• 미생물학회지
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• 제32권2호
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• pp.102-108
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• 1994

ABF1(Autonomously replicating sequence Binding Factor 1)은 효모 genome에서 $RTCRYN_5ACG$의 염기 서열을 가지고 있는 promoter, mating-type silencer, ARS에 결합하는 DNA binding 단백질이다. E. coli 에서 ABF1 유전자를 발현하기 위하여, ABF1 유전자를 pMAL-c2 벡터에 cloning하였다.(pMAHW). pMAHW를 E. coli에 형질전환하여, ABF1 융합단백질을 발현시키고, amylose resin affinity chromatography에 의하여 분리하였다. Factor Xa protease를 이용하여 분리된 융합단백질로부터 maltose binding protein을 잘라낸 후에 gel retardation analysis 방법으로 분리된 ABF1이 ARS1에 결합하는 능력을 지니고 있음을 확인하였다. DNA 결합에 관련된 부위를 찾기 위하여, 비전형적인 zinc finger motif가 위치하는 자리에서 pMAHW의 ABF1 유전자에 His-61을 다른 아미노산으로 치환하였다. DNA binding 부위로 추정되는 ABF1 단백질의 중간지역에 Leu-353, Leu-360를 다른 아미노산으로 치환하였다. Site-specific mutagenesis 를 통해 만들어진 mutant를 gel retardation analysis와 complementation test를 통해서 비전형적인 zinc finger motif이외에 다른 DNA binding motif가 있는 것을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제30권1호
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• pp.127-135
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• 2020

Small RNAs (sRNAs) are widespread and play major roles in regulation circuits in bacteria. Previously, we have demonstrated that transcription of esrE is under the control of its own promoter. However, the regulatory elements involved in EsrE sRNA expression are still unknown. In this study, we found that different cis-regulatory elements exist in the promoter region of esrE. We then screened and analyzed seven potential corresponding trans-regulatory elements by using pull-down assays based on DNA affinity chromatography. Among these candidate regulators, we investigated the relationship between the ferric uptake regulator (Fur) and the EsrE sRNA. Electrophoresis mobility shift assays (EMSAs) and β-galactosidase activity assays demonstrated that Fur can bind to the promoter region of esrE, and positively regulate EsrE sRNA expression in the presence of Fe²⁺.

• 미생물학회지
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• 제30권6호
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• pp.466-471
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• 1992

HTLV-I 의 gag-pro 유전자 중첩영역내에서 -1 ribosomal frameshifting 이 일어나는 자리를 결정하기 위하여 gag-pro 중첩영역의 일부를 SP6 promoter 를 가진 백터내에 클로닝하였다. 그 결과 닭의 prelysozyme 에서 유래한 5개의 아미노산을 코드하는 합성유전자와 141 bp 로된 gag-pro 중첩영역의 뒤에 Straphylococcus aureus 의 protein A 유전자단편이 연결된 hybrid 유전자를 보유한 플라스미드를 제작하였다. 이 DNA 클론을 주형으로 SP6 RNA polymerase 의 작용에 의해 한종류의 mRNA 를 다량으로 합성하였다. Invitro 에서 합성된 mRNA 로 무세포계에서 단백질을 합성한 결과 21 kDal 의 단백질이 생성되었고 IgG-Sepharose 를 사용한 affinity chromatography 로 합성된 단백질을 순수하게 정제할 수 있었다. 본연구에서 설명한 in vitro 실험계는 Gag-Pro transframe 단백질의 신속한 정제 및 일차구조의 결정에 유익하게 사용될 것으로 보이며 이와 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 아니가 pro 유전자의 발현에 필요한 frameshift 를 유도하는 tRNA 의 동정도 가능하게 될 것이다.

