家蚕translin蛋白的初步研究
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摘要
Translin又名转位蛋白,是一种可以与DNA/RNA相结合,分子量大约27 kD,进化保守的蛋白。在细胞内参与染色体重组、修复及细胞分裂等多种生物学活动。在它的三级结构中有一段保守的亮氨酸拉链区,对于形成多聚体是必须的。Translin蛋白与mRNA的3’端非编码序列相结合,进而调节mRNA的转运和翻译。Translin蛋白还参与端粒长度调节,与一些肿瘤发病有关。
从本实验室构建的家蚕cDNA文库中克隆到一条ORF长度为708bp的家蚕translin基因,经NCBI中BLAST比对将其命名为BmTLP(for translin-like protein from Bombyx mori),该基因编码235个氨基酸残基,预测分子量为27.259 kD,等电点为5.87。本实验通过设计PCR引物,克隆目的基因。其中BamH I和Xho I是该基因克隆时的两个酶切位点。将双酶切的PCR产物融合到表达质粒pET-28a(+)相应的酶切位点上,成功构建重组质粒pET-28a(+)-BmTLP。将重组质粒转入BL21(DE3)并诱导表达,表达的重组蛋白以可溶形式存在,经过镍柱亲和层析纯化后,经FPLC反相柱层析进一步纯化,再进行质谱分析证实该蛋白表达的正确性。以纯化的重组蛋白为抗原免疫兔获得该蛋白的多克隆抗体,经ELISA检测,抗体效价可达1:25600以上。为了解该蛋白在家蚕中各时期及各组织中的分布,做了Western blotting和荧光定量PCR相关实验,结果显示五龄幼虫的各组织器官转录表达在头、肠、丝腺、表皮、睾丸和气门中都有分布,而在卵巢、脂肪体及马氏管中未检测到该蛋白。在家蚕的不同发育时期中,转录表达水平最高为蛹期,而卵期则最低。亚细胞定位分析中发现,BmTLP主要分布在细胞质中,在细胞的不同时期分布也不同。
As translocation protein translin is an evolutionarily conserved 27 kD protein that binds to specific DNA and RNA sequences and has diverse cellular roles,such as the chromosome recombination, translocation and cell division. In its tertiary structure there is a conserved leucine zipper area which is necessary for the formation of polymer.Translin can bind mRNA 3’UTR of mRNA and then regulate mRNA transport and translation. Translin protein is also involved in telomere length regulation, with a number of tumor pathogenesis.
We cloned an 708 bp ORF fragment from Bombyx mori pupae which was compared with other known translin genes throngh BLAST in NCBI, then we named it BmTLP(for translin-like protein from Bombyx mori).This gene encoded 235 amino acid residues with the predicted molecular weight of 27.259 kD and isoelectric point of 5.87. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli BL21(DE3). The recombinant protein was purified by Ni~(2+) affinity chromatography. We purify the protein in addition by FPLC reversed-phase column chromatography for further purifying and MS analysis confirmed that the protein expression is correct. The purified fusion protein was used to immunize a male New Zealand rabbit.The titer of the polyclonal antibodies reaches 1:25600 measured by ELISA. Western blotting and real time RT-PCR were performed in different stages and in different issues of the fifth instar larva of Bombyx mori.The analysis suggested that the protein was more expressed in head; bowels; silk gland; epidermis; testicle and stomata,but we didn't detect the expression in ovaries; fat body and tuba Malpighii. The protein transcribe and express more in pupal period but egg stage is lowest. After carrying out subcellular localization about BmTLP, we found that it mainly existed in cytoplasm and the location is difference with the cell cycle.
从本实验室构建的家蚕cDNA文库中克隆到一条ORF长度为708bp的家蚕translin基因,经NCBI中BLAST比对将其命名为BmTLP(for translin-like protein from Bombyx mori),该基因编码235个氨基酸残基,预测分子量为27.259 kD,等电点为5.87。本实验通过设计PCR引物,克隆目的基因。其中BamH I和Xho I是该基因克隆时的两个酶切位点。将双酶切的PCR产物融合到表达质粒pET-28a(+)相应的酶切位点上,成功构建重组质粒pET-28a(+)-BmTLP。将重组质粒转入BL21(DE3)并诱导表达,表达的重组蛋白以可溶形式存在,经过镍柱亲和层析纯化后,经FPLC反相柱层析进一步纯化,再进行质谱分析证实该蛋白表达的正确性。以纯化的重组蛋白为抗原免疫兔获得该蛋白的多克隆抗体,经ELISA检测,抗体效价可达1:25600以上。为了解该蛋白在家蚕中各时期及各组织中的分布,做了Western blotting和荧光定量PCR相关实验,结果显示五龄幼虫的各组织器官转录表达在头、肠、丝腺、表皮、睾丸和气门中都有分布,而在卵巢、脂肪体及马氏管中未检测到该蛋白。在家蚕的不同发育时期中,转录表达水平最高为蛹期,而卵期则最低。亚细胞定位分析中发现,BmTLP主要分布在细胞质中,在细胞的不同时期分布也不同。
As translocation protein translin is an evolutionarily conserved 27 kD protein that binds to specific DNA and RNA sequences and has diverse cellular roles,such as the chromosome recombination, translocation and cell division. In its tertiary structure there is a conserved leucine zipper area which is necessary for the formation of polymer.Translin can bind mRNA 3’UTR of mRNA and then regulate mRNA transport and translation. Translin protein is also involved in telomere length regulation, with a number of tumor pathogenesis.
