clements JD, Arntzen CJ: Oral immunization
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10. Mason HS, Haq TA, Clements JD, Arntzen CJ: Edible vaccine protects

mice against Escherichia coli heat-labile enterotoxin (LT): potatoes
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Allard G, Lemieux R, Vezina LP: Production of a diagnostic
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antibodies in transgenic plants. Res Immunol 1998, 149:603-608.

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therapeutic use. Curr Top Microbiol Immunol 1999, 236:119-137.

25. Hood EE, Jilka JM: Plant-based production of xenogenic proteins.

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Horvath H, Huang J, Wong O, Kohl E, Okita T, Kannangara LG,

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von Wettstein D: The production of recombinant proteins in
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An early paper demonstrating transgenic production using barley.

28. Zhang P, Potrykus I, Puonti-Kaerlas J. Efficient production of

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transgenic cassava using negative and positive selection.
Transgenic Res 2000, 9:405-415.

Cassava may be a useful ‘bioreactor’ in the tropics.

29. Schenk PM, Sagi L, Remans T, Dietzgen RG, Bernard MJ,

Graham MW, Manners JM: A promoter from sugarcane bacilliform
badnavirus drives transgene expression in banana and other
monocot and dicot plants. Plant Mol Biol 1999, 39:1221-1230.

30. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N,

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Paradkar V, Schlittler M, Carroll JA, Spatola L et al.: High-yield
production of a human therapeutic protein in tobacco
chloroplasts. Nat Biotechnol 2000, 18:330-338.

Demonstration by the Monsanto/Agracetus/Calgene group(s) of the utility of
using chloroplasts for high-level expression of transgenic proteins.

31. Henriques L, Daniell H: Expression of cholera toxin B subunit

oligomers in transgenic tobacco chloroplasts. Proc Nat Acad Sci
USA 2001, in press.

32. 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.

33. MacFarlane SA, Popovich AH: Efficient expression of foreign

proteins in roots from tobravirus vectors. Virology 2000, 267:29-35.

416

Protein technologies and commercial enzymes

34. Toth RL, Chapman S, Carr F, Santa Cruz S: A novel strategy for the

expression of foreign genes from plant virus vectors. FEBS Lett
2001, 489:215-219.

35. Petolino JF, Young S, Hopkins N, Sukhapinda K, Woosley A, Hayes C,

Pelcher L. Expression of murine adenosine deaminase (ADA) in
transgenic maize. Transgenic Res 2000, 9:1-9.

36. Cabanes-Macheteau M, Fitchette-Laine AC, Loutelier-Bourhis C,

••

Lange C, Vine ND, Ma JK, Lerouge P, Faye L: N-Glycosylation of a
mouse IgG expressed in transgenic tobacco plants. Glycobiology
1999, 9:365-372.

The first in-depth study of glycosylation of a plantibody.

37.

Fotisch K, Altmann F, Haustein D, Vieths S: Involvement of
carbohydrate epitopes in the IgE response of celery-allergic
patients. Int Arch Allergy Immunol 1999, 120:30-42.

38. Garcia-Casado G, Sanchez-Monge R, Chrispeels MJ, Armentia A,

Salcedo G, Gomez L: Role of complex asparagine-linked glycans
in the allergenicity of plant glycoproteins. Glycobiology 1996,
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39. van Ree R, Aalberse RC: Specific IgE without clinical allergy.

J Allergy Clin Immunol 1999, 103:1000-1001.

40. Mari A, Iacovacci P, Afferni C, Barletta B, Tinghino R, Di Felice G,

Pini C: Specific IgE to cross-reactive carbohydrate determinants
strongly affects the in vitro diagnosis of allergic diseases. J Allergy
Clin Immunol 1999, 103:1005-1011.

41. van der Veen MJ, van Ree R, Aalberse RC, Akkerdaas J,

Koppelman SJ, Jansen HM, van der Zee JS: Poor biologic activity of
cross-reactive IgE directed to carbohydrate determinants of
glycoproteins. J Allergy Clin Immunol 1997, 100:327-334.

42. Bakker H, Bardor M, Molthoff JW, Gomord VV, Elbers II, Stevens LH,

Jordi W, Lommen A, Faye L, Lerouge P, Bosch D: Galactose-
extended glycans of antibodies produced by transgenic plants.
Proc Natl Acad Sci USA 2001, 98:2899-2904.

43. Wandelt CI, Khan MRI, Craig S, Schroeder HE, Spencer D,

Higgins TJV: Vicilin with carboxy-terminal KDEL is retained in the
endosplasmic reticulum and accumulates to high levels in leaves
of transgenic plants. Plant J 1992, 2:181-192.

