ARTICLES Chinese Science Bulletin 2006 Vol. 51 No. 14 1703—1709

DOI: 10.1007/s11434-006-2041-0

Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast YANG Zongqi1,2 LI yinü2, CHEN Feng3, LI Dong1, ZHANG Zhifang2, LIU Yanxin3, ZHENG Dexian3, WANG Yong1 & SHEN Guifang2 1. College of Life Sciences, Nankai University, Tianjin 300071, China; 2. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3. National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China Correspondence should be addressed to Wang Yong (email: wangyong @nankai.edu.cn) Received February 24, 2006; accepted May 17, 2006

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expression vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and cultivation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%―0.67% of the total soluble proteins. These experimental results demonstrated the possibility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals. Keywords: TRAIL (TNF-related apoptosis-inducing ligand), C. reinhardtii chloroplast, chloroplast expression, homologous recombinant.

The use of plants as bioreactors for the expression of www.scichina.com

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therapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruses or bacterial endotoxins[1]. Recently, chloroplast transformation technology has been successfully applied to introduce foreign genes into the chloroplast genome and to express foreign proteins, as chloroplast genetic engineering offers several unique advantages over nuclear transformation, including high-level expression of foreign genes, site-specific integration of transgenes by homologous recombination, the absence of position effect variation and gene silencing, the ability to express multiple genes in a single transformation event, and maternal plastid gene inheritance (in most crop plants) that prevents pollen transmission of foreign DNA[2]. To date, several therapeutic proteins such as cholera toxin B subunit[3], human somatotropin[4], human serum albumin[5] and antimicrobial peptides[6] have been expressed in transgenic tobacco chloroplasts. Genetic transformation systems for the expression of foreign genes in the Chlamydomonas reinhardtii chloroplast have also been developed. Recent success in the expression of aminoglycoside adenine transferase (aadA)[7], β-glucuronidase (GUS)[8] and green fluorescent protein (GFP)[9] proteins in C. reinhardtii chloroplast demonstrated the feasibility of expression of foreign proteins in algae chloroplasts. In 2003, Mayfield et al.[10] achieved the expression of HSV8-lsc protein (the large single-chain antibody against herpes simplex virus ) in C. reinhardtii chloroplasts. C. reinhardtii, also called ‘the green yeast’, contains a single large chloroplast occupying approximately 40% of the cell volume that is easy to transform[11], which makes Chlamydomonas chloroplast ideal candidates for the expression of foreign proteins. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a new member of the TNF (tumor necrosis factor) superfamily. The human TRAIL protein contains 281 amino acid residues and belongs to the type II membrane protein, which includes a N-terminal transmembrane domain (15―40 amino acid residues) and a C-terminal extracellular domain (114― 281 amino acid residues). The extracellular domain of TRAIL is responsible for binding with its native receptors, triggering the internal signaling cascade and lead― ing to the apoptosis of cancer cells[12 14]. TRAIL is widely expressed in most normal tissues and cells. An attractive biological feature of TRAIL is that it induces 1703

ARTICLES selectively apoptosis in various tumor cells, transformed cells and virus-infected cells, but rarely in normal cells[14]. Because of its killing specificity, TRAIL is attracting considerable interest in the pharmaceutical industry and is currently being developed as a therapeutic agent for various cancer remedies. The extracellular domain of TRAIL (114―281 amino acids of C-terminal) can be cleaved by metalloproteinases to form a homotrimeric structure of soluble TRAIL (sTRAIL) which has biological activity of native TRAIL and could bind specially with its receptors inducing the apoptosis of cancer cells[13,14]. We have introduced human sTRAIL encoding region DNA into the C. reinhardtii chloroplast genome by the biolistic method and achieved successfully the expression of the aimed protein. In our knowledge, this is the first report that the human soluble TRAIL is expressed in plant chloroplast. 1

Materials and methods

1.1

Algal strain, bacteria and plasmids

Wild-type C. reinhardtii strain, 137cc, was obtained from Dr E.H. Harris (Duke University, Durham, NC). E. coli DH5α was hold in our lab. Plasmid pTRAIL (114―281), containing 0.53 kb cDNA fragment of sTRAIL (114―281), was obtained from Prof. Zheng Dexian (Chinese Academy of Medical Sciences & Peking Union Medical College, China). Plasmid patpX, containing atpA promoter and the rbcL terminator of C. reinhardtii chloroplast, was kindly provided by Dr Q. Cheng (John Innes Center, UK). Plasmid p64D, containing the homologous fragment of C. reinhardtii chloroplast: clpP-trnL-petB-chlL-rpl23-rpl2 with multiple-cloning sites (EcoR V, BamH I, Sac I, Avr II, Bsm I) within the chlL and the aadA gene cassette, was constructed and kept in our lab. 1.2

