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1.
目的:利用分子生物学的方法克隆梅花鹿超氧化歧化酶(Cu,Zn-SOD)基因,并将其于大肠杆菌(E.coli)中表达。方法:通过设计扩增引物及RT-PCR技术从梅花鹿肝细胞中克隆出Cu,Zn-SOD基因,将其构建于表达质粒pET21a(+)并转入E.coli Rosetta(DE3)。结果:通过IPTG诱导表达,在Rosetta(DE3)中成功表达出具Cu,Zn-SOD活性的梅花鹿超氧化物歧化酶,三角摇瓶发酵水平约257U/ml发酵液;经过纯化后得到比活3092U/mg的梅花鹿超氧化物歧化酶。结论:获得了梅花鹿的Cu/Zn-SOD基因的全序列,并使其得到表达,对研究鹿茸的功效和保护梅花鹿这一物种具有一定意义。 相似文献
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A subunit of molecular weight 18300 has been separated and isolated from seeds of Brassica campestris L. This subunit was cleaved by using cyanogen bromide, trypsin, Staphylococcus aureus V8 protease and chymotrypsin; the fragments obtained from enzymatlc and chemical cleavages were separated and isolated by polyacrylamide gel electrophoresis and gel filtration. The amino acid analyses were carried out. The complete amino acid sequence of the subunit containing 172 amino acid residues has been established by manual Edman method. 相似文献
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S. Przedborski† V. Jackson-Lewis V. Kostic E. Carlson‡ C. J. Epstein‡ J. L. Cadet 《Journal of neurochemistry》1992,58(5):1760-1767
Copper/zinc-superoxide dismutase (CuZn-SOD) transgenic mice overexpress the gene for human CuZn-SOD. To assess the effects of the overexpression of CuZn-SOD on the brain scavenging systems, we have measured the activities of manganese-SOD (Mn-SOD), catalase, and glutathione peroxidase (GSH-Px) in various regions of the mouse brain. In nontransgenic mice, cytosolic CuZn-SOD activity was highest in the caudate-putamen complex; this was followed by the brainstem and the hippocampus. The lowest activity was observed in the cerebellum. In transgenic mice, there were significant increases of cytosolic CuZn-SOD activity in all of these regions, with ratios varying from a twofold increase in the brainstem to 3.42-fold in the cerebellum in comparison with nontransgenic mice. Particulate Mn-SOD was similarly distributed in all brain regions, and its levels also were significantly increased in superoxide dismutase (SOD)-transgenic mice. In the brains of nontransgenic mice, cytosolic catalase activity was similar in all brain regions except the cortex, which showed less than 50% of the activity observed in the other regions. In transgenic mice, cytosolic catalase activity was significantly increased, with the cortex showing the greatest changes (133%) in comparison with nontransgenic mice. The smallest increases were observed in the hippocampus (34%). In contrast to what was observed for SOD and catalase, there were no significant changes in cytosolic GSH-Px activity in any of the brain regions examined. The present results indicate that, in addition to displaying marked increases in the levels of brain CuZn-SOD activity, SOD-transgenic mice also exhibit increases in other enzymes that scavenge oxygen-based radicals.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Masanori Kohmura Noriki Nio Yasuo Ariyoshi 《Bioscience, biotechnology, and biochemistry》2013,77(9):2219-2224
The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation. 相似文献
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Luciana Di Ciero Maria L. V. Oliva Ricardo Torquato Peter Köhler Jürgen K. P. Weder José Camillo Novello Claudio A. M. Sampaio Benedito Oliveira Sergio Marangoni 《Journal of Protein Chemistry》1998,17(8):827-834
Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64). 相似文献
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Ryuji Kobayashi Tadashi Yoshimoto Daisuke Tsuru 《Bioscience, biotechnology, and biochemistry》2013,77(10):2737-2749
The neutral protease of Bacillus subtilis var. amylosacchariticus was cleaved chemically or digested with proteolytic enzymes, and the resultant peptides were separated and purified by high performance liquid chromatography. The sequence analyses of these peptides by the manual Edman procedure established the complete amino acid sequence of the enzyme. The neutral protease consisted of 300 amino acid residues with Ala and Leu as its amino- and carboxyl-termini, respectively, and the molecular weight was calculated to be 32,633. The sequence was found to be identical to that of B. subtilis 1A72 neutral protease, which was deduced from nucleotide sequencing. Comparison of the sequence with those of other Bacillus proteases revealed that the putative active site amino acid residues, Zn-binding ligands, and two Ca-binding sites were well conserved among them, as compared with those of thermolysin. 相似文献
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Sakamoto Atsushi; Ohsuga Hiroyuki; Wakaura Makoto; Mitsukawa Norihiro; Hibino Takashi; Masumura Takehiro; Sasaki Yukiko; Tanaka Kunisuke 《Plant & cell physiology》1993,34(6):965-968
A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH2-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach.
1Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A. 相似文献
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Luciana Di Ciero Maria L. V. Oliva Ricardo Torquato Peter Köhler Jürgen K. P. Weder José Camillo Novello Claudio A. M. Sampaio Benedito Oliveira Sergio Marangoni 《The protein journal》1998,17(8):827-834
Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64). 相似文献
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Complete Amino Acid Sequence of a Polypeptide from Zea mays Similar to the Pathogenesis-Related-1 Family
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A polypeptide serologically related to the tobacco pathogenesis-related-1 family of proteins has been purified from the root tissue of maize (Zea mays L.), and the complete amino acid sequence has been determined. The mature protein has a calculated molecular weight of 14,970 and isoelectric point of 4.2. The maize protein shows 66 to 68% amino acid identity with the tobacco pathogenesis-related-1 family and 55% identity with the tomato p14 protein. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(2):386-389
The complete amino acid sequence of gladiolus bulb chitinase-a (GBC-a) was determined. First the tryptic peptides from GBC-a after it was reduced and S-carboxymethylated were sequenced and then the peptides were further studied by chemical cleavage of the enzyme. GBC-a consisted of 274 amino acid residues and had a molecular mass of 30,714 Da. Two consensus sequences essential for chitinase activity by plant class III chitinases were conserved in GBC-a, although its sequence similarity with plant class III chitinases was less than 20%. Sequence comparison of GBC-a with sequences of other proteins in a protein identification resource (PIR) showed that the GBC-a sequence was 33% similar to that of narbonin, a seed storage 2S globulin from narbon beans. 相似文献
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董乐 《基因组学与应用生物学》2012,31(4):349-354
为获得高表达杨梅(Morella rubra)铜锌超氧化物歧化酶(MrCu/Zn-SOD1)的工程菌,本实验采用RT-PCR技术从杨梅果实中分离扩增了MrCu/Zn-SOD1的cDNA序列(456bp),将该基因重组到原核表达载体pGEX-2T中,酶切、测序分析表明,重组质粒pGEX-MrCu/Zn-SOD1结构正确。重组质粒转化大肠杆菌BL21(DE3)进行诱导表达。IPTG诱导表达分子量约41kD融合蛋白GST-MrCu/Zn-SOD1。诱导表达后的菌体超声裂解液经谷胱甘肽亲和层析纯化,得到高纯度的GST-MrCu/Zn-SOD1。采用氯化硝基四氮唑蓝法和黄嘌呤氧化法分析其活性。结果表明,GST-MrCu/Zn-SOD1具有特异性SOD酶活性。 相似文献
13.
陆地棉叶绿体铜锌超氧化物歧化酶基因的克隆与表达 总被引:1,自引:0,他引:1
以陆地棉‘CRI36'的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1 043 bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%~74%之间。基因的表达谱分析显示:棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实:该基因主要在苗期表达,以后表达逐渐减少。用pET-21a(+)构建了原核表达载体,在大肠杆菌BL21(DE3)的表达结果显示:表达后得到一个29.0 kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。 相似文献
14.
