CURRENT MICROBIOLOGY Vol. 38 (1999), pp.

335–341

An International Journal
R Springer-Verlag New York Inc. 1999

Expression of Human Growth Hormone by the Eukaryotic Alga, Chlorella

Richard L. Hawkins,* Michi Nakamura
Future Project Division, Toyota Motor Corporation, 1, Toyota-cho, Toyota, Aichi 471-8572, Japan

Received: 5 November 1998 / Accepted: 25 January 1999

Abstract. A method to use Chlorella to express a recombinant heterologous protein that can be recovered
from the extracellular medium has been developed. Plasmids are constructed with an extracellular
secretion signal sequence inserted between a promoter region and a gene for human growth hormone
(hGH). The plasmids also contain a Kanr region which confers resistance to the antibiotic G418.
Protoplasts are prepared by enzymatic treatment, and the plasmid is introduced by incubation of the
protoplasts with polyethylene glycol and dimethyl sulfoxide. Cells are then grown in the presence of
G418, and the medium is collected from 6 days after transfection. hGH is measured by immunoassay, and
values for expressed hGH of about 200–600 ng/ml are obtained.

Eukaryotic algal species have not been extensively
utilized for the purposes of protein expression, although
they do have characteristics that would be desirable as a
potential system for expression of cloned genes. Besides
the capability of high-level expression of protein, other
factors of importance include cost and availability of
media components for growth, ease of culturing under
various growth conditions, energy efficiency, a minimal
negative environmental influence, and ease of purifica-
tion to meet safety and effectiveness regulations for use
by the consumer. A group of eukaryotic alga, Chlorella,
has the potential to meet many of these criteria. Chlorella
can be grown to achieve 1.5–2.5 g/day/L if grown in
shallow tanks [26], with protein representing 50–60% [5,
10]. This organism is easily grown in an inexpensive,
defined medium of simple salts with KNO3 or ammonium
chloride as a nitrogen source [27, 35] and atmospheric
CO2 as the carbon source. Since Chlorella can also be
used for human consumption [5], bioengineered proteins
from Chlorella should more readily be able to pass
regulatory inspection, because impure contaminant algal
proteins should be better tolerated than contaminants
from potentially pathogenic bacteria.
Since Chlorella has not been extensively described
for use in protein expression, we decided to screen
*Present address: Shinshia Houei, Suigen-cho 1-23-5, Toyota, Aichi
471-0822, Japan.
Correspondence to: R.L. Hawkins
several strains of Chlorella, optimize growth conditions,
and try a variety of plasmid constructs in order to
maximize expression. We used the hGH gene as a marker
for heterologous expression, since it has been previously
used to characterize other protein expression systems [7,
18] and can be readily assayed by enzyme-linked immu-
nosorbent assay (ELISA) as a secreted protein in culture
media. It has recently been reported [13] that the genome
of Chlorella vulgaris has a family of repetitive sequences
existing in multiple copies of non-viral LINE-like retro-
transposons called Zepp elements, primarily located in
the sub-telomeric regions, although they have also been
found to be distributed throughout the genome. Thus,
Zepp sequences may be ideal homologous targets for
co-insertion of heterologous DNA with minimal perturba-
tion of important endogenous genes. The sub-telomeric
location makes them especially interesting, since Iglesias
et al. [14] report that stable transgene expression in
tobacco is correlated with insertion at sites located
toward the telomeres, whereas DNA insertion into para-
centromeric regions tends to be expressed at low levels
and be unstable over time. We also constructed plasmids
with a Chlorella Virus promoter sequence that has been
reported to lead to higher expression in transformed
plants than the CaMV35S promoter [24]. Thus, we
synthesized a Chlorella Virus promoter element, inserted
it upstream of a hGH gene, and compared it with
CaMV35S and other promoters for hGH expression by
Chlorella.
336
Materials and Methods
Gene manipulation and cloning methods follow standard protocols.
Restriction enzyme digests, alkaline phosphate treatment, Western and
Southern blot analyses are given in Sambrook et al. [29], except for