Molecular biology reagents

Restriction endonucleases, Klenow Fragment, T4 DNA Ligase, Mung Bean Nuclease and random-primed DNA labeling kit were from Promega, LA Taq DNA polymerase was from TaKaRa Biotechnology (Dalian) Co., NucleoTrap Gel Extrction Kit was from ClonTech Co., PCR primers were synthesized and DNA sequencing was accomplished by Shanghai Sangon Co. Ltd., TRAIL standard antigen and goat anti-TRAIL polyclonal antibodies were from Santa Cruz Biotechnology Inc. 1704

1.3 Chloroplast transformation and selection of transgenic C. reinhardtii Algal cells were grown in Tris-acetate-phosphate (TAP) medium. Cultures were incubated at 20―25℃, 12 h:12 h light periods, on a gyratory shaker at 160 rpm[15]. 1.5 mL algal cells grown to late log phase (approximately 7 d) in TAP medium were harvested by centrifugation at 5000 g at 20℃ for 5 min. The supernatant was decanted, the cells were resuspended in 200 µL TAP medium, plated on solid TAP medium and incubated under illumination of 3000 lux for 2―3 d. Gold particles coated with plasmid DNAs were shot into Chlamydomonas cells on the agar plate approximately 2―3 cm in diameter using the biolistic device PDS1000/He (BioRad). Bombardment parameters: vacuum, 28 inches Hg, a distance of 9 cm, rupture discs of 1100 psi, 1.0 µm gold particles. Wild-type C. reinhardtii cells were bombarded with Gold particles coated with p64TRAIL DNA. Following incubation at 25℃ for 8 h, cells were washed with TAP liquid medium and plated on TAP solid selective medium (containing 100 mg/L spectinomycin). After incubating under constant illumination of 3000 lux for 10 d at 25℃, individual transformed C. reinhardtii were selected and grown in 50 mL TAP liquid selective medium (containing 100 mg/L spectinomycin) for 7 d for further molecular analyses. Homoplasmic lines were isolated by propagating single colonies in liquid cultures and solid medium containing spectinomycin repeatedly. 1.4 Polymerase chain reaction (PCR) analysis of sTRAIL coding region Total DNA of C. reinhardtii was isolated as described in reference [16] . To confirm integration of sTRAIL coding region DNA into C. reinhardtii chloroplast genome, primers for sTRAIL coding region were designed: P1: 5′-TCACCATGGTGAGAGAAAGAGG-3′, P2: 5′-TTAGCCAACTAAAAAGGCCCCG-3′. C. reinhardtii DNAs isolated from the wild type and the C. reinhardtii transformants were used as template for PCR amplification. The PCR was performed at 94℃ for 1 min, 55℃ for 1 min, and 72℃ for 1 min, for 30 cycles. 1.5 PCR analysis of homoplasmy of C. reinhardtii transformants and PCR-Southern blot analysis In order to determine if sTRAIL coding region had Chinese Science Bulletin

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ARTICLES been integrated into the site-specific areas of C. reinhardtii chloroplast genome and evaluate the degree of homogeneity of C. reinhardtii transformants, a pair of primers were designed, which respectively landed on both sides of the integration site of foreign gene. P3: 5′-GGTTTGCCGAACAATGTTTTTATTCCTG-3′, P4: 5′-AGAGGAAAGTATTTAAAGCTGCTTATTC-3′. PCR was performed by using the LA Taq DNA polymerase. After an initial 5 min denaturation at 95℃, samples were carried through 30 PCR cycles under the following condition: 95℃ for 1 min, 50℃ for 2 min and 72℃ for 4 min. The PCR products of homogeneity were electrophoresed on 0.9% agarose gel, then the gel was transferred to a nylon filter using the Bio-Rad Model 785 Vacuum blotter system. The 0.53 kb sTRAIL coding region was labeled with the random-primed DNA labeling kit and used as probe for Southern blot. 1.6

