• Journal of Life Science
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• v.15 no.6 s.73
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• pp.935-940
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• 2005

Fluorescence change of coumarin labeled phosphate binding protein (PBP-MDCC) was monitored to measure the amount of released inorganic phosphate ($P_{i}$) during nucleoside triphosphate (NTP) hydrolysis reaction. After purification of PBP-MDCC, fluorescence emission spectra showed that fluorescence responded linearly to $P_{i}$ up to about 0.7 molar ratio of $P_{i}$ to protein. The correlation of fluorescence signal and $P_{i}$ standard was measured to obtain [$P_{i}$] - fluorescence intensity standard curve on the stopped-flow instrument. When T7 bacteriophage helicase, double-stranded DNA unwinding enzyme using dTTP hydrolysis as an energy source, reacted with dTTP, the change of fluorescence was able to be converted to the amount of released $P_{i}$ by the $P_{i}$ standard curve. $P_{i}$ release results showed that single-stranded Ml3 DNA stimulated dTTP hydrolysis reaction several folds by T7 helicase. Instead of end point assay in NTP hydrolysis reaction, real time $P_{i}$-release assay by PBP-MDCC was proven to be very easy and convenient to measure released $P_{i}$.

• BMB Reports
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• v.38 no.6
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• pp.676-682
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• 2005

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of $NaBH_4$, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.

• Journal of Radiation Protection and Research
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• v.20 no.2
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• pp.71-78
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• 1995

To develop methods to reduce radiation risk and apply such knowledge to improvement of radiation protection, the effects of cobaltous chloride known as bioantimutagen on the function of E. coli RecA protein involved in the repair of DNA damage were examined. The results demonstrated two distinct effects of cobaltous chloride on the RecA protein function necessary for the strand exchange reaction. Cobaltous chloride enhanced the ability of RecA protein to displace SSB protein from single-stranded DNA and the duplex DNA-dependent ATPase activity. RecA protein was preferentially bound with UV-irradiated supercoiled DNA as compared with nonirradiated DNA The binding of RecA protein to UV-irradiated supercoiled DNA was enhanced in a dose-dependent manner. It is likely that studies on the factors affecting repair efficiency and the DNA repair proteins may provide information on the repair of ionizing radiation-induced DNA damage and the mechanism for DNA radioprotection.

• Journal of Industrial Technology
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• v.28 no.B
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.298-304
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• 2007

Salmonella is facultative intracellular pathogen that can survive and replicate in macrophages even though these cells are equipped with a plethora of anti-microbial mechanisms. To survive in this hostile intracellular environment, Salmonella has evolved numerous defense mechartisms. An approximately 20 kDa protein was detected as a stationary-phase specific protein band in cytosolic fraction. It was identified as a DNA binding protein in stationary phase (Dps) by analysis of MALDI-TOF assay. It has been known that Dps, the protein produced in the stationary phase of bacteria, allows DNA to form chromatin by binding to DNA nonspecifically and protects DNA from reactive oxidative species (ROS). For further study, Dps specific polyclonal antibodies were generated by injection of purified Dps protein into rabbit. To examine the Finfluence of several regulatory proteins in the expression dps gene, Dps protein level in various S. typhimurium mutants defecting regulatory proteins were investigated by Westernblot using Dps specific polyclonal antibodies.

• Biomolecules & Therapeutics
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• v.31 no.3
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• pp.340-349
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• 2023

Mad2B (Mad2L2), the human homolog of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares sequence similarity with the mitotic checkpoint protein Mad2A. Previous studies on Mad2B have concluded that it is a mitotic checkpoint protein that functions by inhibiting the anaphase-promoting complex/cyclosome (APC/C). Here, we demonstrate that Mad2B is activated in response to cisplatin-induced DNA damage. Mad2B co-localizes at nuclear foci with DNA damage markers, such as proliferating cell nuclear antigen and gamma histone H2AX (γ-H2AX), following cisplatin-induced DNA damage. However, unlike Mad2A, the binding of Mad2B to Cdc20 does not inhibit the activity of APC/C in vitro. In contrast to Mad2A, Mad2B does not localize to kinetochores or binds to Cdc20 in spindle assembly checkpoint-activated cells. Loss of the Mad2B protein leads to damaged nuclei following cisplatin-induced DNA damage. Mad2B/Rev7 depletion causes the accumulation of damaged nuclei, thereby accelerating apoptosis in human cancer cells in response to cisplatin-induced DNA damage. Therefore, our results suggest that Mad2B may be a critical modulator of DNA damage response.