The amino acid sequence of triculamin is established from the amino acid composition of the peptides, which have been obtained by partial acid hydrolysis. It is a branched peptides, and a possible structure of the branched parts is proposed. 相似文献
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The Complete Amino Acid Sequence of Green Turtle (Chelonia mydas) Egg White Ribonuclease 总被引:1,自引:0,他引:1
Egg white ribonuclease was first found in green turtle eggs. This enzyme has been purified by CM-toyopearl cation exchange. Two isoforms (GTRNase-1 and GTRNase-2) were further separated by RP-HPLC, with the same M.W. (13 kDa) and activity. These isoforms carried one amino acid exchange of Ser and Leu at the position 37. The N-terminal sequence, ETRYEKF, was determined for the transblotted protein. Internal sequences were analyzed by protein sequencer and ESI-Q-TOF mass spectrometry for tryptic peptides (Ts). The overlapping sequences were obtained from chymotryptic peptides, CNBr fragments and ISD-MS/MS analysis. The C-terminal Ile was identified by CPase-Y. The established sequence composed of 119 residues with the molecular mass of 12,942.1 Da for GTRNase-1 and 12,967.8 Da for GTRNase-2. The comparison of sequence with known pancreatic RNases, 27 positions including catalytic residues at the position 11 and 114 were conserved. Also basic residues contributed to phosphate binding residues were conserved with the exception of Lys 66. One insertion at the position 14, and 3 deletions at the position-1, between position 64–65, and 110 and 111 were found. Two Cys residues at position 65 and 72 that form a disulfide bond in mammalian RNase were deleted and exchanged. All these difference in the sequence were similar to reptile pancreatic RNase.Data deposition: The sequence reported in this paper has been submitted to the UniProt Knowledgebase under accession No. P84844. 相似文献
18.
Neves-Ferreira AG de Andrade CM Vannier-Santos MA Perales J Nascimento HJ da Silva Junior JG 《The protein journal》2004,23(1):71-77
Omp-28 isolated from Salmonella enterica serovar typhi presented a subunit molecular mass of 9,632 Da by MALDI-TOF MS. It was denatured, S-alkylated, and 1) directly submitted to Edman sequencing, 2) cleaved with CNBr, and 3) hydrolyzed either with endoproteinase Glu-C or Asp-N. The major CNBr peptide containing the C-terminal portion of Omp-28 was isolated by tricine-SDS-PAGE and electroblotted whereas Omp-28 enzymatic peptides were isolated by C18-RP-HPLC. All peptides were sequenced. This approach allowed the elucidation of the complete primary structure of Omp-28. Its amino acid sequence is identical to that deduced from part of the DNA of the putative periplasmic transport protein of either S. enterica serovar typhimurium and a multiple drug resistant S. enterica serovar typhi. Omp-28 homologous protein sequences were also deduced from Escherichia coli and Yersinia pestis genomic DNA. All proteins had their secondary structures predicted. Immunogold cytochemistry indicated that Omp-28 is found on the bacterium outer membrane. 相似文献
19.
海枣曲霉木聚糖酶的纯化及末端序列研究 总被引:7,自引:1,他引:7
海枣曲霉麸曲经水浸提、硫酸铵盐析、凝胶过滤、离子交换层析及HPLC分子排阻层析制备了PAGE,SDS-PAGE、PAGE酶谱及HPLC纯的木聚糖酶。 相似文献
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3-Nitropropionic Acid Neurotoxicity Is Attenuated in Copper/Zinc Superoxide Dismutase Transgenic Mice 总被引:1,自引:1,他引:1
M. Flint Beal Robert J. Ferrante Ross Henshaw Russell T. Matthews †Pak H. Chan Neil W. Kowall †Charles J. Epstein Jörg B. Schulz 《Journal of neurochemistry》1995,65(2):919-922
Abstract: The mitochondrial toxin 3-nitropropionic acid (3-NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3-NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non-Tg littermate controls. The Tg-SOD mice showed a pronounced attenuation of Nissl-stained striatal lesions compared with non-Tg mice. Systemic administration of 3-NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid. This production was attenuated significantly in Tg-SOD mice. In a similar way, 3-NP produced significant increases in 3-nitrotyrosine/tyrosine, a marker for peroxynitrite-mediated damage, which were significantly attenuated in Tg-SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3-NP neurotoxicity. 相似文献