2001–2002 - California Sea Grant - UC San Diego

of HSV8-lsc varies considerably depending on the growth conditions.
Expre sion under the control of the rbcL promoter5 UTR
resolved on SDSPAGE, and HSV8-lsc visualized by Western
blo ting with anti Flag antibody. A shown in Fig. 6, a cumulation
anti-flag antibody (Right). (B) Proteins fromE. coli and C. reinhardt i expre sing the Hsv8-lsc antibody were separated intosoluble and insoluble pe lets by
centrifugation. Twenty micrograms ofprotein were either stained with C oma sie blue (Left) orbloted to nitroce lulose membrane and decorated with an
Fig. 4. Expre sion of HSV8-lsc proteins in bacteria and chloroplast. (A)Twenty microgramsofcrude protein from E. coli,WT C.reinhardt i,andtransgenic lines
10-6-3 and 20-4-4 were separated bySDSPAGE and either stained with C oma sie blue (Left) orbloted to nitroce lulose membrane and decorated with an
the light phase, regardle s of the ce l density. Alternatively, The
atpA promoter5 UTR in strain 10-6-3 drives fairly constant levels
in strain 20-4-4, shows a marked increase in the a cumulation of
antibody a the end of the dark phase or immediately after entering
anti-flag antibody (Right). (C) Soluble proteins from C. reinhardt i transgenic line 10-6-3 were eithertreated with (Bme) or without(no Bme) reducing agent
before separation onSDSPAGE. Proteins were blo ted tonitroce lulose membrane and decorated with anti-flag antibody.
of HSV8-lsc production at 10 6 ce ls per ml in a lightdark cycle, yet
shows a tremendous increase in lsc a cumulation on entering the
were resuspended in Tris-bu fered saline, and the ce ls lysed by
sonication. Soluble proteins were separated from insoluble
chloroplast-expre sed protein along with that of the bacterialexpre
sed HSV8-lsc. HSV8-lsc transgenic bacteria and algae
at 10 6 ce ls per ml than at 10 7 ce ls per ml. These data clearly show
that a cumulation of HSV8-lsc in chloroplast of C. reinhardt i can
light phase when ce ls are cultured at 10 7 ce ls per ml. When grown
under continuous light, both strains exhibited higher a cumulation
by SDSPAGE, and HSV8-lsc proteins visualized by Western
blot analysis. As shown in Fig. 4B, 60% of the HSV8-lsc
proteins by centrifugation. Equal amounts of protein from the
soluble fractions and from the insoluble pe lets were separated
be optimized, dependent on the light regime used to culture the
ce ls, the phase in the cycle at which ce ls are harvested, and the
promoterUTR used to driv expre sion.
and nonreducing gels. Under nonreducing conditions any disulfide
bonds formed betw en the two heavy chain moieties of the antibody
produced in bacteria partitioned to the insoluble fraction,
whereas the HSV8-lsc produced in chloroplast was found exclusively
in the soluble fraction.
To determine whether chloroplast-expre sed antibodies contained
any pos translational modifications we first examined the
antibodies by SDSPAGE and Western blot analysis on reducing
conditions chloroplast-expre sed HSV8-lsc runs as a much larger
protein of 140 kDa, the size expected of a dimer. Treatment with
should remain intact a lowing the antibody to migrate as a larger
species. As shown in Fig. 4C (a rowhead), under nonreducing
A PLIED BIOLOGICAL
SCIENCES
the monomer at 68 kDa. To ascertain whether any other pos translational
modifications might be present in the chloroplast-expre sed
BMe, to reduce disulfide bonds, results in the migration of the
chloroplast HSV8-lsc proteins a the predicted molecular ma s of
Fig.5. Characterization of HSV8-lscbinding to HSV8viralprotein via ELISA.
Affinity-purified HSV8-lsc from the transgenic C. reinhardt i strains (10-6-3
transgeni chloroplast was functional, w examined the ability of
chloroplast-expre sed HSV8-lsc to bind HSV8 proteins. HSV8-lsc
proteins, we characterized the bacterial- and chloroplast-expre sed
proteins by ma spectrometry. The ma spectra of peptide fragments
from both the E. coli-and chloroplast-expre sed protein have
an almost identical pa tern, indicating that no additional modifications
are made to the chloroplast protein (data not shown).
Fina ly, to determine whether the HSV8-lsc a cumulating in the
hsv8-lsc were incubated inmicrotiter platescoated withaconstant amountof
viral protein.Protein concentrationsinthese affinity-purifiedextractswere 13
ngl, of which 10% was HSV8-lsc as judged by C oma sie staining. Equal
volumes ofWT C. reinhardt i proteins were used as a negative control (concentration
1 gl).
and 16-3)were scr ened in an ELISA a say against HSVproteinsprepared from
virus-infectedce ls. A totalof1 0,80,70, 60,30, 20,10,or5l of Flag-purified
was purified from transgenic chloroplast by using an anti-flag
a finity resin. As shown in Fig. 5, the chloroplast produced antibody
recognized HSV8 proteins in ELISA a says in a robust manner.