44. Leiter H, Mucha J, Staudacher E, Grimm R, Glossl J, Altmann F:

Purification, cDNA cloning, and expression of GDP-

L

-Fuc:Asn-

linked GlcNAc

αα

1,3-fucosyltransferase from mung beans. J Biol

Chem 1999, 274:21830-21839.

45. Vaucheret H, Fagard M: Transcriptional gene silencing in plants:

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targets, inducers and regulators. Trends Genet 2001, 17:29-35.

Most recent review of gene silencing in plants.

46. Kanno T, Naito S, Shimamoto K: Post-transcriptional gene silencing

in cultured rice cells. Plant Cell Physiol 2000, 41:321-326.

47.

de Wilde C, Van Houdt H, de Buck S, Angenon G, de Jaeger G,
Depicker A: Plants as bioreactors for protein production: avoiding the
problem of transgene silencing. Plant Mol Biol 2000, 43:347-359.

48. Allen GC, Spiker S, Thompson WF: Use of matrix attachment

regions (MARs) to minimize transgene silencing. Plant Mol Biol
2000, 43:361-376.

49. Mallory AC, Ely L, Smith TH, Marathe R, Anandalakshmi R, Fagard M,

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Vaucheret H, Pruss G, Bowman L, Vance VB: HC-Pro suppression of
transgene silencing eliminates the small RNAs but not transgene
methylation or the mobile signal. Plant Cell 2001, 13:571-583.

Grafting experiments indicate that HC-Pro prevents the plant from respond-
ing to the mobile silencing signal but does not eliminate its ability to produce
or send the signal.

50. Llave C, Kasschau KD, Carrington JC: Virus-encoded suppressor of

••

posttranscriptional gene silencing targets a maintenance step in
the silencing pathway. Proc Natl Acad Sci USA 2000,
97:13401-13406.

An interesting and important paper investigating the mechanism of post-tran-
scriptional gene silencing.

51. Mittelsten Scheid O, Afsar K, Paszkowski J: Release of epigenetic

gene silencing by trans-acting mutations in Arabidopsis. Proc Natl
Acad Sci USA 1998, 95:632-636.

52. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming:

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production of antibodies, biopharmaceuticals and edible vaccines
in plants. Trends Plant Sci 2001, 6:219-226.

An excellent review of molecular pharming.

53. Mostov KE, Altschuler Y, Chapin SJ, Enrich C, Low SH, Luton F,

Richman-Eisenstat J, Singer KL, Tang K, Weimbs T: Regulation of
protein traffic in polarized epithelial cells: the polymeric
immunoglobulin receptor model. Cold Spring Harb Symp Quant
Biol 1995, 60:775-781.

54. Hood E, Witcher D, Maddock S, Meyer T, Baszczynski C, Bailey M,

Flynn P, Register J, Marshall L, Bond D et al.: Commercial production
of avidin from transgenic maize: characterization of transformant,
production, processing, extraction and purification. Mol Breed
1997, 3:291-306.

55. Chong DK, Langridge WH: Expression of full-length bioactive

antimicrobial human lactoferrin in potato plants. Transgenic Res
2000, 9:71-78.

56. Ruggiero F, Exposito JY, Bournat P, Gruber V, Perret S, Comte J,

•

Olagnier B, Garrone R, Theisen M: Triple helix assembly and
processing of human collagen produced in transgenic tobacco
plants. FEBS Lett 2000, 469:132-136.

Production of a high molecular weight protein with potential for both cos-
meceutical and pharmaceutical use.

57.

Rooijen V, Moloney M: Structural requirements of oleosin domains
for subcellular targeting to the oilbody. Plant Physio/ 1995,
109:1353-1361.

58. Fooks AR: Development of oral vaccines for human use. Curr Opin

Mol Ther 2000, 2:80-86.

59. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ:

Human immune responses to a novel Norwalk virus vaccine
delivered in transgenic potatoes. J Infect Dis 2000, 182:302-305.

60. Belanger H, Fleysh N, Cox S, Bartman G, Deka D, Trudel M,

Koprowski H, Yusibov V: Human respiratory syncytial virus vaccine
antigen produced in plants. FASEB J 2000, 14:2323-2328.

61. Gil F, Brun A, Wigdorovitz A, Catala R, Martinez-Torrecuadrada JL, Casal I,

Salinas J, Borca MV, Escribano JM: High-yield expression of a viral
peptide vaccine in transgenic plants. FEBS Lett 2001, 488:13-27.

