C H A P T E R
18
Production of Biopharmaceuticals
in Microalgae
Bernardo Ba~nuelos-Hernandez, Josue I. Beltran-Lopez,
Sergio Rosales-Mendoza
Universidad Aut
onoma de San Luis Potosí, Laboratorio de Biofarmaceuticos Recombinantes,
Facultad de Ciencias Químicas, San Luis Potosí, Mexico
1. INTRODUCTION
Recombinant DNA and hybridoma technolo-
gies made it possible to produce, at large scale,
proteins acting as drugs, which are referred to
as biodrugs, biologics, or biopharmaceuticals
(BFs). Many disorders are currently treated
with such BFs, which comprise cytokines, mono-
clonal antibodies, clotting factors, hormones,
and enzymes. BFs differ from traditional drugs
in terms of manufacturing processes, structure,
and action. BFs are much more complex due to
their high molecular weight, specific tridimen-
sional structure (or even quaternary structures),
and heterogeneity given by the number of post-
translational modifications to which they can be
subjected (Schellekens, 2004; WHO, 2013).
Because BFs are of key importance in the fight
against a myriad of both infectious and noncom-
municable diseases, they are typically produced,
at a commercial scale, using mammalian cells,
yeast, or bacteria as the expression host. The
election of the production platform mainly
depends on the requirements determined by the
Handbook of Marine Microalgae
http://dx.doi.org/10.1016/B978-0-12-800776-1.00018-2
BF complexity (e.g., required specific glycosyla-
tion patterns or oligomeric structure formation).
Therefore, BFs with high complexity are pro-
duced in appropriate eukaryotic platforms such
as mammalian cells, whereas simpler molecules
that do not require complex posttranslational
modifications, such as insulin and some interleu-
kins, are typically produced in bacterial systems.
Production of BFs in mammalian cells is
characterized by the most appropriate post-
translational processing, yielding high-quality
proteins with optimum functionality and proper
half-life (Almo and Love, 2014). However, the
limitations of this system comprise the required
scaling-up procedure and the potential contami-
nation with pathogenic viruses or prions. The
latter factor demands strict purification processes
and quality control procedures, leading to costly
BFs. On the other hand, although the production
of recombinant proteins in bacteria cells is more
economical than in mammalian cells, the system
is limited by the lack of capability to perform
complex posttranslational modifications and the
frequent production of nonsoluble proteins that
281 © 2015 Elsevier Inc. All rights reserved.
282 18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE
require refolding processes, which in fact do not
always guarantee functionality (Overton, 2014;
Hochkoeppler, 2013).
Given this outlook, the development of new
BFs production platforms is a relevant aim in
this area. Proper functionality, safety, and low
cost are the main desired attributes, considering
that low-income countries have an urgent need
for these drugs to be more accessible. In recent
years, progres in genetic modification procedures
in combination with plant biotechnology has led
to propose next-generation platforms for the pro-
duction of BFs. Among them, plants have the po-
tential to serve as a low-cost and robust platform.
Microalgae have also been proposed as a potential
platform because these organisms offer some of
the strengths of the mammalian and bacterial sys-
tems. The characteristics identified in microalgae
for the potential BFs production include: (1) high
growth rates can be achieved in low-cost media
culture, (2) efficient genetic modification technol-
ogies are available, (3) technologies for bioreactor-
based production are available, (4) many species
possess the machinery to perform complex post-
translational modifications, and (5) numerous
species are edible and thus may serve as oral
delivery vehicles of certain BFs.
The exploration of this topic has been mainly
performed using the freshwater microalga spe-
cies Chlamydomonas reinhardtii, with promising
results in terms of yield and functionality. A sub-
stantial number of BFs have been produced in
Chlamydomonas thus far, comprising mainly vac-
cines and antibodies. These cases have generated
a solid expectation of the potential impact of this
technology in the pharmaceutical industry
(Specht and Mayfield, 2014; Rosales-Mendoza,
2013; Specht et al., 2010). This chapter analyzes
the current outlook on the use of microalgae as
expression hosts of BFs, emphasizing the poten-
tial of marine algae as convenient platforms, in
particular the species Phaeodactylum tricornutum,
Dunaliella salina, and Nannochloropsis spp., for
which genetic transformation procedures are
currently available.
2. CURRENT OUTLOOK OF BFs
PRODUCED IN MICROALGAE
Since genetic engineering tools are avai-
lable for a number of algae species, the notion of
using them as recombinant protein production
platforms emerged in the early 1990s (Kindle,
1990; Kindle et al., 1991). Figure 1 presents a gen-
eral outlook of the methodology that currently al-
lows for the development of genetically
engineered algae clones. Following this type of
procedure, approximately 13 vaccine prototypes
have been produced in microalgae during the
last decade, targeting both infectious and non-
communicable pathologies of relevance in
humans, such as hypertension (Soria-Guerra
et al., 2014), hepatitis B virus (Geng et al., 2003),
and human papillomavirus infection/cervical
cancer (Demurtas et al., 2013), among others. In
the veterinary field, these approaches have also
been applied, such as in the case of the VP28
protein-based vaccine against the spread of white
spot syndrome virus (WSSV), which is of rele-
vance in shrimp farms (Feng et al., 2014a).
Twelve of these candidate vaccines have been
produced in C. reinhardtii and only one in D. sal-
ina. The characterization of most of these vaccines
comprised the detection and quantification of the
recombinant antigen in algae candidate clones
and preclinical studies conducted in mice to eval-
uate their immunogenic potential in terms of
antibody titers and polarization of the immune
response (see Table 1).
On the other hand, antibodies have consti-
tuted another key target in this field. Although
antibodies are effective therapeutics against a va-
riety of human diseases, the high cost of such BFs
remains a limiting issue for their massive use.
C. reinhardtii has been used for the production
of different types of antibodies, which range
from large single-chain antibodies directed
against the glycoprotein D of herpes simplex
virus (HSV) (Mayfield et al., 2003) to immuno-
toxins that consist of chimeric antibodies
comprising variable regions that recognize a
 2. CURRENT OUTLOOK OF BFs PRODUCED IN MICROALGAE 283

FIGURE 1 A general workflow for the development of genetically engineered microalgae clones for recombinant protein
production.
284 18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE

FIGURE 1 cont’d.
TABLE 1 An Overview of the Recombinant Vaccines Expressed in Microalgae Species

Features of vaccine

Transformation Integration Route of

Target antigen Expression host method site Yields administration Adjuvant Findings References