regimes on the a cumulation of antibody in the two transgenic
strains, 10-6-3 and 20-4-4. Cultures of each strain were maintained
Modulation of HSV8-lsc A cumulation in Transgenic Algae. To determine
whether we could modulate the expre sion of HSV8-lsc in C.
reinhardt i chloroplast, we examined the e fect of di ferent growth
Mayfield et al. PNAS January 21, 2 03 vol. 1 0 no. 2 41
at 10 6 or 10 7 ce ls per ml and grown either in a 1212 lightdark cycle
(5, 0 lux) or under continuous light (5, 0 lux). Ce ls were
harvested by centrifugation and 20 g of soluble protein was
communicating science to the public…
From March 1, 2000 to February 28,
2002, California Sea Grant Communications
produced 33 publications
that describe on-going research projects
and present the results of Sea Grant research
and outreach activities. These
publications were widely distributed to
both scientific and lay audiences at state
and national levels. Another 97 publications
(including journal reprints, dissertation
abstracts, technical reports, and
conference proceedings)
resulting from Sea Grantsponsored
research and
activities were made
available to user communities
and advertised via
newsletters, publication
lists and the web site.
Two outreach projects
each produced a video; a
CALIFORNIA
SEA GRANT
Summary
third project produced an
electronic publication for
Introduction
the Internet; and a fourth
produced a workbook for secondary
school science teachers. Communications
produced 31 additional
publications, including newsletters, news
releases, award and publication announcements,
and brochures that were distributed
to target audiences throughout the state
and nationally.
The Plant Journal (2002) 30(6), 733±744
TECHNICAL ADVANCE
Development of a GFP reporter gene for Chlamydomonas
reinhardtii chloroplast
Program
Directory
2002–2003
The California Sea Grant Extension
Program partnered with other agencies
to produce a 592-page comprehensive
guide, “California’s Living
Marine Resources: A Status Report.”
The West Coast Ballast Outreach
Project published and distributed
to the shipping industry 13,000
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Much of California Sea Grant’s
funded research is disseminated to
the scientific community through
General Public
21%
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16%
Government
29%
Education/Library
34%
40
publication in peer-reviewed,
scientific journals. The
Communications staff supplements
this by advertising
journal reprints to all its
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gain even broader dissemination,
increasingly Sea Grant is
making its publications
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notifying constituents of their
availability.
Scott Franklin, Binh Ngo, Ekem Efuet and Stephen P. May®eld *
Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 N.Torrey Pines Road, La Jolla, CA 92037, USA
Received 18 January 2002; revised 5 March 2002; accepted 6 March 2002.
* For correspondence (fax +1 858 784 9840; e-mail sefrankl@scripps.edu).
Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of
recombinant protein expression have been reported. Reporter genes have also been used in the
chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared
to be very low. We hypothesized that the inability to achieve high levels of recombinant protein
expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii
chloroplast genome. To test this hypothesis, we synthesized a gene encoding green ¯uorescent protein
(GFP) de novo, optimizing its codon usage to re¯ect that of major C. reinhardtii chloroplast-encoded
proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the
codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5¢- and 3¢-UTRs. We
compared this expression with the accumulation of GFP in C. reinhardtii transformed with a nonoptimized
GFP cassette (GFPncb), also under the control of the rbcL 5¢- and 3¢-UTRs. We demonstrate
that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate »80-fold more GFP than
GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under
control of the rbcL 5¢- and 3¢-UTRs, is suf®ciently robust to report differences in protein synthesis based
on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C.
reinhardtii chloroplast gene expression.
Keywords: Chlamydomonas, GFP, reporter gene, codon bias.
Reporter genes have greatly enhanced our ability to
monitor gene expression in a number of biological organisms.
In chloroplasts of higher plants, b-glucuronidase
(uidA, Staub and Maliga, 1993); neomycin phosphotransferase
(nptII, Carrer et al., 1993); adenosyl-3-adenyltransferase
(aadA, Svab and Maliga, 1993); and the green
¯uorescent protein of Aequorea aequorea (gfp, Reed et al.,
2001; Sidorov et al., 1999) have been used as reporter
genes (Heifetz, 2000). Each of these genes has attributes
that makes it a useful reporter of chloroplast gene expression,
such as ease of analysis, sensitivity, or the ability to
examine expression in situ. Based on these studies, other
heterologous proteins have been expressed in the chloroplasts
of higher plants, such as Bacillus thuringiensis Cry
toxins, conferring resistance to insect herbivory (Kota
et al., 1999), or human somatotropin (Staub et al., 2000), a
potential biopharmaceutical.
Several reporter genes have been expressed in the
chloroplast of the eukaryotic green alga Chlamydomonas
reinhardtii, although with varying degrees of success.
These include aadA (Goldschmidt-Clermont, 1991; Zerges
and Rochaix, 1994); uidA (Ishikura et al., 1999; Sakamoto
et al., 1993); Renilla luciferase (Ren Luc, Minko et al., 1999);
and the amino glycoside phosphotransferase from
Acinetobacter baumanii, aphA6 (Bateman and Purton,
2000). The amount of recombinant protein produced was
reported for the uidA gene only (Ishikura et al., 1999), and
from these reports, and based on Western blot analysis or
ã 2002 Blackwell Science Ltd 733
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