Southern blots; probes were random primer labeled with biotin and
visualized colorimetrically with alkaline phosphatase-coupled Strepta-
vidin according to manufacturer’s directions (NEN). Ligation reactions
used the Toyobo DNA ‘‘Ligation High’’ ligation kit. Competent
bacterial cells were purchased from either Promega (JM109) or
Stratagene (Epicurian coli) and transformed according to supplier’s
protocols. Plasmid DNA was isolated with the Kurabo PI-100 automatic
plasmid isolation system. Genomic DNA was isolated by the method of
Aljanabi and Martinez [2] or as given by Rochaix et al. [28].
Plasmids utilized include the pBK-CMV phagemid (Stratagene),
which has the Neor/Kanr gene conferring resistance to the antibiotic
G418 in eukaryotic cells. We obtained the pPZP111 vector from R. Jefferson
(CAMBIA MGRS, Canberra, Australia), which was designed by Pal
Maliga for plant transformation [9]. The plasmid pBI221 (Clontech)
contains the CaMV 35S promoter. The rbcS2 (small subunit of the
ribulose bisphosphate carboxylase/oxygenase) gene from the alga,
Chlamydomonas reinhardtii, has been isolated [8] and kindly supplied
to me by M. Goldschmidt-Clermont. As a marker for protein expres-
sion, the human growth hormone gene (hGH) was obtained in the
plasmid pGH-L9 [15]. Recombinant DNA plasmids were constructed
starting from either pPZP111 or pBK-CMV. A recombinant human
growth hormone (hGH) gene was inserted into the multiple cloning site,
and a synthetic extracellular secretion signal sequence was inserted
upstream of the hGH gene. A general consensus eukaryotic signal
sequence, MAR(L) 10PLAALG [21], which is capable of directing the
extracellular export of a wide variety of proteins, was examined. A
71-bp (including ClaI restriction sites on either end) oligonucleotide
DNA sequence was synthesized (Applied Biosystems 392 DNA synthe-
sizer), and the correct sequence was confirmed by DNA sequence
analysis (Applied Biosystems 373 DNA sequencer). Codon usage was
chosen to reflect the G 1 C rich codon bias of Chlorella [34]. A 68-bp,
plant-specific sequence (MANKSLLLLLLLGSLASG) directing the
extracellular secretion of a-amylase [17] and a combination of the two
signal sequences (MANKLLLLLLLLLLPLAASG) were also synthe-
sized and tested. Promoter sequences, Cauliflower Mosaic Virus-35S
promoter, Chlamydomonas reinhardtii rbcS2 promoter either alone or
in combination, or a synthetic Chlorella virus promoter sequence were
inserted upstream of the secretion signal sequence/hGH gene.
Chlorella was obtained either from the American Type Culture
Collection (Chlorella sorokiniana, ATCC-22521) or Chlorella vulgaris
C-27, supplied by M. Sugiura of Nagoya University. Results from these
two species were similar and data from both were averaged and are
hereafter referred to as the generic Chlorella.
The growth medium was originally formulated based on informa-
tion as given by Harris [12] for Chlamydomonas. Each component of
this medium was then tested for maximal growth either mixotrophically
or heterotrophically in C. vulgaris C-27 or C. sorokiniana. The optimal
mixotrophic medium consists of 10 mM NH4NO3, 50 mM K phosphate,
2 mM MgSO4 · 7H2O, 0.1 mM CaCl2 · 2H2O, 8 mM NaCl, 17.4 mM
acetic acid, 1 ml/L Hutner’s solution (see [12]) and pH adjusted to 6.8
with KOH. The heterotrophic medium is the same except that NH4NO3
is 5 mM, acetic acid is 2.5 mM, and glucose is added to 5 g/L after
autoclaving.
Chlorella cells are grown to mid-log phase either heterotrophi-
cally or autotrophically. Chlorella cell walls can be removed by
enzymatic treatment [1, 3, 33]. Several enzyme mixtures were tested,
and two enzyme mixtures were found to give good protoplast yields (as
tested by sensitivity for trypan blue uptake when cultured in hypotonic
medium) without significant toxicity to the cells. The enzyme mixtures
CURRENT MICROBIOLOGY Vol. 38 (1999)
are (A) 4% cellulase (Wako Chemical Co., from Trichoderma viride),
2% macerase (Calbiochem, from Rhizopus), 4% hemicellulase (Sigma
Chemical Co., from Aspergillus niger), and 1% pectinase (Fluka
Chemical Co., from Mould fungus) or (B) 4% hemicellulase and 2%
Driselase (Kyohwa Fermentation Co.). To prepare protoplasts, 1–3 ml
Chlorella cells (grown to about 1–2 3 107/ml to give a total number of
cells of 2 3 107 ) are collected by centrifugation at 15,000 g for 5 min.