White spot Dunaliella salina Glass beads Nuclear Approximately Oral e Immunogenicity Feng
syndrome virus genome 780 mg/L of not assessed et al. (2014b)
VP28 protein recombinant VP28

2. CURRENT OUTLOOK OF BFs PRODUCED IN MICROALGAE

protein
Angiotensin II Chlamydomonas Agrobacterium Nuclear Up to 0.05% of TSP e e Immunogenicity Soria-Guerra
fused to reinhardtii tumefaciens genome not assessed et al. (2014)
hepatitis B
virus capsid
antigen
(HBcAg)
Human C. reinhardtii Glass beads Chloroplast Up to 0.12% of TSP Subcutaneus Quil A Vaccination in mice Demurtas
papillomavirus genome 0.02% of TSP for the induced high titers et al. (2013)
type 16 E7 His-tagged version of specific IgGs
protein
(attenuated
mutant, E7GGG)
Plasmodium C. reinhardtii Biobalistic Chloroplast Up to 0.09% TSP Intraperitoneal Aluminum Intraperitonial Gregory
falciparum surface genome and oral salt immunization et al. (2013)
protein Pfs25 elicits IgG
fused to cholera responses that
toxin B subunit recognized Pfs25.
Oral vaccination
elicits secretory
IgA responses
C-terminal C. reinhardtii Biobalistic Chloroplast Not quantified; e e Pfs48/45 protein Jones
domain of the P. genome detected by Western was expressed et al. (2013)
falciparum surface blot after affinity and properly
protein Pfs48/45 purification folded;
immunogenicity
not assessed

285
Continued
 286
TABLE 1 An Overview of the Recombinant Vaccines Expressed in Microalgae Speciesdcont’d

Features of vaccine

18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE

Transformation Integration Route of
Target antigen Expression host method site Yields administration Adjuvant Findings References

P. falciparum C. reinhardtii Biobalistic Chloroplast Not quantified; Intraperitoneal Freund’s Both candidates Gregory
surface genome detected by Western are immunogenic et al. (2012)
proteins 25 (Pfs25) blot and Coomassie in mice inducing
and 28 (Pfs28) blue staining after antibodies against
affinity purification the target pathogen
form. Pfs25 elicits
transmission
blocking antibodies.
Chimeric protein C. reinhardtii Glass beads Nuclear 0.2e1 mg of protein Oral LTB Immunogenic in Dauvillee et al.
comprising the genome per mg of purified mice inducing (2010)
Intraperitoneal Freund’s
granule bound starch systemic protective
starch synthase immune responses.
(GBSS) and either
the C-terminal
domains from the
Apical Major
Antigen (AMA1)
or Major Surface
Protein (MSP1)

Chimeric protein C. reinhardtii Biobalistic Chloroplast Up to 0.7% of TSP Oral e Immunogenic in Dreesen et al.
comprising the genome mice inducing (2010)
cholera toxin B mucosal and
subunit and the systemic immune
D2 fibronectin- responses. Induced
binding domain immunoprotection
of Staphylococcus against S. aureus
aureus challenge
VP28 protein of C. reinhardtii Biobalistic Chloroplast Variable, ranging e e Immunogenicity Surzycki et al.
the White spot genome from 0.2% to 20.9% not assessed (2009)
syndrome virus of total cellular
protein (0.1e10.5%
TSP)
Human glutamic C. reinhardtii Biobalistic Chloroplast 0.25e0.3% of TSP e e Showed a positive Wang et al.
acid genome reactivity with (2008)
decarboxylase 65 sera from diabetic
patients and is
immunogenic in
nonobese diabetic

2. CURRENT OUTLOOK OF BFs PRODUCED IN MICROALGAE

mice
Structural protein C. reinhardtii Biobalistic Chloroplast 1.5e2% of TSP Subcutaneous Freund’s Immunogenic in He et al. (2007)
E2 of the classical genome mice inducing
swine fever virus Oral e systemic immune
responses only
when
subcutaneously
administered.
Hepatitis B D. salina Electroporation Nuclear 1.64e3.11 ng/mg e e Immunogenicity Geng et al. (2003)
surface genome of TSP not assessed
antigen
(HBsAg)
Chimeric protein C. reinhardtii Biobalistic Chloroplast 3e4% of TSP e e Displays antigenic Sun et al. (2003)
comprising the genome determinants from
cholera toxin B both components;
subunit foot and immunogenicity not
the mouth assessed
disease virus
VP1 protein

287
288 18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE
relevant cell-specific target (e.g., CD22) fused to a
toxin (e.g., gelonin toxin) that is intended as a ther-
apeutic agent against neoplastic diseases (e.g.,
chronic B-cell lymphoid leukemia; Tran et al.,
2013). Genetic engineering procedures at the
chloroplast compartment have allowed for the
successful assembly of these antibodies, which
have shown proper reactivity (see Table 2).
Besides vaccines and antibodies, many recom-
binant proteins have been expressed successfully
in microalgae, including reporter genes such as
luciferase (Matsuo et al., 2006; Mayfield and
Schultz, 2004; Minko et al., 1999), green fluores-
cent protein (GFP) (Franklin et al., 2002), and
b-glucuronidase (Ishikura et al., 1999). In addi-
tion, selection markers such as aminoglycoside
transferase (aphA) (Bateman and Purton, 2000)
and the adenine aminoglycoside transferase
(aadA) (Goldschmidt-Clermont, 1991) have
been expressed properly, allowing for the selec-
tion of transformed clones.
Other BFs that have been expressed in micro-
algae include the Kunitz trypsin inhibitor of soy-
bean (SKTI), which is used to downregulate
inflammatory activity (Chai et al., 2013); the
mammary-associated serum amyloid A protein
(M-SAA), which stimulates mucin production
in gut epithelial cells to protect the bowel in new-
borns (Manuell et al., 2007); and the human tu-
mor necrosis factor-related apoptosis-inducing
ligand (TRAIL) for cancer therapy (Yang et al.,
2006). Although most of these proteins have
been expressed in C. reinhardtii, a remarkable in-
terest in expressing recombinant proteins in
other promising microalgae species, such as
P. tricornutum and D. salina, has been reflected
in the recent literature (see Table 3).
3. OTHER MICROALGAE AS
PLATFORMS TO PRODUCE
BIOPHARMACEUTICALS
Although C. reinhardtii has been the main
microalga applied for BFs production (Rasala
and Mayfield, 2014; Mayfield et al., 2003), ma-
rine algae species are also being explored for
this purpose. The attributes that justify the use
of new species include efficient protein secretion
mechanisms, higher yields, use of seawater to
avoid interference with potable or agriculture
water sources, and the feasibility of growing
algae under full containment favoring biosafety.
The following sections describe these cases
and the potential for using them as new
microalgae-based platforms in the biopharming
field.
3.1 Phaeodactylum tricornutum
Phaeodactylum tricornutum is a diatom that has
emerged in the biotechnology field for various
applications, such as a biofuel precursor and re-
combinant protein expression host due to its
biosynthetic capacity and high growth rates.
Both biolistic and electroporation-mediated ge-
netic engineering methods have been reported
for P. tricornutum (Hempel et al., 2011; Hempel
and Maier, 2012; Zaslavskaia et al., 2000; Apt
et al., 1996; Niu et al., 2012; Miyahara et al.,
2013). Initial attempts comprised the use of vec-
tors containing selection markers targeting the
nuclear genome via a biolistic method. Apt
et al. (1996) showed that the sh-ble (phleomy-
cin-resistance gene) and cat (chloramphenicol
acetyltransferase) genes serve as appropriate se-
lection markers. Another important genetic engi-
neering tool applied in this species consists of the
use of reporter genes. Zaslavskaia et al. (2000)
developed P. tricornutum clones expressing
both the GFP and GUS proteins, which are the
main reporter genes used to monitoring either
transient or stable transgene expression.
An important advance related to BFs produc-
tion consisted of the production of functional an-
tibodies in P. triconutum. Hempel et al. (2011)
expressed a monoclonal human immunoglob-
ulin (Ig) G antibody against the hepatitis B
surface protein. Assembling and binding proper-
ties of the antibodies produced were positively
TABLE 2 An Overview of Antibodies Expressed in Microalgae Species