62. Huang Z, Dry II, Webster D, Strugnell R, Wesselingh S: Plant-derived

measles virus hemagglutinin protein induces neutralizing
antibodies in mice. Vaccine 2001, 19:2163-2171.

63. Tuboly T, Yu W, Bailey A, Degrandis S, Du S, Erickson L, Nagy E:

Immunogenicity of porcine transmissible gastroenteritis virus
spike protein expressed in plants. Vaccine 2000, 18:2023-2028.

64. Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M,

Lisowa O, Yusibov V, Koprowski H, Plucienniczak A, Legocki AB: A
plant-derived edible vaccine against hepatitis B virus. FASEB J
1999, 13:1796-1799.

65. Wigdorovitz A, Carrillo C, Dus Santos MJ, Trono K, Peralta A,

Gomez MC, Rios RD, Franzone PM, Sadir AM, Escribano JM,
Borca MV: Induction of a protective antibody response to foot and
mouth disease virus in mice following oral or parenteral
immunization with alfalfa transgenic plants expressing the viral
structural protein VP1. Virology 1999, 255:347-353.

66. Pollock DP, Kutzko JP, Birck-Wilson E, Williams JL, Echelard Y,

Meade HM: Transgenic milk as a method for the production of
recombinant antibodies. J Immunol Methods 1999, 231:147-157.

67.

Meade H, Ziomek C: Urine as a substitute for milk? Nat Biotechnol
1998, 16:21-22.

68. Hens JR, Amstutz MD, Schanbacher FL, Mather IH: Introduction of

••

the human growth hormone gene into the guinea pig mammary
gland by in vivo transfection promotes sustained expression of
human growth hormone in the milk throughout lactation. Biochim
Biophys Acta 2000, 1523:161-171.

Rapid expression of recombinant proteins in milk is important to ascertain its
stability and qualitative aspects before embarking on a costly campaign to
produce transgenic animals. This article illustrates one technique by which
this can be accomplished through the transfection of mammary tissue with a
nonviral expression vector to produce adequate levels of recombinant pro-
tein in milk suitable for analysis.

69. Shiga K, Fujita T, Hirose K, Sasae Y, Nagai T: Production of calves

by transfer of nuclei from cultured somatic cells obtained from
Japanese black bulls. Theriogenology 1999, 52:527-535.

70. Kuhholzer B, Baguisi A, Overstrom EW: Long-term culture and

characterization of goat primordial germ cells. Theriogenology
2000, 53:1071-1079.

Producing proteins in transgenic plants and animals Larrick and Thomas 417

71. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH: Viable

offspring derived from fetal and adult mammalian cells. Nature
1997, 385:810-813.

72. Kishi M, Itagaki Y, Takakura R, Imamura M, Sudo T, Yoshinari M,

Tanimoto M, Yasue H, Kashima N: Nuclear transfer in cattle using
colostrum-derived mammary gland epithelial cells and ear-
derived fibroblast cells. Theriogenology 2000, 54:675-684.

73. Kato Y, Tani T, Tsunoda Y: Cloning of calves from various somatic

cell types of male and female adult, newborn and fetal cows.
J Reprod Fertil 2000, 120:231-237.

74.

Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J,
Forsythe T, Golueke P, Jurgella G, Koppang R et al.: Production of
cloned pigs from in vitro systems. Nat Biotechnol 2000,
18:1055-1059.

75. Ogura A, Inoue K, Takano K, Wakayama T, Yanagimachi R: Birth of

mice after nuclear transfer by electrofusion using tail tip cells. Mol
Reprod Dev 2000, 57:55-59.

76. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y,

••

Boone J, Walker S, Ayares DL et al.: Cloned pigs produced by
nuclear transfer from adult somatic cells. Nature 2000, 407:86-90.

For some reason pigs have been more difficult to clone than cows, sheep or
goats. In this article, a novel technique using a double nuclear transfer result-
ed in viable piglets. This is a procedure that relies on in vivo activation and
minimizes in vitro manipulation. It will be interesting to determine if this pro-
cedure reduces the abnormalities in other species arising from nuclear trans-
fer into oocytes activated in vitro.

77.

McCreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE,

••

Kind AJ: Production of gene-targeted sheep by nuclear transfer
from culture somatic cell. Nature 2000, 405:1066-1069.

One of the promises of nuclear transfer is the rapid scale-up of animals
reproducibly expressing the recombinant protein of interest. This article is
the first demonstrating the generation of cloned sheep that express the
recombinant protein, human

α

1-antitrypsin, using targeted gene insertion.