Protein extraction and Western blot analysis

Proteins of C. reinhardtii was isolated as described in reference[9]. 10 mL algal cells grown to late log phase in TAP medium were harvested by centrifugation, and resuspended in 200 µL protein extraction buffer containing 750 mmol/L Tris-Cl pH 8.0, 15% sucrose (w/v), 100 mmol/L β-mercaptoethanol and 1 mmol/L phenylmethylsulfonylfluoride (PMSF). Samples were then centrifuged for 20 min at 13000 g at 4℃, with the resulting supernatant used in Western blot analysis. Protein concentrations were determined by Bradford protein assay. Approximately 10 μg total soluble protein from each sample was boiled for 5 min and loaded in the wells of a 12% polyacrylamide gel. The protein bands were separated by polyacrylamide gel electrophoresis, and transferred onto a methanol-treated PVDF membrane by electroblotting with a semi-dry blotter (BioRad Inc.). The PVDF membrane was blocked with 5% milk in TBST buffer (10 mmol/L Tris/HCl pH 8, 150 mmol/L NaCl and 0.05% Tween 20) for at least 1 h at room temperature. The blocked membrane was incubated at 4℃ overnight in TBST solution containing a 1:500 dilution of a commercially available goat anti-TRAIL polyclonal antibodies (Santa Cruz Biothchnology), and washed with 0.1% TBST for 10 min, three times, and then incubated with 1:2000 TBST dilution of a alkaline phosphatase-conjugated rabbit anti-goat IgG (Santa Cruz Biothchnology) for 1 h. Signal was detected using ECL Western Blotting Dewww.scichina.com

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tection Reagents kit (Amersham Biosciences). According to reference[17], the expressed human TRAIL protein was quantified on the western blots by densitometric analysis with Image Master VDS (Phamacia Biotech). 1.7 Phenotype analysis of C. reinhardtii transformants The C. reinhardtii transformants and the wild type were plated on TAP solid medium, incubated at 25℃ in the dark and observed phenotype after 10 d. 2

Results

2.1 Construction of vector for C. reinhardtii chloroplast transformation The constructing process is shown in Fig. 1. The plasmid patpX, which contains the atpA promoter and the rbcL terminator of C. reinhardtii chloroplast, was digested with Noc I and then the resulting end was blunted by digestion with Mung Bean Nuclease to remove the ATG codon. After ligation of the linearized patpX, a plasmid without Noc I site, the plasmid patpY was obtained; Then digestion of the plasmid with Xba I, blunting with DNA polymerase I large fragment (Klenow) and digestion with BamH I was followed orderly to create the designed site for the sTRAIL coding region subcloning. The aimed 0.53 kb of sTRAIL coding region was obtained by digesting plasmid pTRAIL (114―281) with Hind III blunting with the Klenow and then digesting with BamH I, and subcloned into the plasmid patpY to get the plasmid patpY-TRAIL; Subsequently a 1.2 kb fragment containing the sTRAIL expression cassette was excised from the patpY-TRAIL by digesting the plasmid with EcoR V and Not I. The C. reinhardtii chloroplast expression vector, the p64TRAIL, was finally produced by inserting this fragment (after blunting) into the EcoR V digested p64D. 2.2 Obtainment of transgenic algae in C. reinhardtii and PCR identification The wild-type C. reinhardtii was bombarded with Gold particles coated with p64TRAIL DNA as described in sec. 1 of this paper. Following incubation at 25℃ for 8 h, cells were washed with TAP liquid medium and plated onto TAP solid selective medium containing 100 mg/L spectinomycin. After 7―10 d, green algal cells were observed, while cells bom1705

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Fig. 1. Construction of C. reinhardtii chloroplast transformation vector p64TRAIL.

barded with Gold particles without p64TRAIL DNA were all bleached. Out of 10 shots, 6 putative transformants resistant to spectinomycin were obtained. These putative transformants survived after second round of selection, which indicated that sTRAIL coding region DNA and the aadA selective marker gene might be transferred into Chlamydmonas chloroplast genome. C. reinhardtii transformants were initially identified by PCR using sTRAIL coding sequence specific primers P1, P2. C. reinhardtii DNAs isolated from the wild type and the putative transformants were used as template for PCR amplification. After PCR amplification, 3 of the 6 putative transformants yielded the 0.53 kb product, the same size as that amplified by using p64TRAIL as the template (Fig. 2). No DNA band was observed for the non-transformed C. reinhardtii. 2.3 PCR assays of homogeneity of C. reinhardtii transformants Although C. reinhardtii contains a single large chloroplast, each chloroplast possesses approximately 80 identical copies of the genome. It is impossible to transform all plastid genome copies simultaneously. The primary plastid transformation event involves the change of only a single or at most a few plastid genome copies within the chloroplast. So primary transplastomic cell lines contain a mixed population of wildtype and transformed plastid genomes. Such cells are also referred to as heteroplasmic. For Chlamydomonas, homoplasmy can be achieved by allowing for a sufficient number of cell divisions under high selective pressure as exerted by high concentrations of the selecting antibiotic spectinomycin[18]. The 3 above-identified transformants were subjected to 6 rounds of spectinomycin selection (growing algal 1706