• Journal of the Korean Magnetic Resonance Society
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• v.15 no.1
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• pp.69-79
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• 2011

HP0827 has two RNP motif which is a very common protein domain involved in recognition of a wide range of ssRNA/DNA.We acquired 3D NMR spectra of HP0827 which shows well dispersed and homogeneous signals which allows us to assign 98% of all $^1H_N$, $^{15}N$, $^{13}C_{\alpha}$, $^{13}C_{\beta}$ and $^{13}C$=O resonances and 90% of all sidechain resonances. The sequence-specific backbone resonance assignment of HP0827 can be used to gain deeper insights into the nucleic acids binding specificity of HP0827 in the future study. Here, we report secondary structure prediction of HP0827 derived from NMR data. Additionally, ssRNA/DNA binding assay studies was also conducted. This study might provide a clue for exact function of HP0827 based on structure and sequence.

• BMB Reports
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• v.37 no.6
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• pp.663-670
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• 2004

ZNF 333 is a new and sole gene containing two KRAB domains which has been identified currently. It is a member of subfamilies of zinc finger gene complex which had been localized on chromosome 19p13.1. The ZNF333 gene mainly encodes a 75.5 kDa protein which contains 10 zinc finger domains. Using the methods of random oligonucleotide selection assay, electromobility gel shift assay and luciferase activity assay, we found that ZNF333 recognized the specific DNA core binding sequence ATAAT. Moreover, these data indicated that the KRAB domain of ZNF333 really has the ability of transcriptional repression.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.39-44
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• 2014

We isolated highly-expressed genes in the posterior silk glands of silkworm on a previously study, which one of these was identified as RNA binding protein-1 homologue (RBP-1) gene. In this study, we investigated gene expressional characteristics of the RBP-1 depending on silkworm development stages and several tissues of the larvae, respectively. Northern blot hybridization analysis showed that the RBP-1 gene was expressed high in larval and pupal periods, and highly expressed than endogenous internal control gene (BmA3) on all tested larval tissues. In addition, we isolated and analyzed a phage DNA having 1,660 bp-long promoter region of the RBP-1 gene from a genomic DNA library. To study the RBP-1 gene promoter activity, RBP-1 (-740/+ 30) was amplified by PCR and subcloned into a pGL3 basic vector to generate pGL-RBP1. A luciferase report vector carrying RBP-1 gene promoter (770 bp) was tested by luciferase assay in Sf9 cells. In the result, the RBP-1 gene promoter was more efficient than constitutive promoter (BmA3) by approximately ten percent.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.97-102
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• 1996

To investigate whether human $\beta$$_2$-adrenergic receptor devoid of the C-terminal two transmembrane helices retain its ligand binding activity and specificity, 5'780-bp DNA fragment of the receptor gene which encodes amino acid 1-260 of human $\beta$$_2$-adrenergic receptor was subcloned into the bacterial fusion protein expression vector and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was expressed as a membrane bound form which was verified by SDS-PAGE and Western blot. The fusion protein expressed in this study specifically bound $\beta$-adrenergic receptor ligand [$^3$H] Dihydroalprenolol. In saturation ligand binding assay, the $K_{d}$ value was 7.6 nM which was similar to that of intact $\beta$$_2$-adrenergic receptor in normal animal tissue ( $K_{d}$=1~2 nM) and the $B_{max}$ value was 266 fmol/mg membrane protein. In competition binding assay, the order of binding affinity of various adrenergic receptor agonists to the fusion protein was isoproterenol》epinephrine norepinephrine, which was similar to that of intact receptor in normal animal tissue. These results suggest that N-terminal five transmembrane helices of the $\beta$$_2$-adrenergic receptor be sufficient to determine the ligand binding activity and specificity, irrespective of the presence or absence of the C-terminal two transmembrane helices.s.s.s.