Description of Transformation
antibody Expression host method Integration site Findings Yields References

Immunotoxin Chlamydomonas Biobalistic Chloroplast Functional aCD22 accumulates Tran et al. (2013)
protein containing reinhardtii genome immunotoxin is at approximately
a ribosome expressed in the 0.7% of TSP.

3. OTHER MICROALGAE AS PLATFORMS TO PRODUCE BIOPHARMACEUTICALS

inactivating protein chloroplast of aCD22Gel
named gelonin with C. reinhardtii, having accumulates at
variable regions of as perspective the approximately
heavy and light use of therapies in 0.2e0.3% of TSP
chains from an neoplastic diseases aCD22CH23Gel
antibody (scFv) that such as B-cell chronic accumulates at
recognizes CD22 lymphocytic leukemia approximately
0.1e0.2% of TSP
Human monoclonal Phaeodactylum Biobalistic Nuclear genome Functional IgG IgG antibodies were Hempel and Maier
IgG antibody tricornutum antibody was secreted to the (2012)
CL4mAb against the expressed and medium at levels
hepatitis B virus secreted into the of up to 2.5 mg/mL
surface protein culture medium.
Human monoclonal P. tricornutum Biobalistic Nuclear genome Functional antibody Monoclonal IgG Hempel et al. (2011)
IgG1 antibody is expressed and antibody is
CL4mAb against the assembled in the accumulated at
hepatitis B virus endoplasmatic up to 8.7% of TSP.
surface antigen reticulum of P. HBsAg is
tricornutum using accumulated at
the endopasmic up to 0.7% TSP
reticulum retention
signal (DDEL) at the
C-terminus of both
antibody chains and
use a nitrate-inducible
promoter system
HBsAg was expressed
and accumulated in P.
tricornutum more
efficient than in other
plant systems, such as
N. tabacum

289
Continued
 290
TABLE 2 An Overview of Antibodies Expressed in Microalgae Speciesdcont’d

18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE

Description of Transformation
antibody Expression host method Integration site Findings Yields References

Human IgG1 C. reinhardtii Biobalistic Chloroplast Human antibody was Not quantified; Tran et al. (2009)
monoclonal antibody genome successfully expressed detected by Western
83K7C against the PA83 and the levels of blot
anthrax antigen expression are similar
to the same antibody
expressed in
mammalian cells

Single-chain variable C. reinhardtii Biobalistic Chloroplast Functional Antibody is Mayfield and Franklin
regions antibody genome immunotoxin protein accumulated at up to (2005)
against the Herpes is expressed in C. 0.25% of TSP
simplex virus reinhardtii chloroplast
glycoprotein D
(HSV8-scFv)
Large single-chain (lsc) C. reinhardtii Biobalistic Chloroplast HSV8-lsc antibody is Antibody is Mayfield et al. (2003)
antibody directed genome produced in a accumulated at >1%
against herpes simplex functional manner TSP
virus (HSV) in C. reinhardtii
glycoprotein D chloroplast
(HSV8-lsc)
TABLE 3 An Overview of Other Biopharmaceuticals and Reporter/Marker Genes Expressed in Microalgae Species

Description of Transformation Integration

recombinant protein Expression host method site Findings Yields References

Soybean Kunitz trypsin Dunalliela salina LiAc transformation Nuclear SKTI gene was successfully 0.68% of TSP Chai et al. (2013)
inhibitor (SKTI) method genome expressed in D. salina cells
as an approach to down
regulate inflammatory

3. OTHER MICROALGAE AS PLATFORMS TO PRODUCE BIOPHARMACEUTICALS

activity.

Chloramphenicol P. tricornutum Electroporation Nuclear CAT gene was successfully Not quantified; detected Niu et al. (2012)
acetyltransferase (CAT) genome expressed in P. tricornutum by Western blot
cells. An interesting strategy
consisted of the use of an
NaNO3-inducible promoter.
Mammary-associated serum Chlamydomonas Biobalistic Chloroplast Robust expression of 12.5% of TSP Manuell et al. (2007)
amyloid A (M-SAA) reinhardtii genome bioactive M-SAA was
achieved, with the potential
to protect against intestinal
bacterial and viral infection
in newborns.