Fig. 2. PCR analysis of C. reinhardtii transformants of sTRAIL coding region. Lane M, 1.0 kb DNA markers (Promega); lane 1, the wild type as the negative control; lane 2, p64TRAIL as the positive control; lanes 3― 5, transformed cell lines.

transformants on both TAP solid selective medium and TAP liquid selective medium counted as one round of spectinomycin selection). To evaluate the degree of homogeneity of C. reinhardtii transformants, and as well as to confirm further that the integration of foreign genes had occurred in the chloroplast genome at the directed site by homologous recombination, primers P3 and P4 were designed, which respectively locates on the 5′ upstream and 3′ downstream of C. reinhardtii chloroplast genomic chlL gene (Fig. 3(a) and (b)). According to the design, a 3.6 kb DNA band should, and only be obtained if all the copies of chloroplast genomes possess the integrated sTRAIL coding region DNA, in comparison, a 1.0 kb DNA will be amplified using the wild-type DNA as template. The results confirmed this prediction (Fig. 3(c)) and suggested that these C. reinhardtii transformants all reached homogeChinese Science Bulletin

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Fig. 4. Southern blot analysis of the PCR products probed with sTRAIL coding region. Lane 1, The 0.53 kb sTRAIL coding region as the positive control; lane 2, the wild type as the negative control; lanes 3―5, C. reinhardtii transformant lines.

Fig. 3. PCR analysis of homogeneity of C. reinhardtii transformants. (a) Schematic diagram of PCR amplification using DNA of the wild-type C. reinhardtii chloroplast; (b) schematic diagram of PCR amplification using DNA of transformants; (c) electrophoretic analysis of PCR products from the template DNAs of the wild type and transformed C. reinhardtii after six rounds of spectinomycin selection. Lane M, 1.0 kb DNA markers (Promega); lane 1, the wild type; lanes 2―4, C. reinhardtii transformant lines.

neitic state after 6 rounds of spectinomycin selection. 2.4 PCR-Southern blot analysis of the C. reinhardtii transformants To further verify integration of sTRAIL coding region DNA into C. reinhardtii chloroplast genome, the electrophoreticly separated PCR products were blotted onto nylon membrane and probed with the P32-labeled 0.53 kb sTRAIL coding region (Fig. 4). The results showed that the 3.6 kb PCR product could be hybridized to the probe but no hybridized band was detected in wild-type C. reinhardtii, which indicated that the sTRAIL coding region was indeed inserted into the chlL gene of C. reinhardtii chloroplast genome. 2.5 Phenotype analysis of C. reinhardtii transformants Different from other plants, green algae have a light-independent pathway for chlorophyll biosynthesis www.scichina.com

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in which chlorophyll can be synthesized in complete darkness, in addition to the light-dependent chlorophyll biosynthesis pathway[19,20]. Three chloroplast genes termed chlL, chlN, and chlB coding light-independent protochlorophyllide reductase are required for this alternative chlorophyll biosynthesis pathway. Disruption of any of the three genes can inactivate the dark-dependent pathway of chlorophyll biosynthesis leading to the ‘yellow-in-the-dark’ phenotype. In contrast, the wild-type strain has a distinct ‘green-in- the-dark’ phenotype[19,20]. The sTRAIL coding region and aadA gene cloned in the plasmid p64TRAIL should replace the 1.0 kb fragment of chlL structure gene if integration occurs through homologous recombination, and inactivation of chlL results in yellow-in-the-dark phenotype. After incubation in the dark for 10 d, the obtained transformants turned yellow but wild-type C. reinhardtii was green, which indicated that the sTRAIL coding region and aadA gene had replaced the 1.0 kb fragment of chlL structure gene and were integrated into the C. reinhardtii chloroplast genome through homologous recombination, and that the chlL gene was disrupted (Fig. 5(a) and (b)). 2.6 Western blot analysis of sTRAIL in the C. reinhardtii transformants To determine whether human soluble TRAIL protein accumulated in the C. reinhardtii transformants, total proteins from wild-type C. reinhardtii and the three C. reinhardtii transformants were subjected to 1707

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Fig. 5. Phenotypes of C. reinhardtii transformants (a) and the wild type (b) under dark growth conditions.

