• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.150-155
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• 1995

Carboxypeptidase Z(CPZ) cDNA of Absidia zychae was experssed in Saccharomyces cerevisiae. The expressed CPZ(YCPZ) was secreted about 30 mg/l into the medium and has a little higher molecular weight than the wild type CPZ in SDS-PAGE. By the result of N-terminal amino acid sequencing, YCPZ has additional 15 amino acids residues in N-terminus of CPZ. But YCPZ shows no difference with CPZ in enzyme activity and substrate specificity. For the identification of processing mechanism of YCPZ, 36-Arg was changed to 36-Thr by site specific mutagenesis. Mutant YCPZ does not processed at 36-Thr. It was, therefore, concluded that the YCPZ was processed by KEX2. According to endo F treatment, high amount of carbohydrate was N-glycosylated in YCPZ.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.5
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• pp.442-448
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• 2003

Melanin concentrating hormone (MCH) regulating color change of fish skin was identified from brain cDNA library of Olive flounder (Paralichthys olivaceus) during the analysis of Expressed Sequence Tags (ESTs). Olive flounder MCH gene consisted of 598 nucleotides encoding 150 amino acids. Olive flounder MCH protein revealed to contain signal peptide of 19 amino acid residues, pro-MCH of 131 amino acids being processed to biologically active and mature form of hormone with 25 amino acid residues at the carboxyl terminus. A comparative structural analysis revealed that Olive flounder MCH precursor had low sequence identity with other fish species and mammalian counterparts, while the amino acid sequences of mature hormone had a relatively high identity and more conserved. RT-PCR analysis revealed that olive flounder MCH precersor gene was expressed spectically only in the brain and not in other tissues.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.328-334
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• 2011

Leucine zipper motif consists of multiple periodic leucine residues, which forms amphipathic alpha helix. The hydrophobic nature of leucine zipper motif can dimerize proteins which contain this motif. Leucine zipper motif addition at C-terminus of single-chain Fv (ScFv) antibody induces its dimerization. Since the dimeric ScFv antibody contains two antigen binding sites (bivalency) like Y-shaped complete antibody, it could increase avidity. As a result, it could show higher antigen binding activity than monomeric ScFv antibodies. Based on this concept, monomeric chicken 8C3 ScFv antibody previously developed from chicken hybridoma was dimerized by the addition of leucine zipper motif at C-terminus of ScFv antibody. The dimeric 8C3 ScFv antibody specifically reacted with Eimerian sporozoite which causes Avian Coccidiosis. As expected, dimeric 8C3 ScFv antibody showed 3-folds higher antigen binding activity than monomer due to increased avidity. In addition, protien yields of dimer expression were 2-folds higher than monomer.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.139-147
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• 1999

Peptides are formed in the rumen as the result of microbial proteinase activity. The predominant type of activity is cysteine ptoteinase, but others, such as serine proteinases, are also present. Many species of protozoa, bacteria and fungi are involved in ptoteolysis; large animal-to-animal variability is found when proteinase activities in different animals are compared. The peptides formed from proteolysis are broken down to amino acids by peptidases. Different peptides are broken down at different rates, depending on their chemical composition and particularly their N-terminal structure. Indeed, chemical addition to the N-terminus of small peptides, such as by acetylation, causes the peptides to become stable to breakdown by the rumen microbial population; the microorganisms do not appear to adapt to hydrolyse acetylated peptides even after several weeks exposure to dietary acetylated peptides, and the amino acids present in acetylated peptides are absorbed from the small intestine. The amino acids present in some acetylated peptides remain available in nutritional trials with rats, but the nutritive value of the whole amino acid mixture is decreased by acetylation. The genus Prevotella is responsible for most of the catabolic peptidase activity in the rumen, via its dipeptidyl peptidase activities, which release dipeptides rather than free amino acids from the N-terminus of oligopeptides. Studies with dipeptidyl peptidase mutants of Prevotella suggest that it may be possible to slow the rate of peptide hydrolysis by the mixed rumen microbial population by inhibiting dipeptidyl peptidase activity of Prevotella or the rate of peptide uptake by this genus. Peptides and amino acids also stimulate the growth of rumen microorganisms, and are necessary for optimal growth rates of many species growing on tapidly fermented substrates; in rich medium, most bacteria use pre-formed amino acids for more than 90% of their amino acid requirements. Cellulolytic species are exceptional in this respect, but they still incorporate about half of their cell N from pre-formed amino acids in rich medium. However, the extent to which bacteria use ammonia vs. peptides and amino acids for protein synthesis also depends on the concentrations of each, such that preformed amino acids and peptides are probably used to a much lesser extent in vivo than many in vitro experiments might suggest.

• Journal of Microbiology
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• v.40 no.4
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• pp.295-300
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• 2002

Previously, we reported induced expression of developmentally regulated CTP-binding protein 2 (DRG2) in fish cells at the late stage of rhabdovirus infection. To investigate the biological role of fish DRG2 (fDRG2), we transfected CHSE-214 cells with an expression vector containing complete fDRG2 fused to the N-terminal end of an enhanced green fluorescent protein (EGFP). Low level expression of fDRG2-EGFP did not induce morphological change or cell death. However, a high level expression of fDRG2-EGFP induced cell rounding and caused depletion of the cell population in FACS analysis. Several truncated fragments were fused to EGFP. FACS analysis was conducted to determine the presence of cells expressing high levels of the resulting chimera. While cells expressing a high level of N-terminus were detected, those expressing high levels of the C-terminal fragment 243-290 containing the G4 motif were absent in FACS analysis. Based on these observations, we propose that overexpression of fDRG2 may induce cell rounding, a representative cytopathic effect of virus-infected cells in the late stage of infection and the C-terminus of the fDRG2 is essential for this function.