Firefly luciferase C. reinhardtii Biobalistic Chloroplast Firefly luciferase was Not quantified Matsuo et al. (2006)
genome successfully used as a
reporter gene that will be
useful in chloroplast gene
regulation studies

Human tumor necrosis C. reinhardtii Biobalistic Chloroplast Chlamydomonas-made 0.43e0.67% of TSP Yang et al. (2006)
factor-related apoptosis- genome TRAIL was functional,
inducing ligand (TRAIL) with possible use for
therapies in viral disease
and cancer.
Human metallothionine-2 C. reinhardtii Biobalistic Chloroplast The hMT-2 human gene Not quantified; visible by Zhang et al. (2006)
genome was integrated into the Western blot
chloroplast genome of C.
reinhardtii and successfully
expressed in the
transplastomic alga. hMT-2
transplastomic algae were
resistant to UV-B radiation
exposure.

291
Continued
TABLE 3 An Overview of Other Biopharmaceuticals and Reporter/Marker Genes Expressed in Microalgae Speciesdcont’d

292
Description of Transformation Integration
recombinant protein Expression host method site Findings Yields References

Allophycocyanin C. reinhardtii Biobalistic Chloroplast Successful expression of 2%e3% (W/W) of TSP Su et al. (2005)
genome polycistronic arrays of
prokaryotic cyanobacteria
in C. reinhardtii.
Bacterial luciferase C. reinhardtii Biobalistic Chloroplast A codon-optimized Not quantified; detected Myfield and Schultz
genome bacterial luciferase gene by Western blot (2004)
was successfully used as

18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE

a reporter gene that will be
useful in chloroplast gene
regulation studies.
Green fluorescent C. reinhardtii Biobalistic Chloroplast A codon-optimized green 0.5 of TSP Franklin et al. (2002)
protein genome fluorescent protein gene
was successfully used as
a reporter gene that will be
useful in chloroplast gene
regulation studies.
Aminoglycoside C. reinhardtii Biobalistic Chloroplast The aphA-6 gene serves as Not quantified Bateman and Purton
phosphotransferase genome a selectable marker that (2000)
confers kanamycin and
amikacin resistance in
chloroplast-transformed
Chlamydomonas clones.

