• Journal of Life Science
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• v.13 no.6
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• pp.805-813
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• 2003

cDNA encoding somatolactin (SL) was obtained by RT-PCR from pituitary glands of rock bream (Oplegnathus fasciatus). The full length cDNA of rock bream somatolactin (rbSL) is 1636 bp long. It contains a 696 bp open reading frame encoding a signal peptide of 24 amino acids (an) and a mature protein of 207 aa. rbSL has seven cysteine residues$(Cys^{5},\; Cys^{15},\; Cys^{42},\; Cys^{65},\; Cys^{181},\; Cys^{198}\; $and $Cys^{206})$ and two potential N-glycosylation sites at positions $Asn^{121}$and $Asn^{153}$. The rbSL shares 61.1∼92.6% amino acid sequence similarities and 63∼92.6% nucleotide sequence identities with other teleost SLs, except for goldfish and channel catfish SL. Amino acid sequence alignment revealed that rbSL has four conserved domains $(A_{SL},\; B_{SL},\; C_{SL}and\; D_{SL})$ common to all SLs. Out of these domains, $(A_{SL},\; B_{SL},\; C_{SL}and\; D_{SL})$, are also conserved in all teleost growth hormones and prolactins. The cDNA of rbSL has been cloned into pET expression vector in order to produce recombinant rbSL in E. coli BL2l(DE3) cells. The recombinant protein showed a molecular weight of 27 kDa in SDS-PAGE.

• Applied Biological Chemistry
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• v.37 no.5
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• pp.361-369
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• 1994

To study the light-induced expression mechanism and protein transport into the chloroplast, a rice genomic clone (GrbcS) for the small subunit of ribulose 1,5-bisphosphate carboxylase (rbcS) was isolated and its nucleotide sequence was determined. Nucleotide sequence analysis of GrbcS revealed that the gene consists of two exons interrupted by an intron, encoding a protein of 175 amino acids including a transit peptide of 47 amino acids. These structural features of GrbcS are consistent with those of other rbcS genes from monocot species. Genomic Southern blot analysis suggested that the rbcS genes are present as a relatively small multigene family in the rice genome. Comparison of the nucleotide and deduced amino acid sequences to other rice rbcSs shows close sequence similaritiy. Conserved DNA sequences present in other light-responsive genes are also found in the 5’ upstream region of GrbcS such as G-box, 3AF1-binding site and GATA site. The possible function of these putative regulatory elements are discussed.

• Journal of agriculture & life science
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• v.45 no.6
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• pp.201-212
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• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.448-452
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• 2018

The full genome sequence of Bacillus safensis KCTC 12796BP which had been isolated from the marine sponge in the seawater of Jeju Island, was determined by Pac-Bio next-generation sequencing system. A circular chromosome in the length of 3,935,874 bp was obtained in addition to a circular form of plasmid having 36,690 bp. The G + C content of chromosome was 41.4%, and that of plasmid was 37.3%. The number of deduced CDSs in the chromosome was 3,980, whereas 36 CDS regions were determined in a plasmid. Among the deduced CDSs in chromosome, 81 tRNA genes and 24 rRNA genes in addition to one tmRNA were allocated. More than 30 CDSs for sporulation, 16 CDSs for spore coat, and 20 CDSs for germination were also assigned in the chromosome. Several genes for capsular polysaccharide biosynthesis and for flagella biosynthesis and chemotaxis in addition to genes for osmotic tolerance through glycine-choline betaine pathway were also identified. Above all, the biosynthetic gene cluster for anti-allergic compounds seongsanamides were found among two non-ribosomal peptide synthetase (NRPS) gene clusters for secondary metabolites.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1459-1464
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• 2004