We cloned an 708 bp ORF fragment from Bombyx mori pupae which was compared with other known translin genes throngh BLAST in NCBI, then we named it BmTLP(for translin-like protein from Bombyx mori).This gene encoded 235 amino acid residues with the predicted molecular weight of 27.259 kD and isoelectric point of 5.87. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli BL21(DE3). The recombinant protein was purified by Ni~(2+) affinity chromatography. We purify the protein in addition by FPLC reversed-phase column chromatography for further purifying and MS analysis confirmed that the protein expression is correct. The purified fusion protein was used to immunize a male New Zealand rabbit.The titer of the polyclonal antibodies reaches 1:25600 measured by ELISA. Western blotting and real time RT-PCR were performed in different stages and in different issues of the fifth instar larva of Bombyx mori.The analysis suggested that the protein was more expressed in head; bowels; silk gland; epidermis; testicle and stomata,but we didn't detect the expression in ovaries; fat body and tuba Malpighii. The protein transcribe and express more in pupal period but egg stage is lowest. After carrying out subcellular localization about BmTLP, we found that it mainly existed in cytoplasm and the location is difference with the cell cycle.
引文
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[2] Chiaruttini C.Vicario A. Li Z. Dendritic trafficking of BDNF mRNA is mediated by translin and blocked by the G196A (Val66Met) mutation[J]. Proc Natl Acad Sci U S A.,2009,22:16481-6.
[3]Wu, X. Q. Lefrancois, S. Morales, C. R..Protein–protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain[J].Biochemistry,1999,38: 11261–11270.
[4] Orly Laufman.Ron Ben Yosef. Noam Adir1.Cloning and characterization of the Schizosaccharomyces pombe homologs of thehuman protein Translin and the Translin-associated protein TRAX.[J]. Nucleic Acids Research,2005,13:4128–4139.
[5] Sugiura, I..Sasaki, C. Hasegawa, T,etal. Structure of human translin at 2.2 A ? resolution[J]. Acta Cryst. D.2004,60:674–679.
[6]Pascal,J.M.Hart,P.J.Hecht,N.B.Crystal Structure of TB-RBP, a novel RNA-binding and regulating protein[J]. J. Mol. Biol,2002,319:1049–1057.
[7] X Q Wu. L Xu.N B Hecht.Dimerization of the testis brain RNA-binding protein (translin) is mediated through its C-terminus and is required for DNA- and RNA-binding[J]. Nucleic Acids Res,1998,26(7): 1675–1680.
[8] K. Aoki.K. Suzuki.R. Ishida. The DNA binding activity of Translin is mediated by a basic region in the ring-shaped structure conserved in evolution[J].FEBS Lett,1999, 443:363–366.
[9]Wu, R.F..Osatomi, K. Terada, L.S. Uyeda, K. Identification of Translin/Trax complex as a glucose response element binding protein in liver[J]. Biochim. Biophys. Act,2003,1624:29–35.
[10]Stefania.Stingo.Mariorosario.Masullo.Eugenia.Polverini.The N-Terminal Domain of 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Harbors a GTP/ATP Binding Site.[J] Chem Biol Drug Des,2007,70:502–510.
[11] VanLoock, M. S. Yu, X.Kasai, M. Egelman, E. H. Electron microscopic studies of the translin octameric ring[J].J. Struct. Biol,2001,135:58–66.
[12] John M. Pascal. P. John Hart.Norman.B. Hecht.Crystal Structure of TB-RBP, a Novel RNA-binding and Regulating Protein[J]. J. Mol. Biol,2002,319:1049–1057
[13]Okuda A. Kishi T.Okochi T. Translin-Associated Factor X Gene (TSNAX) may be Associated with Female major Depressive Disorder in the Japanese Population[J]. Neuromolecular Med. ,2009,10:1559-1568.
[14]Yang, S.Cho, Y.S.Chennathukuzhi, V.M. Underkoffler, L.A. Loomes, K. and Hecht, N.B. Translin-associated factor X is post-transcriptionally regulated by its partner protein TB-RBP, and both are essential for normal cell proliferation[J]. J. Biol. Chem,2004, 279:12605–12614.
[15] Patricia M. FinkenstadtWha-Sun KangMihee JeonEiichi TairaWeizhu TangJay M. Baraban. Somatodendritic Localization of Translin, a Component of the Translin/Trax RNA Binding Complex[J] Journal of Neurochemistry,2002,75:1754– 1762.
[16]Aharoni,A.Baran,N.Manor,H. Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA[J]. Nucleic Acids Res,1993,21:5221–5228.
[17] Millar, J. K. Wilson-Annan, J. C.Anderson, S.. Disruption of two novel genes by a translocation cosegregating with schizophrenia[J]. Hum. Mol. Genet,2000, 9:1415–1423.
[18]V. M. Chennathukuzhi.Y. Kurihara, J. D. Bray, J. Yang and N. B. Hecht. Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype[J]. Nucleic Acids Res,2001,29:4433–4440.
[19] Katsunori Aoki. Reiko Ishida. Masataka Kasai. Isolation and characterization of a cDNA encoding a Translin-like protein, TRAX.[J]FEBS,2007,401:109-112.
[20]Cho,Y.S.Chennathukuzhi,V.M.Handel,M.A. Eppig,J. The relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells[J]. J. Biol. Chem,2004,279:31514–31523.
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