b-Glucuronidase C. reinhardtii Biobalistic Chloroplast Expression of GUS, a Not quantified Ishikura et al. (1999)
genome functional reporter gene
using different promoters
in C. reinhardtii.
Renilla luciferase C. reinhardtii Biobalistic Chloroplast Renilla luciferase was Not quantified; detected Minko et al. (1999)
genome successfully used as a by Western blot
reporter gene that will be
useful in chloroplast gene
regulation studies.
Aminoglycoside adenine C. reinhardtii Biobalistic Chloroplast Functional aadA gene is Not quantified Goldschmidt-
transferase (aadA) genome expressed in C. reinhardtii Clermont (1991)
and allowed for the
selection of transformed
clones.
 3. OTHER MICROALGAE AS PLATFORMS TO PRODUCE BIOPHARMACEUTICALS
evidenced by enzyme-linked immunosorbent
assay (ELISA). In terms of production, recombi-
nant protein yields were of up to 8.7% of the total
soluble protein, which represents approximately
1.6 mg of antibody per liter. The expression sys-
tem used in this approach was driven by an
inducible promoter from the nitrate reductase,
which is activated by the presence of nitrate
and downregulated by ammonia. Another char-
acteristic of this system was given by the inclu-
sion of an endoplasmic reticulum retention
signal to avoid the complex glycosylation pat-
terns that occur in the Golgi apparatus.
In 2012, Hempel and Maier made a modifica-
tion in the expression system that removed the
endoplasmic reticulum retention signal. This
modification allowed for the antibody to be
secreted by the diatom into the culture medium.
Because P. tricornutum essentially does not secrete
endogenous proteins, the secreted recombinant
protein can be easily purified, which makes the
production extremely cost efficient (Hempel and
Maier, 2012). Importantly, the expression levels
in this system were higher (2250 ng per mL)
than those observed when the protein was sub-
jected to endoplasmic reticulum retrieval.
3.2 Dunaliella salina
Dunaliella salina is a unicellular, biflagellate,
naked green alga that is morphologically similar
to Chlamydomonas, with the main difference
being the lack of a rigid cell wall (Emeish, 2013).
The first description of D. salina was made in
1832 by Dunal, who initially considered it as a
variety of Haematococcus salinus. In 1905, Teodor-
esco designated it as a new genera of microalgae.
The Dunaliella genus currently comprises many
algae species (Oren, 2005; Borowitzka and Siva,
2007). An important feature of D. salina is the
ability to be grown in high salt concentrations
of up to 35% w/v (Brown and Borowitzka,
1979). This property diminishes the contamina-
tion problems that are frequently present in
293
other species, such as Chlamydomonas. In addi-
tion, D. salina is able to grow under high-light
intensity, high temperatures, and a wide pH
range (Cifuentes et al., 1996). Another important
characteristic is the absence of a rigid cell wall,
which creates a natural protoplast, facilitating
the DNA transfer step during genetic transforma-
tion procedures (Ben-Amotz and Avron, 1990).
Several methods have been applied to
transform D. salina: electroporation, particle
bombardment, Agrobacterium tumefaciens, glass
beads, and the lithium acetate/polyethylene gly-
col (LiAc/PEG)-mediated method, with the last
two methods being the most efficient (Feng
et al., 2014b; Feng et al., 2009). D. salina has been
cultivated for several decades for pigment pro-
duction at industrial levels (Olaizola, 2003;
Gomez et al., 2003; Ben-Amotz, 1993), but in
recent years it has also emerged as a new plat-
form for BFs production. Hepatitis B surface an-
tigen (HBsAg), VP28 from WSSV, and Soybean
Kunitz trypsin inhibitor are important recombi-
nant proteins produced in D. salina. These ap-
proaches are presented in the following sections.
3.2.1 HBsAg
The first nuclear stable transformation of D.
salina was accomplished by Geng et al. 2003.
Microalga was transformed by an electropora-
tion method, obtaining approximately 50 col-
onies per plate in a period of 3 weeks. The cat
gene that confers chloramphenicol resistance
was used as a selection marker. The transformed
colonies were subcultivated 60 times, and then
the presence of the transgene was evaluated.
All colonies were positive, which reflected a
highly stable transgene insertion. The best pro-
ductivity was of 3.11 ng HBsAg/mg of soluble
protein (Geng et al., 2003). The expression of
HBsAg is important because of the epidemio-
logic relevance of the hepatitis B virus. In addi-
tion, this antigen can also serve as an
immunogenic carrier of genetically fused, nonre-
lated epitopes from other pathogens.
294 18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE
3.2.2 VP28 from WSSV
WSSV is a serious problem affecting shrimp
yields (Lightner and Redman, 1998), and vacci-
nation with plant-made vaccines has been
applied to reduce lost production (Thagun
et al., 2012). Feng et al. (2014b) obtained trans-
genic D. salina clones expressing the Vp28 pro-
tein of WSSV. A nuclear transformation was
achieved through the glass bead method, and
the vector pUU-GUS designed by Geng in 2003
was used to drive the expression. By means of
ELISA, the Vp28 protein productivity was esti-
mated at levels of up to 3.04 ng/mg of total pro-
tein in D. salina clones. In terms of production by
volume of culture, approximately 780 mg of re-
combinant VP28 protein was produced per liter.
Lysates from these transgenic microalgae clones
were used to orally immunize shrimp, as was re-
ported by Fu et al. (2010). This immunization
approach was able to diminish shrimp mortality
by 40% with respect to the nonvaccinated
shrimp population (Feng et al., 2014b). It is envi-
sioned that this algae-based vaccine will be a
breakthrough in resolving aquaculture
problems.
3.2.3 Soybean Kunitz Trypsin Inhibitor
Soybean Kunitz trypsin inhibitor (SKTI) is a
serine proteinase inhibitor against the activities
of both trypsin and chymotrypsin, which leads
to anti-inflammatory and anticarcinogenic activ-
ities (Hsieh et al., 2010; Lippman and Matrisian,
2000; Ribeiro et al., 2010). Kobayashi et al. (2004)
showed that SKTI can suppress ovarian cancer
cell invasion by blocking urokinase upregula-
tion. Trypsin inhibitors are mainly extracted
from human urine, soybean, and pumpkin. To
diminish production costs, the expression of
SKTI in D. salina has been attempted. Using the
expression vector pCAM2201, in which the
transgene skti is under the control of the 35S pro-
moter, Chai et al. (2013) transformed D. salina via
the lithium acetate/polyethylene glycol method.
Expression levels of SKTI in algae clones were
calculated at about 0.68% of total soluble protein,
and the stability of transgene insertion was posi-
