• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.spc1
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• pp.318-333
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• 2004

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.291-297
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• 1988

Three alleles of rbsB gene, rbsB, rbsB103, and rbsB106 from the wild type, the mutant and the revertant strain, respectively, were cloned for overproduction of proteins under the control of lambda $P_{L}$ promoter. Five different species of precursor and mature ribose-binding proteins were purified to homogeneity using DEAE-Sephadex column chromatography, osmotic shock pocedure, CM-Sephadex column chromatography, and Chromatofocusing column chromaography. pI of the precursor proteins and mature proteins were determined and found to be pH 8.0 and 7.5, respectively. The purified proteins were subjected to amino acid sequencing. The results confirmed the amino acid changes deduced from the DNA sequencing.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.235-236
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• 2001

We developed a DNA probe analytical system with a patterned array of oligonucleotide molecules immobilized on glass surfaces. The detection capability of the system depended mainly on the way the capture probes were attached to the support as wen as the sequence. We optimized major variables to graft DNA molecules onto a glass support and the DNA probe assay was eventually accomplished without purification of the PCR product.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.55-55
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• 1993

Farnesyl Protein transferase(FPT)는 발암유전자 ras의 단백질 산물인 p$^{21}$의 post-translational modification의 첫 단계인 ras-farnesylation에 관여하는 효소로 본 연구에서는 정제된 FPT와 E. coli에서의 발현 system을 이용하여 FPT의 구조와 기능을 밝히고 이를 FPT 방해제의 설계에 이용하고자 한다. Bovine testis에 존재하는 FPT를 30%-50%의 Ammonium sulfate로 fractionation하고, DEAE-Sephacel, Sephacryl S-300 column을 통과시킨 후 peptide(KKCVIM) affinity column을 이용하여 순수 정제하였다. 정제된 효소의 분자량은 gel-filtration에 의해 100KDa으로 추정되었고 SDS-PAGE 결과 49KDa과 46KDa의 두 subunit로 구성되었음이 확인되었다. 효소활성에는 $Mg^{2＋}$ 와 $Zn^{2＋}$가 필수적이며 최적 pH는 7.0이었다. Yeast의 FPT의 두 subunit 유전자는 Yeast genomic DNA를 template로 사용하고 각 subunit에 specific한 합성된 primer들과 vent polymerase를 이용하여 Polymerase chain reaction을 통하여 얻었다. 두 유전자를 pBluescriptII SK＋ vector를 변형시킨 두 vector, pBSK＋4와 pBChl＋4에 재조합 시킨 후 E.coli에 transformation시켜 발현시켰다. 현재 정제된 Bovine FPT와 E. coli에서 발현된 Yeast FPT의 chemical modification과 site-directed mutagenesis를 통하여 FPT의 active site와 substrate binding site에 관한 연구를 진행시키고 있다.

• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.17-23
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• 2009

The gene encoding Thermus thermophilus HJ6 DNA polymerase (Tod) was cloned and sequenced. The open reading frame (ORF) of the Tod gene was composed of 2,505 nucleotides and encoded a protein (843 amino acids) with a predicted molecular weight of 93,795 Da. The deduced amino acid sequence of Tod showed 98% and 86% identities to the Thermus thermophilus HB8 DNA pol and Thermus aquaticus DNA pol, respectively, The Tod gene was expressed under the control of the bacteriophage $\lambda$ promoters PR and PL on the expression vector pJLA503 in Escherichia coli strain BL21 (DE3) codon plus. The expressed enzyme was purified by heat treatment, $HiTrap^{TM}$ Q column, and $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 column chromatographies. The optimal temperature and pH for DNA polymerase activity were found to be $75{\sim}80^{\circ}C$ and 9.0, respectively. The optimal concentrations of $Mg^{2+}$ and $Mn^{2+}$ were 2.5 mM and 1 mM, respectively. The enzyme activity was activated by divalent cations, and was inhibited by monovalent cations. The result of the PCR experiment with Tod DNA polymerase indicates that this enzyme might be useful in DNA amplification and PCR-based applications.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 1998.06a
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• pp.363.2-364
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• 1998

• Journal of Plant Biology
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• v.32 no.4
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• pp.331-341
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• 1989

DNA methyltransferase was purified 282.6-fold from Chlamydomonas reinhardtii 21gr (mt+) gametic cell to examine the effect of polyamine on the enzyme acctivity. Polyacrylamide gel electrophoresis(PAGE) revealed at least three bands(1 major band, 2 minor bands). Among these, the major band represents DNA methyltransferase. Polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecylsulfate(SDS-PAGE) revealed a major band with M.W. 60,000. DNA methyltransferase activity was inhibited more effectively by spermine than by spermidine, and the inhibition by putrescine was smaller than spermine and spermidine. DNA methyltransferase activity was inhibited by 40% and 53% at 5mM and 20mM spermine, respectively. In the case of spermidine, the inhibition was 35% at 10mM and 44% at 20mM. However, the inhibition by putrescine appeared only above 5mM and reached about 25% at 20mM.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

• KSBB Journal
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• v.11 no.1
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• pp.37-45
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• 1996

Marine bacterial strain, highly effective agar degrading, was isolated from south sea of Korea and was identified as Pseudomonas sp. This strain was named Halophilic Pseudomonas sp. W7 and accumulated an extracellular agarase which showed a high level of enzyme activity in the presence of agar and agarose. This extracellular agarase was purified by anion-exchange chromatography and gel filtration. Purified agarase showed a single protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 89KDa. The agarase gene was cloned into Escherichia coli JM83 using the plasmid vector pUC19. DNA fragments(3.7, 3.0Kb) of Hind III-digested chromosomal DNA of Pseudomonas sp. W7 was inserted into the Hind III site of pUC19. Selected transformants, E. coli JM83/pSWl 000000and E. coli JM83/pSW3, produced agarase and this agarase was accumulated In the cytoplasmic space.

• Proceedings of the Korean Information Science Society Conference
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• 2003.10a
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• pp.67-69
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• 2003

바이오네트웍 모델링은 유전자네트웍, 단백질네트웍, 대사회로, 신호전달회로네트웍등에 대하여 각 요소간의 관계를 그래프이론을 통하여 표현하는 작업을 말한다. 특히 조절네트웍의 모델링은 다양한 생물학적 실험 데이터로부터 단백질들간의 활성과 불활성 관계를 유추해내는 것을 말한다. 현재 조절네트웍 모델링을 위한 다양한 알고리즘들이 개발되어 있으나 응용적인 측면에서 유추된 네트웍은 활용성이 부족하다. 본 논문에서는 In Vitro상에서 DNA 컴퓨팅을 이용하여 간단한 연산을 수행함으로서 유전자 조절 기전을 모델링하고자 한다. 이러한 방법의 장점은 DNA컴퓨팅의 연산이 세포의 현재 또는 다음 상태를 In Vivo 상에서 구현되어 진단 등의 문제에 응용될 수 있다는 가능성을 제시해 준다는 것이다.