|
|
|
| Preparation and Preliminary Application of Polyclonal Antibody of NdhO Protein in the Cyanobacterium Synechocystis sp. Strain PCC 6803 |
| ZHAO Jiao-hong, GAO Fu-dan, WEI Lan-zhen, WANG Quan-xi, MA Wei-min |
| College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China |
|
|
|
Abstract Cyanobacterial NADPH dehydrogenase (NDH-1) is an important photosynthetic membrane protein complex, and is essential to CO2 uptake, cyclic electron transport around photosystem I and cellular respiration. This enzyme accepts electrons from NADPH and consists of at least 17 subunits, i.e., NdhA to NdhQ. Recently, an ndhO gene inactivation mutant, ΔndhO, has also successfully been obtained. However, little is known regarding the functional roles of NdhO subunit in cyanobacteria. Therefore, the encoding gene, ndhO, was PCR amplified from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the expression plasmid pET32a(+)-ndhO was constructed and transformed into BL21(DE3)pLysS, and the expression of NdhO protein was induced by IPTG. After purification, the fusion protein pET-NdhO was used to immunize Japanese white rabbit to obtain the polyclonal antibody. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting. The titer of polyclonal antibody was found to be up to 1 ∶ 1 025 000, and thus possessed a high specificity. Further, immunoblotting results using the polyclonal antibody showed the presence of NdhO in active NDH-1 mediumcomplex, and not active NDH-1 supercomplex. Therefore, the antibody of NdhO obtained will further help us to reveal the functional roles of cyanobacterial NdhO subunit.
|
|
Received: 03 February 2011
Published: 28 June 2011
|
|
|
|
Corresponding Authors:
Weimin Ma
E-mail: wma@shnu.edu.cn
|
|
|
| [1] Berger S, Ellersiek U, Steinmuller K. Cyanobacteria contain a mitochondrial complex I-homologous NADH-dehydrogenase. FEBS Lett, 1991, 286(1-2): 129-132.
[2] Berger S, Ellersiek U, Kinzelt D, et al. Immunopurification of a subcomplex of the NAD(P)H-plastoquinone-oxidoreductase from the cyanobacterium Synechocystis sp. PCC6803. FEBS Lett, 1993, 326(1-3): 246-250.
[3] Ogawa T. A gene homologous to the subunit-2 gene of NADH dehydrogenase is essential to inorganic carbon transport of Synechocystis PCC 6803. Proc Natl Acad Sci USA, 1991, 88: 4275-4279.
[4] Mi H, Endo T, Schreiber U, et al. Electron donation from cyclic and respiratory flows to the photosynthetic intersystem chain is mediated by pyridine nucleotide dehydrogenase in the cyanobacterium Synechocystis PCC 6803. Plant Cell Physiol, 1992, 33: 1233-1237.
[5] Mi H, Endo T, Ogawa T, et al. Thylakoid membrane-bound pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the cyanobacteria Synechocystis PCC 6803. Plant Cell Physiol, 1995, 36: 661-668.
[6] Prommeenate P, Lennon A M, Markert C, et al. Subunit composition of NDH-1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclear-encoded homologues in the chloroplast Ndh complex. J Biol Chem, 2004, 279(27): 28165-28173.
[7] Zhang P, Battchikova N, Paakkarinen V, et al. Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1. Biochem J, 2005, 390(2): 513-520.
[8] Battchikova N, Zhang P, Rudd S, et al. Identification of NdhL and ssl1690 (NdhO) in NDH-1L and NDH-1M complexes of Synechocystis sp. PCC 6803. J Biol Chem, 2005, 280(4): 2587-2595.
[9] Nowaczyk M M, Wulfhorst H, Ryan C M, et al. NdhP and NdhQ: two novel small subunits of the cyanobacterial NDH-1 complex. Biochemistry, 2011, 50(7):1121-1124.
[10] Ohkawa H, Pakrasi H B, Ogawa T. Two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis sp. strain PCC6803. J Biol Chem, 2000, 275(41): 31630-31634.
[11] Pieulle L, Guedeney G, Cassier-Chauvat C, et al. The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of Synechocystis PCC6803. FEBS Lett, 2000, 487(2):272-276.
[12] 龙宗娟, 赵娇红, 魏兰珍, 等. 利用同源重组构建蓝藻集胞藻6803 ndhO基因突变株及其分子鉴定. 中国生物工程杂志, 2010, 30(9): 31-35. Long Z J, Zhao J H, Wei L Z, et al. China Biotechnology, 2010, 30(9): 31-35.
[13] Ma W, Deng Y, Ogawa T, et al. Active NDH-1 complexes from the cyanobacterium Synechocystis sp. strain PCC 6803. Plant Cell Physiol, 2006, 47(10): 1432-1436.
[14] Ma W, Mi H. Effect of exogenous glucose on the expression and activity of NADPH dehydrogenase complexes in the cyanobacterium Synechocystis sp. strain PCC 6803. Plant Physiol Biochem, 2008, 46(8-9): 775-779.
[15] Ma W, Deng Y, Mi H. Redox of plastoquinone pool regulates the expression and activity of NADPH dehydrogenase supercomplex in Synechocystis sp. strain PCC 6803. Curr Microbiol, 2008, 56: 189-193.
[16] Allen M M. Simple conditions for growth of unicellular blue-green algae on plates. J Phycol, 1968, 4: 1-4.
[17] Cai Y P, Wolk C P. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol, 1990, 172(6): 3138-3145.
[18] 张劲松, 陈李萍, 高复旦, 等. 集胞藻6803藻胆体藻蓝蛋白多克隆抗体制备及其初步应用. 广东农业科学, 2010, 37(11): 1-5. Zhang J S, Chen L P, Gao F D, et al. Guangdong Agricultural Sciences, 2010, 37(11): 1-5.
[19] 孔卫青, 李海芳, 刘涛, 等. 人DFF45蛋白多克隆抗体制备及其初步应用. 中国生物工程杂志, 2010, 30(2): 54-59. Kong W Q, Li H F, Liu T, et al. China Biotechnology, 2010, 30(2): 54-59.
[20] Ma W, Mi H. Expression and activity of type-1 NAD(P)H dehydrogenase at different growth phases of cyanobacterium, Synechocystis PCC6803. Physiol Plant, 2005, 125: 135-140.
[21] Davis B J. Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci, 1964, 121:404-427.
[22] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680-685. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