tively evaluated after 35 subcultures (Chai et al.,
2013).
3.3 Haematococcus pluvialis
Hematococus pluvialis is an autotrophic fresh
water microalgae that has been applied for in-
dustrial production of pigments (Fabregas
et al., 2000; Sarada et al., 2002; Olaizola, 2000).
Although H. pluvialis is a freshwater microalgae,
it is able to grow in extreme environments and
survive extreme fluctuations in light, tempera-
ture, and salt concentration (reviewed by Saei
et al. (2012)). An important feature in H. pluvialis
is its codon usage, which is highly similar to
codon usage in Homo sapiens, as reported by
Saei et al. (2012). This is an important trait
because codon optimization is not necessary in
the case of human origin BFs to avoid the low
productivity associated to codon usage issues.
The development of genetic engineering
tools for H. pluvialis has been reported. Gutierrez
et al. (2012) have developed a chloroplast-
transforming vector, designed using the endoge-
nous 5’ rbcL as promoter and 30 rbcL as
terminator. These elements were used to express
the aadA selection marker. The construct was
introduced into the chloroplast through the bio-
listic method. The transgene insertion was stable
through 40 subcultivation steps in three trans-
genic lines, although only one line became ho-
moplastic after the selection rounds. Another
important advantage of H. pluvialis is the avail-
ability of nuclear transformation protocols via
agro-inoculation. Kathiresan et al. (2009) used
agrobacterium and elements from the pCAMBIA
vector to express the reporter genes gfp and uidA.
The integration of the transgene was maintained
after 2 years of subcultivation. These genetic
engineering tools will be critical to explore the
use of H. pluvialis as a new BF production
platform.
 4. PERSPECTIVES
3.4 Nannochloropsis spp.
Nannochloropsis spp. are microalgae living in
freshwater and seawater that are related to dia-
toms and brown algae (Sukenik et al., 2009;
Andersen et al., 1998). Nannochloropsis species
have been used for several decades to produce
nutraceuticals and feed supplements (Rodolfi
et al., 2009). Genetic engineering tools for nuclear
transformation have been recently developed for
this species. Kilian et al. (2011) established a pro-
tocol for nuclear transformation of Nannochlorop-
sis via homologous recombination with a high
transformation efficiency. These tools increase
the possibilities of implementing robust BF pro-
duction platforms based in this species.
4. PERSPECTIVES
Green microalgae are potential expression
hosts for the production of biomolecules of high
biotechnological value because these organisms
have unique advantages, such as low production
costs, high growth rates, high yields, and the abil-
ity to accomplish posttranslational modifications.
They are compatible with high biosafety proce-
dures, with some of them considered Generally
Recognized as Safe by the US Food and Drug
Administration. Among green algae, C. reinhard-
tii is the eukaryotic green microalga mainly
used as an expression host in this field. The adop-
tion of these approaches by the industry high-
lights the potential of algae-based platforms.
For example, Triton Algae Innovations is a com-
pany that produces proteins, enzymes, and other
biologics in Chlamydomonas. One of the products
currently in the market is the mammary-
associated amyloid, which is contained in the
colostrum of mammals and can be used to treat
intestinal disease in livestock, companion ani-
mals, and humans. In addition, antibacterials, an-
tioxidants, biosurfactants, DNA repair enzymes,
antimicrobials, intestinal health proteins, growth
factors, bone growth enhancers, and vaccines
295
are in the pipeline. All of these candidates will
have implications on the development of phar-
maceuticals, nutraceuticals, cosmetics, and hu-
man and animal health nutrition products;
applications in agricultural and other retail mar-
kets are also envisioned.
Although current biologicals produced in
Chlamydomonas are promising and interesting
cases, relevant perspectives related to the expan-
sion of this field can be also outlined. Improve-
ments in distinct aspects can be identified as a
need in the Chlamydomonas-based approaches.
For example, Chlamydomonas cultures are prone
to contamination, which causes frequency delays
in clone propagation steps. On the other hand,
many BFs require posttranslational modifica-
tions, such as glycosylation; in this case,
following a nuclear-based expression, it is neces-
sary to direct the protein to endoplastmic reticu-
lum and Golgi apparatus or even the secretory
pathway. However, nuclear-based expression
in Chlamydomonas has shown generally low
yields (Rasala et al., 2012). In addition, the use
of seawater to cultivate algae is desirable in
this process to avoid the use of freshwater, which
is also used as potable water or for agricultural
purposes. It is envisioned that these pitfalls
would be overridden by the use of marine algae
species with distinct characteristics.
The identified marine algae species in this
chapter, for which culture and genetic modifica-
tion tools are available, constitute relevant hosts
to generate new BFs production platforms with
improved features, including the following:
low-contamination events due to cultivation un-
der extreme culture conditions (e.g., pH, and
high salinity); higher protein yields; the use of
seawater instead fresh water; and the use of the
secretion machinery to facilitate downstream
processing in the case of BFs requiring parenteral
administration. The coming years will be critical
to assess the potential of these candidate species
to determine their performance with a wide
number of specific BFs.
296 18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE
References
Almo, S.C., Love, J.D., 2014. Better and faster: improvements
and optimization for mammalian recombinant protein
production. Curr. Opin. Struct. Biol. 7, 39e43.
Andersen, R.A., Brett, R.W., Potter, D., Sexton, J.P., 1998.
Phylogeny of the Eustigmatophyceae based upon 18S
rDNA, with emphasis on Nannochloropsis. Protist 149,
61e74.
Apt, K.E., KrothPancic, P.G., Grossman, A.R., 1996. Stable
nuclear transformation of the diatom Phaeodactylum
tricornutum. Mol. Gen. Genet. 252, 572e579.
Bateman, J.M., Purton, S., 2000. Tools for chloroplast transfor-
mation in Chlamydmonas: expression vectors and a new
dominant selectable marker. Mol. Gen. Genet. 263,
404e410.
Ben-Amotz, A., 1993. Production of b-carotene and vitamin
by the halotolerant algae Dunaliella. In: Ahaway, A.,
Zabrosky, O. (Eds.), Marine Biotechnology. Plenum Press,
New York, pp. 411e417.
Ben-Amotz, A., Avron, M., 1990. The biotechnology of culti-
vating the halotolerant alga Dunaliella. Trends Biotechnol.