Cells are resuspended in 400 µl growth medium and 600 µl 1 M
mannitol/sorbitol (0.5 M each) in heterotrophic growth medium. After
overnight culture at 37°C, cells were collected by centrifugation at
600 g, 5 min in a swing-out rotor, transferred to enzyme solution A and
kept at 37°C for 8 h, at which time enzyme solution B was added, and
the cells were kept at 37°C overnight. Cells were washed once in 600 µl
growth medium, 400 µl 1 M mannitol/sorbitol. The cells were then
washed once in heterotrophic growth medium (with glucose) and
resuspended in growth medium (no glucose) and heat shocked at 45°C
for 5 min. Cells were added to tubes containing 50 mg of Sea Sand
(15–20 mesh, Nacalai Tesque Chemical Co.) and a recombinant DNA
plasmid in a solution of 4.5% dimethyl sulfoxide (DMSO), 500 µM
chloroquine, 240 mM CaCl2, and 14 mM MgSO4 and shaken for 15 s on
an Eppendorf shaker. After 1–2 h of incubation at 31°C, a solution of
40% polyethylene glycol (PEG-6000) containing 2.25% DMSO was
added, and the cells were incubated for another 3–4 h. The cells were
added to a solution containing geneticin (G418) at a final concentration
of 1 mg/ml in growth medium as the selective agent. After 2–3 days,
half the medium was removed and replaced with growth medium
containing 500 µg/ml G418.
hGH expression was measured by ELISA. Samples of culture
media were withdrawn and centrifuged at 15,000 g for 5 min; 100 µl of
the supernatants was aliquoted into 96-well ELISA plates. Plates were
incubated overnight at 37°C. Plates were washed once with Tris-
buffered saline, pH 7.0 (TBS), and then 200 µl of 1% bovine serum
albumin (BSA) in TBS was incubated at 37°C for 1 h. Plates were
washed three times with TBS, and 100 µl of rabbit anti-hGH IgG
(Biostride, Inc., 2.2 mg/ml) at 1:2000 dilution in TBS was added and
incubated at 37°C for 1 h. Plates were washed three times with TBS, and
100 µl of goat anti-rabbit IgG-alkaline phosphatase conjugate (Southern
Biotech Assoc.) at 1:3000 dilution in TBS/1% BSA was added and
incubated at 37°C for 1 h. Plates were washed twice with TBS and twice
with diethanolamine buffer, pH 9.8, containing 49 mg/L MgCl2. 100 µl
of disodium para-nitrophenylphosphate (Wako) in diethanolamine
buffer was added, and color was allowed to develop at room tempera-
ture for 30 min. Absorbance was measured at 405 nm with an ELISA
plate scanner.
Transfection of Chlorella was also tested with an electroporation
method [23] at 600 V/cm for 525 msec (Shimadzu GTE-10).
Results
The two strains of Chlorella used (C. vulgaris, strain
C-27, and C. sorokiniana, ATCC-22521) were chosen on
the basis of having a high growth rate (doubling time of
approximately 9–14 h), removal of cell walls by enzyme
treatment, and a high basal level of extracellular protein
secretion (10–20 µg/ml/day). The absorbance spectrum
for Chlorella shows maximal absorbance at 685 nm, and
it was found that measurement of absorbance accurately
parallels the increase in cell mass with time and also the
secretion of extracellular protein. Therefore, growth was
typically assessed by absorbance measurements, which
R.L. Hawkins, M. Nakamura: Expression of hGH by Chlorella
are particularly useful for screening multiple cultures in
microwell plates.
Stable transfection of cells with plasmids bearing an
antibiotic resistance gene can be selected for by culturing
in the presence of an antibiotic that is lethal to cells
without the plasmid. Therefore, the sensitivity of Chlor-
ella to various antibiotics was checked in order to find an
antibiotic that would be highly inhibitory to growth of
untransformed cells. High concentrations (10 mg/ml) of
ampicillin or kanamycin had minimal effect, and spectino-
mycin or streptomycin were only partially inhibitory.
Chloramphenicol (2 mg/ml) was more inhibitory, but
there was still some growth of Chlorella at this concentra-
tion. G418 (geneticin) was tested and was found to be
completely inhibitory at 1 mg/ml.
Electroporation was found to produce transfection
results similar to those with the PEG method, but
generally was more toxic to the cells, so the PEG method
was adopted for convenience as the standard method.
Heat shock treatment was found to enhance transfec-
tion; 5 min and 10 min of heat shock were found to be
essentially similar. Examination of temperatures between
37°C and 57°C found that 45°C was clearly the best