The purpose of this research was to investigate the potential use of cheese whey protein (CWP), a cheese by-product. The physiological activity of calcium-binding peptides in CWP may be used as a food additive that prevents bone disorders. This research also examined the characteristics of calcium-binding peptides. After the CWP was heat treated, it was hydrolyzed by trypsin. Then calcium-binding peptides were separated and purified by ion-exchange chromatography and reverse phase HPLC, respectively. To examine the characteristics of the purified calcium-binding peptides, amino acid composition and amino acid sequence were analyzed. Calcium-binding peptides with a small molecular weight of about 1.4 to 3.4 kDa were identified in the fraction that was flowed out from 0.25 M NaCl step gradient by ion-exchange chromatography of tryptic hydrolysates. The results of the amino acid analysis revealed that glutamic acid in a calcium-binding site took up most part of the amino acids including a quantity of proline, leucine and lysine. The amino acid sequence of calcium-binding peptides showed Phe-Leu-Asp-Asp-Asp-Leu-Thr-Asp and Ile-Leu-Asp-Lys from $\alpha$-LA and Ile-Pro-Ala-Val-Phe-Lys and Val-Tyr-Val-Glu-Glu-Leu-Lys from ${\beta}$-LG.

• Bulletin of the Korean Chemical Society
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• v.33 no.10
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• pp.3233-3240
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• 2012

We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

• BMB Reports
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• v.28 no.4
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.91-95
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• 2005

In the multidimensional protein identification technology of high-throughput proteomics, we use one-dimensional gel electrophoresis and after the separation by two-dimensional liquid chromatography, the sample is analyzed by tandem mass spectrometry. In this study, we have analyzed the Pseudomonas Putida KT2440 protein. From the protein identification, the protein database was combined with its reversed sequence database. From the peptide selection whose error rate is less than 1%, the SEQUEST database search for the tandem mass spectral data identified 2,045 proteins. For each protein, we compared the molecular weight calibrated from 1D-gel band position with the theoretical molecular weight computed from the amino acid sequence, by defining a variable MW$_{corr}$ Since the bacterial proteome is simpler than human proteome considering the complexity and modifications, the proteome analysis result for the Pseudomonas Putida KT2440 could suggest a guideline to build the protocol to analyze human proteome data.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.27-34
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• 1990

Two macrolide-lincosamide-streptogramin B (MLS) antibiotic resistance genes, one expressed inducibly and the other expressed constitutively were recognized from a single Staphylococcus aureus DH1 strain. The inducible MLS resistance gene was isolated and cloned from the R-plasmid pDE1(7.4kb) and the constitutive gene was from chromosomal DNA. Base sequence of the inducible MLS resistance gene (1.2kb) was determined and found as same that of pE194. The restriction map of the cloned constitutive MLS resistance gene was compared with that of the inducible gene. Two genes have same restriction map except leader region. In the constitutive gene there is no leader region which is doing major role in inducible expression.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.652-658
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• 1995

We attempted simultaneous expression of genes coding for endoglucanase and $\beta $-glucosidase from Pseudomonas sp. by using a synthetic two-cistron svstem in Escherichia coli and Saccharomyces cerevisiae. Two-cistron system, 5'--tac promoter-endoglucanase gene--$\beta $-glucosidase gene-- 3', 5'-tac promoter--$\beta $-glucosidase gene--endoglucanase gene--3' and 5'-tac promoter--endoglucanase gene--SD sequence--$\beta $-glucosidase gene--3, were constructed, and expressed in E. coli and S. cerevisiae. The E. coli and S. cerevisiae contained two-cistron system produced simultaneously endoglucanase and $\beta $-glucosidase. The recombinant genes contained the bacterial signal peptide sequence produced low level of endoglucanase and $\beta $-glucosidase in S. cerevisiae transformants: Approximately above 44% of two enzymes was localized in the intracellular fraction. The production of endoglucanase and $\beta $-glucosidase in veast was not repressed in the presence of glucose or cellobiose. The veast strain contained recombinant DNA with two genes hydrolyzed carboxvmethyl cellulose, and these endoglucanase and $\beta $-glucosidase degraded CMC synergistically to glucose, cellobiose and oligosaccharide. This result suggests the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.