• BMB Reports
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• 제28권3호
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• pp.249-253
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• 1995

The nonenzymatic glycation of copper, zinc-superoxide dismutase (Cu,Zn-SOD) led to inactivation and fragmentation of the enzyme. The glycated Cu,zn-SOD was isolated by boronate affinity chromatography. The formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in calf thymus DNA and the generation of strand breaks in pBhiescript plasmid DNA by a metal-catalyzed oxidation (MCO) system composed of $Fe^{3+}$, $O_2$, and glutathione (GSH) as an electron donor was enhanced more effectively by the glycated CU,Zn-SOD than by the nonglycated enzyme. The capacity of glycated Cu,Zn-SOD to enhance damage to DNA was inhibited by diethylenetriaminepentaacetic acid (DETAPAC), azide, mannitol, and catalase. These results indicated that incubation of glycated CU,Zn-SOD with GSH-MCO may result in a release of $Cu^{2+}$ from the enzyme. The released $Cu^{2+}$ then likely participated in a Fenton-type reaction to produce hydroxyl radicals, which may cause the enhancement of DNA damage.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.291-297
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• 1998

Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

• Molecules and Cells
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• 제20권2호
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• pp.297-302
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• 2005

The integrity and proper functioning of telomeres require association of telomeric DNA sequences with specific binding proteins. We have characterized PLP-1, a $PUR{\alpha}$ homolog encoded by F45E4.2, which we previously identified as a candidate double stranded telomere binding protein, by affinity chromatography followed by mass spectrometry. PLP-1 bound double-stranded telomeric DNA in vitro as shown by competition assays. Core binding was provided by the third and fourth nucleotides of the TTAGGC telomeric repeat. This is quite different from the binding sequence of CEH-37, another C. elegans telomere binding protein, suggesting that multiple proteins may bind nematode telomeric DNA simultaneously in vivo.

• BMB Reports
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• 제33권2호
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• pp.155-161
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• 2000

The saccharomyces cerevisiae Rad27, a structure-specific endonuclease for the okazaski fragment maturation has been known to interact genetically and biochemically with Dna2, an essential enzyme for DNA replication. In an attempt to define the significance of the interaction between the two enzymes, we expressed and purified both Dna2 and Rad27 proteins. In this report, Rad27 could not form a complex with Dna2 in the three different analyses. The analyses included glycerol gradient sedimentation, protein-column chromatography, and coinfection of baculoviruses followed by affinity purification. This is in striking contrast to the previous results that used crude extracts. These results suggest that the interaction between the two proteins is not sufficiently stable or indirect, and thus requires an additional protein(s) in order for Rad27 and Dna2 to form a stable physical complex. This result is consistent with our genetic findings that Schizosaccharomyces pombe Dna2 is capable of interacting with several proteins that include two subunits of polymerase $\delta$, DNA ligase I, as well as Fen-1. In addition, we found that the N-terminal modification of Rad27 abolished its enzymatic activity. Thus, as suspected, we found that on the basis of the structure determination, N-terminal methionine indeed plays an important role in the nucleolytic cleavage reaction.

• BMB Reports
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• 제32권3호
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• pp.306-310
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• 1999

The partial cDNA of the MT-c clone encoding snake venom metalloprotease was subcloned and expressed in E. coli. The expressed metalloprotease was purified by affinity chromatography in the presence of urea, and then successfully refolded into its functional form, retaining metalloprotease activity that hydrolyzes fibrinogen. The simple and convenient refolding strategy established in this work was highly efficient in recovering the recombinant enzyme activity. Experimental evidence suggests that the C-terminal amino acid stretch of 16 residues is a critical sequence for proper folding of the metalloprotease domain.

• BMB Reports
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• 제34권6호
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• pp.526-530
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• 2001

The translationally controlled tumor protein (TCTP), also known as the IgE-dependent histamine releasing factor (HRF), was used in the yeast two-hybrid system to screen the interacting molecules. We obtained the N-terminus truncated rat fast myosin alkai light chain from the rat skeletal muscle cDNA library in the screening. Since either TCTP/HRF or the myosin light chain is known to be associated with histamine secretion from RBL-2H3 cells, we investigated the possible interaction between rat TCTP/HRF and nonmuscle myosin light chain in these cells. We used affinity chromatography and coimmunoprecipitation. Our data suggests that HRF and the myosin light chain interact, which may play an important role in histamine release in RBL-2H3 cells.

• BMB Reports
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• 제38권6호
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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.