temperature.
Co-precipitation of plasmid DNA with Ca1 can
enhance uptake of plasmids by eukaryotic cells [29].
Since plant (and algal) cells also have uptake mecha-
nisms for Mg1 for chlorophyll synthesis, co-precipitation
with both Ca1 and Mg1 was tested. A combination of
Ca1 and Mg1 was found to markedly improve transfec-
tion, with 14 mM Mg1 and 240 mM Ca1 being best.
Chloroquine is an agent that is reported to inhibit
lysosomal hydrolases [29] and therefore can protect
against degradation of introduced plasmid DNA. A range
of chloroquine concentrations was tested, and 250 µM
final concentration of chloroquine was found to almost
double transfection response.
Vortexing briefly with glass beads has been reported
to enhance transformation of Chlamydomonas reinhardtii
[20]. Vortexing with 0.5-mm glass beads for 20 s
produced a small increase in expression of hGH. Com-
bined treatment with PEG produced a further small
increment. PEG was more effective when added after the
vortex step rather than being included in the vortex
medium. Vortexing longer than 20 s led to cell damage
and death. Shaking with glass beads or Sea Sand was
examined further. Sea Sand A produced the best results,
followed by 180-µm glass beads (150–212 µm acid-
washed from Sigma Chemical). 250-µm or 500-µm glass
beads or Sea Sand B (30–50 mesh) were not as effective.
Shaking on an Eppendorf shaker was better (less cell
damage) than vortexing. Best duration of shaking was 15
s for Sea Sand A or 10 s for 175-µm beads. For Sea Sand
337
A, 50 mg/50 µl of cell/plasmid suspension was best,
while for 180-µm beads, 25 mg/50 µl was best.
The CaMV35S promoter and the rbcS2 promoter
were excised from their supplied plasmids and inserted
into either the pBK-CMV phagemid vector or the pPZP111
vector. The pBK-CMV vector is supplied with a Plac
promoter and a cytomegalovirus promoter upstream from
the MCS into which the CaMV35S and rbcS2 promoters
were inserted. Removal of both the Plac and cytomegalo-
virus promoters was found to increase expression. The
pPZP111 plasmid was found to be better expressed than
the pBK-CMV phagemid and was subsequently used as
the standard vector. The addition of an extracellular
secretion signal sequence allowed for hGH secretion into
the extracellular medium (Fig. 1). Both the 68-bp se-
quence and the 71-bp sequence were about equally
effective. hGH in the cell pellet was found to be at very
low levels. The pPZP111 plasmid has a Kanr gene in a
CaMV35S expression cassette located adjacent to the
multiple cloning site [9]. Interestingly, plasmids with a
hGH and signal sequence insert in the multiple cloning
site showed hGH expression without an added promoter,
possibly owing to expression promoted from the adjacent
CaMV35S cassette. Addition of another CaMV35S pro-
moter immediately upstream of the hGH signal sequence
insert or addition of an rbcS2 promoter yielded better
hGH expression, and a combination of the two promoters
(with CaMV35S promoter upstream of the rbcS2 pro-
moter) was slightly more effective (Fig. 1). This effect of
the CaMV35S promoter to act as a strong enhancer of
heterologous promoters or in tandem with itself, has been
reported [19]. Addition of a NOS-ter termination se-
quence downstream of the hGH construct did not affect
activity. Cells transformed with the rbcS2 promoter
constructs were cultured either in the dark or light, using
either glucose or acetate in the medium, but although this
promoter is reported to be regulated by light and acetate
[8], we did not observe any difference in expression by
cells cultured under these various conditions.
The inserted promoter–signal sequence–hGH DNA
was also tested in both orientations (left to right and right
to left) relative to the left and right T-DNA border regions
of the pPZP111 plasmid. No significant difference was
found for orientation within the plasmid. Linearization of
the plasmid at either a Nru1 site or an AscI site within the
rbcS2 sequence did not affect expression, nor did linear-
ization at several unique sites within the rest of the
pPZP111 plasmid have any effect on expression levels.
Stability of hGH in the extracellular medium was
tested by adding standard hGH to control Chlorella
cultures (which were not secreting hGH). After 1 week,
87.2% of the originally added hGH was detected, about
338 CURRENT MICROBIOLOGY Vol. 38 (1999)