Western blot analysis with goat anti-TRAIL polyclonal antibodies and alkaline phosphatase-conjugated rabbit anti-goat IgG. The three C. reinhardtii transformants were found to express Human Soluble TRAIL proteins with identical molecular weight of the standard sTRAIL protein (~18.5 kD) when probed with antiTRAIL polyclonal antibodies. The protein extract from wild-type C. reinhardtii did not react with the antiTRAIL antibody (Fig. 6). This result showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of western blots with Image Master VDS (Phamacia Biotech) by comparison of the experimental samples with standard sTRAIL protein showed that the human sTRAIL protein expressed in C. reinhardtii transformants accounted for 0.43%―0.67% (w/w) of total soluble protein (about 4.3 ― 6.7 μg of chloroplast-expressed sTRAIL in 1 mg of TSP).

Fig. 6. Western blotting analysis using the total proteins from the wild type and transformants. Lane 1, Standard sTRAIL protein; lane 2, total proteins from the wild type; lanes 3―5, total proteins from the C. reinhardtii transformant lines.

3

Discussion

C. reinhardtii has many specific features that make it an ideal candidate for therapeutic protein production: (1) transgenic algae can be generated quickly, requiring only a few weeks between the generation of initial transformants and their scale up to production volumes; 1708

(2) both the chloroplast and nuclear genome of C. reinhardtii can be genetically transformed, opening the possibility of producing a variety of transgenic proteins in a single organism; (3) a wide variety of promoters that are regulated by factors such as light or specific nutrient levels in the medium are available; (4) C. reinhardtii can grow phototrophically or heterotrophically, utilizing acetate as a carbon source in darkness; (5) C. reinhardtii has the ability to be grown on large scales in a cost-effective manner. These attributes make C. reinhardtii a particularly attractive alternative to other systems for the expression of valuable recombinant proteins[10,21]. Studies show that the soluble TRAIL protein has identical biological activities with the native TRAIL in inducing apoptosis in various tumor cells and is currently being developed as a therapeutic agent for various cancer remedies[14]. In this study, we constructed a chloroplast expression vector harboring the sTRAIL coding region (Fig. 1) and introduced it biolistically into C. reinhardtii chloroplast. PCR analysis (Figs. 2 and 3), together with Southern blotting results (Fig. 4) all demonstrated that the sTRAIL coding region had been integrated into chloroplast genome of the transformed C. reinhardtii and that the homogeneity of the transformed chloroplast genome had reached a very high level after 6 rounds of stringent selection on the medium containing spectinomycin as selection pressure. The ‘yellow–in-the-dark’ phenotype (Fig. 5) of the transformed cells showed that the sTRAIL coding region had been inserted into the designed site in the chloroplast genome DNA by homologous recombination, resulting in the loss of function of the light-independent chlorophyll biosynthetic pathway. The expression of sTRAIL protein in the transformed C. reinhardtii chloroplast was finally verified by Western blot analysis (Fig. 6) and the expressed sTRAIL protein accounted for about 0.43%―0.67% of total soluble proteins. Human Soluble TRAIL protein is another important therapeutic protein expressed in C. reinhardtii chloroplast after HSV8-lsc protein (the large single-chain antibody against herpes simplex virus). This is apparently an important progress in this research field. At present, the expression of heterologous proteins in C. reinhardtii chloroplast has been reported to be quite low[21,22]. Early attempts to express recombinant proteins in the C. reinhardtii chloroplast primarily involved reporter proteins and resistant proteins. AtChinese Science Bulletin