8, 121e126.
Borowitzka, M.A., Siva, C.J., 2007. The taxonomy of the
genus Dunaliella (Chlorophyta, Dunaliellales) with
emphasis on the marine and halophilic species. J. Appl.
Phycol. 19, 567e590.
Brown, A.D., Borowitzka, L.J., 1979. Halotolerance of Duna-
liella. In: Levandowsky, M., Hunter, S.H. (Eds.), Biochem-
istry and Physiology of Protozoa, second ed., vol. 1.
Academic Press, NY, pp. 139e190.
Chai, X.F., Chen, H.X., Xu, W.Q., Xu, Y.W., 2013. Expression
of soybean Kunitz trypsin inhibitor gene SKTI in Duna-
liella salina. J. Appl. Phycol. 25, 139e144.
Cifuentes, A.S., Gonzalez, M., Parra, O., Zu~ niga, M., 1996.
Culture of strains of Dunaliella salina (Teodoresco 1905)
in different media under laboratory conditions. Rev.
Chil. Hist. Nat. 69, 105e112.
Dauvillee, D., Delhaye, S., Gruyer, S., Slomianny, C.,
Moretz, S.E., d’Hulst, C., Long, C.A., Ball, S.G.,
Tomavo, S., 2010. Engineering the chloroplast targeted
malarial vaccine antigens in Chlamydmonas starch
granules. PLoS One 5, e15424.
Demurtas, O.C., Massa, S., Ferrante, P., Venuti, A.,
Franconi, R., Giuliano, G.A., 2013. Chlamydmonas-derived
humanpapillomavirus16 E7 vaccine induces specific
tumor protection. PLoS One 8, e61473.
Dreesen, I.A., Charpin-El Hamri, G., Fussenegger, M., 2010.
Heat-stable oral alga-based vaccine protects mice from
Staphylococcus aureus infection. J. Biotechnol. 145,
273e280.
Emeish, S., 2013. Production of natural biopharmaceuticals
from the microalgae living in the dead sea. J. Environ.
Earth Sci. 3, 6e15.
Fabregas, J., Domínguez, A., Regueiro, M., Maseda, A.,
Otero, A., 2000. Optimization of culture medium for the
continuous cultivation of the microalga Haematococcus
pluvialis. Appl. Microbiol. Biotechnol. 53, 530e535.
Feng, S., Feng, W., Zhao, L., Gu, H., Li, Q., Shi, K., Guo, S.,
Zhang, N., 2014a. Preparation of transgenic Dunaliella sal-
ina for immunization against white spot syndrome virus
in crayfish. Arch. Virol. 159, 519e525.
Feng, S., Li, X., Xu, Z., Qi, J., 2014b. Dunaliella salina as a novel
host for the production of recombinant proteins. Appl.
Microbiol. Biotechnol. 98, 4293e4300.
Feng, S., Xue, L., Liu, H., Lu, P., 2009. Improvement of
efficiency of genetic transformation for Dunaliella salina
by glass beads method. Mol. Biol. Rep. 36, 1433e1439.
Franklin, S., Ngo, B., Efuet, E., Mayfield, S.P., 2002. Develop-
ment of a GFP reporter gene for Chlamydmonas reinhardtii
chloroplast. Plant J. 30, 733e744.
Fu, L.L., Shuai, J.B., Xu, Z.R., Li, J.R., Li, W.F., 2010. Immune
responses of Fenneropenaeus chinensis against white spot
syndrome virus after oral delivery of VP28 using Bacillus
subtilis as vehicles. Fish Shellfish Immunol. 28, 49e55.
Geng, D., Wang, Y., Wang, P., Li, W., Sun, Y., 2003. Stable
expression of hepatitis B surface antigen gene in Dunaliella
salina (Chlorophyta). J. Appl. Phycol. 15, 451e456.
Gregory, J.A., Li, F., Tomosada, L.M., Cox, C.J., Topol, A.B.,
Vinetz, J.M., Mayfield, S.P., 2012. Algae-produced Pfs25
elicits antibodies that inhibit malaria transmission. PLoS
One 7, e37179.
Gregory, J.A., Topol, A.B., Doerner, D.Z., Mayfield, S., 2013.
Alga-produced cholera toxin-Pfs25 fusion proteins as oral
vaccines. Appl. Environ. Microbiol. 79, 3917e3925.
Goldschmidt-Clermont, M., 1991. Transgenic expression of
aminoglycoside adenine transferase in the chloroplast: a
selectable marker of site-directed transformation of
Chlamydmonas. Nucleic Acids Res. 19, 4083e4089.
Gomez, P.I., Barriga, A., Cifuentes, A.S., Gonzalez, M.A.,
2003. Effect of salinity on the quantity and quality of ca-
rotenoids accumulated by Dunaliella salina (strain
CONC-007) and Dunaliella bardawil (strain ATCC 30861)
Chlorophyta. Biol. Res. 36, 185e192.
Gutierrez, C.L., Gimpel, J., Escobar, C., Marshall, S.H.,
Henríquez, V., 2012. Chloroplast genetic tool for the green
microalgae Haematococcus pluvialis (chlorophyceae,
volvocales). J. Phycol. 48, 976e983.
He, D.M., Qian, K.X., Shen, G.F., Zhang, Z.F., Li, Y.N.,
Su, Z.L., Shao, H.B., 2007. Recombination and expression
of classical swine fever virus (CSFV) structural protein E2
gene in Chlamydmonas reinhardtii chroloplasts. Colloids
Surf. B. Biointerfaces 55, 26e30.
Hempel, F., Lau, J., Klingl, A., Maier, U.G., 2011. Algae as
protein factories: expression of a human antibody and
the respective antigen in the diatom Phaeodactylum
tricornutum. PLoS One 6, e28424.
 REFERENCES
Hempel, F., Maier, U.G., 2012. An engineered diatom acting
like a plasma cell secreting human IgG antibodies with
high efficiency. Microb. Cell. Fact. 11, 1e6.
Hochkoeppler, A., 2013. Expanding the landscape of recom-
binant protein production in Escherichia coli. Biotechnol.
Lett. 35, 1971e1981.
Hsieh, C.C., Hern andez-Ledesma, B., Jeong, H.J., Park, J.H.,
Ben, O., 2010. Complementary roles in cancer prevention:
protease inhibitor makes the cancer preventive peptide
lunasin bioavailable. PLoS One 5, e8890.
Ishikura, K., Takaoka, Y., Kato, K., Sekine, M., Yoshida, K.,
Shinmyo, A., 1999. Expression of a foreign gene in Chla-
mydmonas reinhardtii chloroplast. Biosci. Bioeng. 87,
307e314.
Jones, C.S., Luong, T., Hannon, M., Tran, M., Gregory, J.A.,
Shen, Z., Briggs, S.P., Mayfield, S.P., 2013. Heterologous
expression of the C terminal antigenic domain of the
malaria vaccine candidate Pfs48/45 in the green algae
Chlamydmonas reinhardtii. Appl. Microbiol. Biotechnol.
97, 1987e1995.
Kathiresan, S., Chandrashekar, A., Ravishankar, A.,
Sarada, R., 2009. Agrobacterium-mediated transforma-
tion in the green alga Haematococcus pluvialis (Chlorophy-
ceae, volvocales). J. Phycol. 45, 642e649.
Kilian, O., Benemann, C.S., Niyogi, K.K., Vick, B., 2011.
High-efficiency homologous recombination in the oil-
producing alga Nannochloropsis sp. Proc. Natl Acad. Sci.
U.S.A. 108, 21265e21269.
Kindle, K.L., 1990. High-frequency nuclear transformation of
Chlamydomonas reinhardtii. Proc. Natl Acad. Sci. USA 87,
1228e1232.
Kindle, K.L., Richards, K.L., Stern, D.B., 1991. Engineering
the chloroplast genome: techniques and capabilities for
chloroplast transformation in Chlamydomonas reinhardtii.
Proc. Natl Acad. Sci. USA 88, 1721e1725.
Kobayashi, H., Suzuki, M., Kanayama, N., Terao, T., 2004. A
soybean Kunitz trypsin inhibitor suppresses ovarian can-
cer cell invasion by blocking urokinase upregulation.
Clin. Exp. Metastasis 21, 159e166.
Lightner, D.V., Redman, R.M., 1998. Shrimp diseases and
current diagnostic methods. Aquaculture 164, 201e220.
Lippman, S.M., Matrisian, L.M., 2000. Protease inhibitors in
oral carcinogenesis and chemoprevention. Clin. Cancer
Res. 6, 4599e4603.
Manuell, A.L., Beligni, M.V., Elder, J.H., Siefker, D.T.,
Tran, M., Weber, A., McDonald, T.L., Mayfield, S.P.,
2007. Robust expression of a bioactive mammalian pro-
tein in Chlamydmonas chloroplast. Plant Biotechnol. J. 5,