Fig. 1. hGH secreted into the culture medium 1 week after transfection was assayed by ELISA. Chlorella cells were transfected with the following
plasmids: pPZP111 with an hGH insert (hGH), hGH with a signal sequence insert (signal 1 hGH), hGH with a CaMV35S promoter inserted
immediately upstream (CaMV 1 hGH), CaMV35S promoter inserted upstream of signal 1 hGH (CaMV 1 signal 1 hGH), rbcS2 promoter inserted
upstream of signal 1 hGH (rbcS 1 signal 1 hGH), or CaMV35S promoter inserted upstream up rbcS 1 signal 1 hGH
(CaMV 1 rbcS 1 signal 1 hGH). Results are expressed as mean 6 SEM.

the same recovery for similarly diluted hGH kept refriger-
ated with no cells for 1 week.
A preliminary Western blot analysis using an anti-
body to hGH found a strong band at about 21 kDa that
migrated similarly to standard hGH. Some minor bands
of lower molecular weight were also observed, indicating
the possibility of some degradation.
Southern blot analysis did not show any hybridiza-
tion of genomic DNA from transfected cells with a
labeled hGH probe. The probe did bind to control plasmid
DNA, but was relatively insensitive, since the detection
limit required approximately 50 pg of target DNA
sequence. The colorimetric assay used was probably not
sensitive enough to detect a single copy gene in the
genomic DNA, since a probe for a 600-bp Chlorella
vulgaris tubulin fragment [34] also did not hybridize to
Chlorella genomic DNA. Alternatively, the lack of hGH
probe binding may indicate that there was no stable
integration of plasmid DNA within the genomic DNA.
We had hoped that insertion of Chlorella homolo-
gous sequences would promote recombination and inte-
gration of the plasmid DNA into the nuclear genome and
hence perhaps yield more stable expression. Zepp se-
quences are retrotransposon-like repeated elements found
in Chlorella vulgaris [13]. We obtained a Zepp sequence
containing cosmid (ZA1) from Prof. Yamada of Hiro-
shima Univ. and isolated two fragments of 1.8 (KpnI-
KpnI) and 1.5 kb (HindIII-HindIII). These fragments
were then ligated into pPZP111 containing the hGH–
signal sequence–rbcS2–CaMV insert (hGH cassette).
The 1.5 ZA1 fragment was found to reliably increase
hGH production. This ZA1 fragment also seemed to
improve stability of expression somewhat, in that plas-
mids without the ZA1 fragment would usually lose
expression after 2–3 weeks, but after transfection with
ZA1 fragment, expression would be maintained for about
6 weeks, although at gradually declining levels. The
1.8-kb ZA1 fragment was found to be less effective.
Using PCR primers, we were also able to obtain a 742-bp
Zepp sequence from Chlorella genomic DNA isolated in
our laboratory. This Zepp fragment gave somewhat better
yields than the 1.5-kb ZA1 fragment (Fig. 2). Results in
Fig. 2 and Fig. 3 are higher than in Fig. 1, since
improvements in the basic transfection protocol (such as
co-precipitation, incubation times, and shaking with Sea
Sand, see above) resulted in better expression levels
attained in later experiments. Zepp sequences were
inserted either upstream or downstream or in tandem at
both ends relative to the hGH expression cassette, but
R.L. Hawkins, M. Nakamura: Expression of hGH by Chlorella
Fig. 2. Chlorella cells were transfected with pPZP111 plasmids with CaMV 1 rbcS 1 signal 1 hGH expression cassettes (no Zepp) or with an
additional insert of either a 1.5-kb HindIII-HindIII fragment of ZA1 (1.5-kb Zepp), a 1.8-kb KpnI-KpnI fragment of ZA1 (1.8-kb Zepp) or a 742-bp
Zepp PCR product from Chlorella genomic DNA (0.74-kb Zepp). Results represent secreted hGH 6 SEM.
results did not differ dependent on the location of
insertion of the Zepp sequence.
Using PCR primers on the basis of the sequence data
of Yamada et al. [34], we were able to isolate a 600-bp
fragment of a-tubulin, covering three exons and two
introns of internal tubulin sequence. It was hoped that, by
using exactly strain-homologous DNA fragments, recom-
bination would be enhanced. Plasmids with the hGH
cassette adjacent to the various tubulin inserts were
tested. A somewhat enhanced hGH expression was found,
similar to the effect of ZA1 insert or 742-bp Zepp
fragment insert (Fig. 3). This fragment of the coding
sequence had about 60% identity with a published
Chlamydomonas reinhardtii sequence [30].
Since the first intronic region of the rbcS2 gene has
been reported to act as a transcriptional enhancer for
heterologous genes in C. reinhardtii [22], we isolated a
136-bp fragment (PleI-BsaI) covering most of the first
intron of the C. reinhardtii rbcS2 gene and inserted it
upstream relative to the hGH cassette. Expression was
not significantly affected by addition of this intronic
sequence (Fig. 3).
A Chlorella Virus promoter insert was constructed
entirely from synthesized oligonucleotides. Using the
protocol of Stemmer et al. [31], we were able to achieve a
339
single-step assembly of a 300-bp cassette comprising
over 200 bp of the published Virus promoter sequence
[25], a 20-amino acid secretion signal sequence (MANK-
LLLLLLLLLLPLAASG), and 28 bp of the N-terminal
region of the hGH gene in which base substitutions were
made to reflect the codon bias of Chlorella while
preserving the amino acid sequence. A HinfI restriction
site allowed easy ligation with the remaining original
downstream hGH sequence inserted into the pPZP111
plasmid. This cassette was inserted into plasmids contain-
ing the hGH sequence only, or in tandem with the CaMV
sequence and also the 1.5-kb ZA1 sequence. Results with
only the hGH (and no added enhancer) sequence showed
hGH production comparable to the best ZA1-containing
plasmid obtained previously (Fig. 3), but addition of
CaMV35S promoter and ZA1 sequences to the Virus
Promoter cassette/hGH cassette did not result in further
improvement.
Discussion
In these experiments, the usual level of hGH production
was about 200–600 ng/ml medium. This compares to
reported production of hGH of 0.5–2.4 µg/ml in E. coli
[7] or B. brevis [18]. Currently, there seems to a problem
340 CURRENT MICROBIOLOGY Vol. 38 (1999)