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ARTICLES tempts to express common reporter proteins, such as β-glucuronidase (GUS)[8] or Renilla luciferase[23], resulted in very low levels of protein accumulation and thus unacceptably high levels of background signal. The expression levels of chloroplast-codon-optimized GFP[9] and HSV8-lsc[10] proteins were reported to be only 0.5% of the total soluble protein. Mayfield and Franklin[21,22] suggested one possible explanation for the inability of heterologous transgenes to express well in the C. reinhardtii genome was the presence of a strong codon bias in the chloroplast, favoring A or T (80%) in the third position and A-T nucleotides making up about 66.3% of the total. Another possible factor that influences the expression levels of foreign genes in C. reinhardtii chloroplasts was the use of promoter and terminator from C. reinhardtii chloropolast genome[21,22]. In this work, we have chosen the strong chloroplast promoter 5′ atpA and the terminator of 3′ rbcL from C. reinhardtii chloroplast genome to regulate transcription and translation of sTRAIL coding region. Therefore, optimizing sTRAIL coding region in C. reinhardtii chloroplast codon bias to increase its expression and studying the accumulation mechanism of foreign proteins in C. reinhardtii chloroplasts will be the focus of next work.

genic bacteria and fungi. Plant Physiol, 2001,127(3): 852―862. 7 Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: A selectable marker for site-directed transformation of Chlamydomonas. Nucleic Acids Res, 1991, 19(15): 4083―4089 8 Ishikura K, Takaoka Y, Kato K, et al. Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast. J Biosci Bioeng, 1999, 87(3): 307―314 9 Franklin S, Ngo B, Efuet E, Mayfield S P. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J, 2002, 30(6): 733―744 10 Mayfield S P, Franklin S E, Lerner R A. Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA, 2003, 100(2): 438―442 11 Kindle K L, Richards K L, Stem D B. Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA, 1991, 88(5): 1721―1725. 12 Wiley S R, Schooley K, Smolak P J, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity, 1995, 3(6): 673―682 13 Pitti R M, Marsters S A, Ruppert S, et al. Induction of apoptosis by Apo22 ligand, a new member of the Tumor Necrosis Factor cytokine family. J Biol Chem, 1996, 271(22): 12687―12690 14 Gura T. How TRAIL kills cancer cells, but not normal cells. Science, 1997, 277(5327): 768―772 15 Harris E. The Chlamydomonas Sourcebook, A Comprehensive Guide to Biology and Laboratory Use. San Diego: Academic Press,

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 30571189) and a grant from Tianjin Municipal Science and Technology Commission (Grant No. 033605111) to Yong Wang.

16 Rochaix J D, Mayfield S, Goldschmidt-Clermont M, et al. Molecu-

References

17 Tregoning J S, Nixon P, Kuroda H, et al. Expression of tetanus

1989. 31 lar biology of Chlamydomonas. In: Shaw C H, ed. Plant Molecular Biology. A Practical Approach. Oxford: IRL Press, 1988. 253― 275

1 Fischer, R, Stoger E, Schillberg S, et al. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol, 2004, 7(2): 152―158 2 Daniell H, Chebolu S, Kumar S, et al. Chloroplast-derived vaccine antigens and other therapeutic proteins. Vaccine, 2005, 23(15): 1779―1783 3 Daniell H, Lee S B, Panchal T, et al. Expression of cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol, 2001, 311(5): 1001―1009 4 Staub J M, Garcia B, Graves J, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol, 2000, 18(3): 333―338

toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res, 2003, 31(4): 1174―1179 18 Bock R. Transgenic plastids in basic research and plant biotechnology. J Mol Biol, 2001, 312(3): 425―438 19 Suzuki J Y, Bauer C E. Light-independent chlorophyll biosynthesis: Involvement of the chloroplast gene chlL (frxC). Plant Cell, 1992, 4(8): 929―940 20 Ford C, Wang W. Three new yellow mutants in Chlamydomonas reinhardtii. Mol Gen Genet, 1980, 179(2): 259―263 21 Mayfield S P, Franklin S E. Expression of human antibodies in eukaryotic micro-algae. Vaccine, 2005, 23(15): 1828―1832

5 Fernandez-SanMillan A, Mingo-Castel A, Miller M, et al. A

22 Franklin S E, Mayfield S P. Prospects for molecular farming in the

chloroplast transgenic approach to hyper-express and purify human

green alga Chlamydomonas reinhardtii. Curr Opin Plant Biol, 2004,

serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J, 2003, 1(2): 71―79 6 DeGray G, Rajasekaran K, Smith F, et al. Expression of an antimicrobial peptide via the chloroplast genome to control phytopatho-

www.scichina.com

www.springerlink.com

7(2): 159―165 23 Minko I, Holloway S P, Nikaido S, et al. Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas. Mol Gen Genet, 1999, 262(3): 421―425

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