78. Hill JR, Roussel AJ, Cibelli JB, Edwards JF, Hooper NL, Miller MW,

Thompson JA, Looney CR, Westhusin ME, Robl JM, Stice SL: Clinical
and pathologic features of cloned transgenic calves and fetuses
(13 case studies). Theriogenology 1999, 51:1451-1465.

79. Peura TT, Lane MW, Lewis IM, Trounson AO: Development of bovine

••

embryo-derived clones after increasing rounds of nuclear
recycling. Mol Reprod Dev 2001, 58:384-389.

One major problem with cloning has been the high incidence of abnormali-
ties occurring in the cloned embryo or after birth. In this article the amount
of in vitro nuclear manipulation was shown to have a significant effect on
embryonic development. This points out the need to understand the mecha-
nisms of genetic reprogramming and how this is influenced by different
manipulations and methodologies.

80. Shiels PG, Kind AJ, Campbell KH, Waddington D, Wilmut I,

Colman A, Schnieke AE: Analysis of telomere lengths in cloned
sheep. Nature 1999, 399:316-317.

81. Xu J, Yang X: Telomerase activity in early bovine embryos derived

from parthenogenetic activation and nuclear transfer. Biol Reprod
2001, 64:770-774.

82. Betts D, Bordignon V, Hill J, Winger Q, Westhusin M, Smith L,

••

King W: Reprogramming of telomerase activity and rebuilding of
telomere length in cloned cattle. Proc Natl Acad Sci USA 2001,
98:1077-1082.

The potentially shortened telomeres in offspring derived senescent adult cell
nuclear donors, as shown with Dolly, raised a significant problem in the long-
term viability of cloned animals. This article shows that telomerase activity is
reprogrammed upon transfer of nuclei with shortened telomeres into oocytes
and that cloned embryos and newborn calves had normal telomere lengths.
It will be important to discover the mechanism for reprogramming of telom-
erase such that lengthening of shortened telomeres is reproducible.

83. Lanza RP, Cibelli JB, Blackwell C, Cristofalo VJ, Francis MK,

Baerlocher GM, Mak J, Schertzer M, Chavez EA, Sawyer N et al.:
Extension of cell life-span and telomere length in animals cloned
from senescent somatic cells. Science 2000, 288:665-669.

84. Van den Hout H, Reuser AJ, Vulto AG, Loonen MC,

Cromme-Dijkhuis A, Van der Ploeg AT: Recombinant human

αα

-glucosidase from rabbit milk in Pompe patients. Lancet 2000,

356:397-398.

85. Nibbering PH, Ravensbergen E, Welling MM, van Berkel LA,

van Berkel PH, Pauwels EK, Nuijens JH: Human lactoferrin and
peptides derived from its N terminus are highly effective against
infections with antibiotic-resistant bacteria. Infect Immun 2001,
69:1469-1476.

86. Yeung PK: Technology evaluation: transgenic antithrombin III

(rhAT-III), Genzyme Transgenics. Curr Opin Mol Ther
2000, 2:336-339.

87.

Kerr DE, Plaut K, Bramley AJ, Williamson CM, Lax AJ, Moore K,

••

Wells KD, Wall RJ: Lysostaphin expression in mammary glands
confers protection against staphylococcal infection in transgenic
mice. Nat Biotechnol 2001, 19:66-70.

One of the uses for transgenic animals is to improve the health of dairy animals.
This article demonstrates the feasibility of conferring resistance to mastitis
through expression of a recombinant antibacterial protein. It will be important
to repeat these studies in dairy animals and to determine if other bioactive
recombinant proteins can be produced concurrently with lysostaphin.

88. Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J,

Williams S, Keen D, Perrin Y, Christou P, Fischer R: Cereal crops as
viable production and storage systems for pharmaceutical scFv
antibodies. Plant Mol Biol 2000 42:583-590.

89. McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK,

Tuse D, Levy S, Levy R: Rapid production of specific vaccines for
lymphoma by expression of the tumor-derived single-chain Fv
epitopes in tobacco plants. Proc Natl Acad Sci USA 1999
96:703-708.

90. Modelska A, Dietzschold B, Sleysh N, Fu Z, Steplewski K, Hooper D,

Koprowski H, Yusibov V: Immunization against rabies with plant-
derived antigen. Proc Natl Acad Sci USA 1998, 96:2481-2485.

Patents

P1. Lindsay S, Mulroy R, Semeniuk D: Expression of secreted human

αα

-fetoprotein in transgenic animals. Patent Number WO040693A2.

Issued July 13, 2000.

418

Protein technologies and commercial enzymes