• Transactions of the Korean Society of Mechanical Engineers A
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• v.30 no.7 s.250
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• pp.841-848
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• 2006

Many researchers are developing computational prediction methods for protein tertiary structures to get much more information of protein. These methods are very attractive on the aspects of breaking technologies of computer hardware and simulation software. One of the computational methods for the prediction is a fragment assembly method which shows good ab initio predictions at several cases. There are many barriers, however, in conventional fragment assembly methods. Argues on protein energy functions and global optimization to predict the structures are in progress fer example. In this study, a new prediction method for protein structures is proposed. The proposed method mainly consists of two parts. The first one is a fragment assembly which uses very shot fragments of representative proteins and produces a prototype of a given sequence query of amino acids. The second one is a global optimization which folds the prototype and makes the only protein structure. The goodness of the proposed method is shown through numerical experiments.

• The Korean Journal of Pesticide Science
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• v.2 no.3
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• pp.79-84
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• 1998

Tandem HPLC and atmospheric pressure chemical ionization(APcI) LC/MS method was used for the determination and confirmation of carbendazim residues in soybean sprout. Fluorescence(FL) detector was connected in tandem with the ultraviolet(UV) detector for dual detection of the carbendazim residue at the excitation and emission wavelength of 280 nm and 310 nm, respectively. The limit of detection for carbendazim was $0.1{\mu}g/kg$ sample. Recoveries of carbendazim from fortified soybean sprout at 0.5, 1.0 and 2.0 ppm were averaged 89.1%. Mass spectrometry using a APcI source confirmed the carbendazim residue in the soybean sprout sample. Fragmentation pattern on the APcI LC/MS spectrum of carbendazim was simpler than that from electron impact(EI) mass spectrum. Carbendazim produced 3 major ions including m/z 133, m/z 159 and m/z 191($M^{+}$). This method was sensitive enough to provide reliable and reproducible results for practical applications.

• Journal of the Korean Chemical Society
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• v.34 no.3
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• pp.288-296
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• 1990

Antielastic fragment in GBⅠ-Ⅱ differ on $P_1$ site with that of antitryptic fragment in LBI. To obtain further understanding of the role of amino acid residue near the reactive site, specificity of $P_1$ site and loop size, Tyr substituted cyclic nonapeptide and cyclic pentapeptide were synthesized and the inhibition constants for some proteinases were calculated by Dixon method. Cyclic nonapeptide showed no inhibition for chymotrypsin but appeared low inhibitory activity for trypsin and elastase and that of cyclic pentapeptide possessed inhibitory activity for chymotrypsin.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.226-229
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• 2004

Histone deacetylase (HDAC) removes acetyl group in lysine residue of histone protein, which is transferred by histone acetylase. HDAC is important in the stabilization and regulation of gene expression in eukaryotic organisms. PCR has been carried out to clone HDAC genes using cDNA library and genomic DNA as the templates from Ganoderma lucidum isolated in Korea. One 470 bp cDNA gene fragment, and 3 genomic HDAC fragments (585 bp, 589 bp, 630 bp) were amplified. When their deduced amino acid sequences were compared with other fungal HDACs, they showed 59-72％ homology.

• Journal of Life Science
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• v.8 no.3
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• pp.235-240
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• 1998

Matrix metalloproteinases(MMPs) are a group of zinc enzymes responsible for degradation of the matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteprocess. Genetically distince MMPs have been characterized and their genes have been cloned thus far from a variaty of species but not from fishes. In this stydy, a mmp cDNA was cloned by using RT-PCR(reverse transcriptase dependent polymerase chain reaction) from Scylliorhinus toraxzame(shark), agroup of cartilaginous fish, abundant in the coast of Pusan, Korea. It has 74% base homologue with membrane type matrix matalloproteinase-3 genes(mt3-mmps) from human, rat and chick, and also shows more than 90% residue homologue with them. In addition, it has cysteine switch domain, zinc binding domain(HExGH motif), propeptide cleavage site, and RRKR motif, which are present in MMPs. This result indicates that cDNA fragment cloned here may be mt3-mmp or its analogous gejne cDNA fragment of Scylliorhinus torzame.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.255-259
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• 1999

A 6.9-kb DNA fragment including the minimal Bacillus pasteurii urease gene cluster was subcloned into a high-copy-number plasmid vector, pUC19, and the recombinant B. pasteurii urease was overexpressed in Escherichia coli. The recombinant urease was purified 25.9-fold by using combinations of anion-exchange and gel-filtration chromatography followed by Mono-Q chromatography on a FPLC. N-terminal peptide sequencing analyses revealed that two distinct smaller peptide bands resolved on a 10-18％ gradient SDS-PAGE corresponded to UreA and UreB peptides, respectively. It was also shown that the ureB gene was translated from a GUG codon and the first methionine residue was post-translationally cleaved off. The native molecular weight of the recombinant urease was 176,000 and 2 nickel atoms were present per catalytic unit. pH stability studies of the purified enzyme showed that the recombinant Bacillus pasteurii urease is stable in alkaline pH range, which is similar to the enzyme of the evolutionarily related bacterium, Sporosarcina ureae.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.534-538
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• 2001

The xynA gene encoding an alikali-tolerant endo-1,4-${\beta}$-xylanase (XYN) was cloned from the alkalophilic Bacillus pumilus A-30. The nucleotide sequence of a 974-bp DNA fragment containing the xynA was determined. An ORF of 684 nucleotides that encoded a protein of 228 amino aicds was detected. Asparagine-71 of XYN from B. Pumilus A-30 showed to be highly conservative in alkaline xylanases of family G/11, upon comparing the amino acid sequences of 17 family G/11 xylanases. Site-directed mutation of N71D of the xynA gene resulted in a decrease of 12.4% in the specific acitivity and a significant decline in the enzyme activity in the alkaline pH range.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.279-285
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• 1993

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.

• Korean Journal of Environmental Agriculture
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• v.34 no.3
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• pp.230-237
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• 2015

BACKGROUND: To identify the sources of inaccuracy in LC/MS/MS methods used in the routine quantitation of small molecules are described and discussed. METHODS AND RESULTS: Various UPLC coupled to triple quadrupole mass spectrometer and time of flight (TOF) were used to identify the potential sources of inaccuracy and inducing the pitfalls of qualification and quntitation during the veterinary drug residue analysis. Some of stable isotope labelled veterinary drugs, which were used as internal standards, presented "cross-talk", regardless of manufactures of mass spectrometer and types of spectrometer. Group of sulfonamides also presented inaccuracy qualification and quantitation due to the multi-residue analytical method with the same fragment ions at the close retention times. CONCLUSION: The phenomena of "cross-talk" occurring between subsequently monitored transition from stable isotope labelled and isotope non-labelled authentic chemical were identified. To prevent errors and achieve more accurate data during the analysis of small molecules by LC/MS/MS SRM method, Followings should be taken care of and kept checking; purity and concentration of stable isotope as an internal standard, prevention of carry-over during the separation in column, minimizing the ion suppression by matrix effect, identification of retention time, precursor ion and product ion, and full knowledge of data processing including smoothing and peak integration.