402e412.
Matsuo, T., Onai, K., Okamoto, K., Minagawa, J., Ishiura, M.,
2006. Real-time monitoring of chloroplast gene expression
by a luciferase reporter: evidence for nuclear regulation of
chloroplast circadian period. Mol. Cell. Biol. 26, 863e870.
297
Mayfield, S.P., Franklin, S.E., Lerner, R.A., 2003. .Expression
and assembly of a fully active antibody in algae. Proc.
Natl. Acad. Sci. U.S.A. 100, 438e442.
Mayfield, S.P., Franklin, S.E., 2005. Expression of human
antibodies in eukaryotic micro-algae. Vaccine 23,
1828e1832.
Mayfield, S.P., Schultz, J., 2004. Development of a luciferase
reporter gene, luxCt, for Chlamydmonas reinhardtii
chloroplast. Plant J. 37, 449e458.
Minko, I., Holloway, S.P., Nikaido, S., Carter, M.,
Odom, O.W., Johnson, C.H., Herrin, D.L., 1999. Renilla
luciferase as a vital reporter for chloroplast gene expres-
sion in Chlamydomonas. Mol. Gen. Genet. 262, 421e425.
Miyahara, M., Aoi, M., Inoue-Kashino, N., Kashino, Y.,
Ifuku, K., 2013. Highly efficient transformation of the
diatom Phaeodactylum tricornutum by multi-pulse electro-
poration. Biosci. Biotechnol. Biochem. 77, 874e876.
Niu, Y.F., Yang, Z.K., Zhang, M.H., Zhu, C.C., Yang, W.D.,
Liu, J.S., Li, H.Y., 2012. Transformation of diatom Phaeo-
dactylum tricornutum by electroporation and establish-
ment of inducible selection marker. BioTechniques 1e3.
Olaizola, M., 2003. .Commercial development of microalgal
biotechnology: from the test tube to the marketplace.
Biomol. Eng. 20, 459e466.
Olaizola, M., 2000. Commercial production of astaxanthin
from Haematococcus pluvialis using 25,000-liter outdoor
photobioreactors. J. Appl. Phycol. 12, 499e506.
Oren, A., 2005. A hundred years of Dunaliella research:
1905e2005. Saline Syst. 1, 1e14.
Overton, T.W., 2014. Recombinant protein production in bac-
terial hosts. Drug Discov. Today 19, 590e601.
Rasala, B.A., Mayfield, S.P., 2014. Photosynthetic bio-
manufacturing in green algae; production of recombinant
proteins for industrial, nutritional, and medical uses.
Photosynth. Res. http://dx.doi.org/10.1007/s11120-014-
9994-7.
Rasala, B.A., Lee, P.A., Shen, Z., Briggs, S.P., Mendez, M.,
Mayfield, S.P., 2012. Robust expression and secretion of
xylanase1 in Chlamydomonas reinhardtii by fusion to a se-
lection gene and processing with the FMDV 2A peptide.
PLoS One 7 (8), e43349.
Ribeiro, J.K., Cunha, D.D., Fook, J.M., Sales, M.P., 2010. New
properties of the soybean trypsin inhibitor: inhibition of
human neutrophil elastase and its effect on acute pulmo-
nary injury. Eur. J. Pharmacol. 644, 238e244.
Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G.,
Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae
for oil: strain selection, induction of lipid synthesis and
outdoor mass cultivation in a low-cost photobioreactor.
Biotechnol. Bioeng. 102, 100e112.
Rosales-Mendoza, S., 2013. Future directions for the develop-
ment of Chlamydmonas-based vaccines. Expert Rev.
Vaccines 12, 1011e1019.
298 18. PRODUCTION OF BIOPHARMACEUTICALS IN MICROALGAE
Saei, A.A., Ghanbari, P., Barzegari, A., 2012. Haematococcus
as a promising cell factory to produce recombinant phar-
maceutical proteins. Mol. Biol. Rep. 39, 9931e9939.
Sarada, R., Bhattacharya, S., Ravishankar, G.A., 2002. Opti-
mization of culture conditions for growth of the green
alga Haematococcus pluvialis. World J. Microbiol. Bio-
technol. 18, 517e521.
Schellekens, H., 2004. When biotech protein go off-patent.
Trends Biotechnol. 22, 406e410.
Soria-Guerra, R.E., Ramírez-Alonso, J.I., Iban~ ez-Salazar, A.,
Govea-Alonso, D.O., Paz-Maldonado, L.M.T., Ba~ nuelos-
Hern andez, B., Korban, S.S., Rosales-Mendoza, S., 2014.
Expression of an HBcAg-based antigen carrying angio-
tensin II in Chlamydmonas reinhardtii as a candidate hyper-
tension vaccine. Plant Cell, Tissue Organ Cult. 116,
133e139.
Specht, E.A., Mayfield, S.P., 2014. Algae-based oral recombi-
nant vaccines. Front. Microbiol. 17, 5e60.
Specht, E.A., Miyake-Stoner, S., Mayfield, S.P., 2010. Micro-
algae come of age as a platform for recombinant protein
production. Biotechnol. Lett. 32, 1373e1383.
Su, Z.L., Qian, K.X., Tan, C.P., Meng, C.X., Qin, S., 2005.
Recombination and heterologous expression of allophy-
cocyanin gene in the chloroplast of Chlamydmonas
reinhardtii. Acta Biochim. Biophys. Sin 37, 709e712.
Sukenik, A., Beardall, J., Kromkamp, J.C., Kopecký, J.,
Masojídek, J., Bergeijk, S.V., Gabai, S., Shaham, E.,
Yamshon, A., 2009. Photosynthetic performance of out-
door Nannochloropsis mass cultures under a wide range
of environmental conditions. Aquat. Microb. Ecol. 56,
297e308.
Sun, M., Qian, K., Su, N., Chang, H., Liu, J., Shen, G., 2003.
Foot-and-mouth disease virus VP1 protein fused with
cholera toxin B subunit expressed in Chlamydmonas rein-
hardtii chloroplast. Biotechnol. Lett. 25, 1087e1092.
Surzycki, R., Greenham, K., Kitayama, K., Dibal, F.,
Wagner, R., Rochaix, J.D., Ajam, T., Surzycki, S., 2009.
Factors effecting expression of vaccines in microalgae.
Biologicals 37, 133e138.
Thagun, C., Srisala, J., Sritunyalucksana, K., Narangajavana, J.,
Sojikul, P., 2012. Arabidopsis-derived shrimp viral-binding
protein, PmRab7 can protect white spot syndrome virus
infection in shrimp. J. Biotechnol. 161, 60e67.
Tran, M., Henry, R.E., Siefker, D., Van, C., Newkirk, G., Kim, J.,
Bui, J., Mayfield, S.P., 2013. Production of anti-cancer
immunotoxins in algae: ribosome inactivating proteins as
fusion partners. Biotechnol. Bioeng. 110, 2826e2835.
Tran, M., Zhou, B., Pettersson, P.L., Gonzalez, M.J.,
Mayfield, S.P., 2009. Synthesis and assembly of a full-
length human monoclonal antibody in algal
chloroplasts. Biotechnol. Bioeng. 104, 663e673.
Wang, X.F., Brandsma, M., Tremblay, R., Maxwell, D.,
Jevnikar, M.A., Huner, N., Ma, S., 2008. A novel expres-
sion platform for the production of diabetes-associated
autoantigen human glutamic acid decarboxylase
(hGAD65). BMC Biotechnol. 8, e87.
World Health Organization, 2013. WHO Guidelines on the
Quality, Safety, and Efficacy of Biotherapeutic Protein
Products Prepared by Recombinant DNA Technology.
World Health Organization. Technical Report Series,
No. 814.
Yang, Z., Li, Y., Chen, F., Li, D., Zhang, Z., Liu, Y., Zheng, D.,
Wang, Y., Shen, G., 2006. Expression of human soluble
TRAIL in Chlamydmonas reinhardtii chloroplast. Chin.
Sci. Bull. 51, 1703e1709.
Zaslavskaia, L.A., Lippmeier, J.C., Kroth, P.G.,
Grossman, A.R., Apt, K.E., 2000. Transformation of the
diatom Phaeodactylum tricornutum (Bacillariophyceae)
with a variety of selectable marker and reporter genes.
J. Phycol. 36, 379e386.
Zhang, Y.K., Shen, G.F., Ru, B.G., 2006. Survival of human
metallothionein- 2 transplastomic Chlamydmonas reinhard-
tii to ultraviolet B exposure. Acta Biochim. Biophys. Sin.
38, 187e193.