Fig. 3. Chlorella cells were transfected with pPZP111 plasmids with either a synthetic virus promoter fragment with a signal sequence and hGH insert
(virus promoter) or with CaMV 1 rbcS 1 signal 1 hGH expression cassettes and additional inserts of 742-bp Zepp fragment with or without a 136-bp
first intronic sequence of the rbcS2 gene, or a 600-bp tubulin fragment PCR product from Chlorella genomic DNA with or without a 136-bp first
intronic sequence of the rbcS2 gene. Results represent secreted hGH 6 SEM.

with instability of the introduced DNA. Even in the face
of strong selection for G418 resistance, the cells usually
start to die after the first week, and hGH production
usually cannot be measured after about 1–2 months and
essentially all the cells have been killed. The results
reported here, in aggregate, used approximately 109 cells
for transfection. Of these cells, in the continuous pres-
ence of G418 selection, less that a dozen stable colonies
were established. However, none of these colonies ex-
pressed hGH, indicating either that plasmid DNA for
Kanr was being expressed while hGH was either lost or
silenced, or, alternatively, that a spontaneous mutation in
the genome led to development of G418 resistance.
Previous attempts to express heterologous proteins
with either Chlorella ellipsoidea or C. saccharophila [16,
23] also were able to achieve only transient expression.
However, stable transformation has recently been re-
ported with a C. vulgaris gene to rescue a nitrate
reductase-deficient C. sorokiniana mutant [6]. Similar
problems have been encountered in work with Chlamydo-
monas, in which stable transformation and expression of
heterologous proteins has been elusive [32], although
there is one report of heterologous expression when
co-transformed with a nitrate reductase gene in a nitrate
reductase C. reinhardtii mutant [11]. As has been found in
higher plants, unstable expression of heterologous genes
in algae may result from transcriptional inactivation by
an epigenetic silencing mechanism [4].
As an expression system, the alga, Chlorella, holds
some promise as a low-cost, environmentally friendly,
eukaryotic organism capable of heterologous protein
synthesis. We have used the hGH gene to demonstrate
that transient expression by Chlorella can be effected
either by a PEG/CaCl2 method. The use of extracellular
secretion signal sequences allows for hGH to be detected
in the culture medium. Several eukaryotic promoters
were evaluated, including the CaMV35S and/or rbcS2
promoters and a synthetic Chlorella Virus promoter,
which did not differ markedly in the expression levels
obtained.
ACKNOWLEDGMENTS
We thank the following for kindly providing us with plasmids: Prof.
Takashi Yamada, Dr. Michel Goldschmidt-Clermont, Rebecca Har-
court, and Prof. M. Ikehara. We also thank Prof. Yamada for his
comments on the manuscript.
R.L. Hawkins, M. Nakamura: Expression of hGH by Chlorella
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