• BMB Reports
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• v.33 no.2
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• pp.120-125
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• 2000

M2PI is an active single chain mini-proinsulin with a 9-residue linker containing the turn-forming sequence 'YPGDV' between the B- and A-chains, but which retains about 50% of native insulin receptor binding activity. The refolding efficiency of M2PI is higher than proinsulin by 20-40% at alkaline pH, and native insulin is generated by the enzymatic conversion of M2PI. The solution structure of M2PI was determined by NMR spectroscopy. The global structure of M2PI is similar to that of native insulin, but the flexible linker between the B- and A-chains perturbed the N-terminal A-chain and C-terminal B-chain. The helix in the N-terminal A-chain is partly perturbed and the ${\beta}$-turn in the B-chain is disrupted in M2PI. However, the linker between the two chains was completely disordered indicating that the designed turn was not formed under the experimental conditions (20% acetic acid). Considering the fact that an insulin analogue, directly cross-linked between the C-terminus of the B-chain and the N-terminus of the A-chain, has negligible binding activity, a flexible linker between the two chains is sufficient to keep binding activity of M2PI, but the perturbed secondary structures are detrimental to receptor binding.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.921-928
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• 2002

Microorganism producing extracellular CFTase was isolated from soil and designated as Bacillus polymyxa MGL21. The gene encoding the CFTase (cft) from B. polymyxa MGL21 was cloned and sequenced. The ORF of the cf gene was composed of 3,999 nucleotides, encoding a protein (1,333 amino acids) with a predicted molecular mass of 149,375 Da. Sequence analysis indicated that CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase ($50\%$ identity, 259 amino acids). Furthermore, CFTase possessed a highly conserved core region, which is considered to be functional for the hydrolysis reaction of inulin. The cft gene was expressed in a His-tagged form in Escherichia coli cells, and the His-tagged CFTase was purified to homogeneity. The optimal temperature and pH for CFTase activity were found to be $50^{\circ}C$ and 9.0, respectively. The enzyme activity was completely inhibited by 10 mM $Ag^+\;and\;Cu^2+$. Thin-layer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction ut also disproportionation and hydrolysis reactions as well.

• Mycobiology
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• v.42 no.2
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• pp.167-173
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• 2014

A ${\beta}$-glucan synthase gene was isolated from the genomic DNA of polypore mushroom Sparassis crispa, which reportedly produces unusually high amount of soluble ${\beta}$-1,3-glucan (${\beta}$-glucan). Sequencing and subsequent open reading frame analysis of the isolated gene revealed that the gene (5,502 bp) consisted of 10 exons separated by nine introns. The predicted mRNA encoded a ${\beta}$-glucan synthase protein, consisting of 1,576 amino acid residues. Comparison of the predicted protein sequence with multiple fungal ${\beta}$-glucan synthases estimated that the isolated gene contained a complete N-terminus but was lacking approximately 70 amino acid residues in the C-terminus. Fungal ${\beta}$-glucan synthases are integral membrane proteins, containing the two catalytic and two transmembrane domains. The lacking C-terminal part of S. crispa ${\beta}$-glucan synthase was estimated to include catalytically insignificant transmembrane ${\alpha}$-helices and loops. Sequence analysis of 101 fungal ${\beta}$-glucan synthases, obtained from public databases, revealed that the ${\beta}$-glucan synthases with various fungal origins were categorized into corresponding fungal groups in the classification system. Interestingly, mushrooms belonging to the class Agaricomycetes were found to contain two distinct types (Type I and II) of ${\beta}$-glucan synthases with the type-specific sequence signatures in the loop regions. S. crispa ${\beta}$-glucan synthase in this study belonged to Type II family, meaning Type I ${\beta}$-glucan synthase is expected to be discovered in S. crispa. The high productivity of soluble ${\beta}$-glucan was not explained but detailed biochemical studies on the catalytic loop domain in the S. crispa ${\beta}$-glucan synthase will provide better explanations.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2649-2655
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• 2010

$PKC{\delta}$-catalytic V5 Heptapeptide (FEQFLDI, FP7) interacts with heat shock protein 27 (HSP27) and inhibits HSP27-mediated resistance to cell death against various stimuli including radiation therapy. Here, we prepared radio-iodinated heptapeptide and further investigated its uptake properties in HSP27 expression cells. Peptide sequence of FP7 and a negative control peptide (WSLLEKR, QP7) was modified by substituting their C-terminus residue to tyrosine (FP6Y and QP6Y) to label radio-iodine. Iodinated peptides were confirmed by LC mass analysis with cold iodine reaction mixture. Accumulation of [$^{125}I$]iodo-FP6Y and [$^{125}I$]iodo-QP6Y in NCI-H1299 cell line, with higher level of HSP27, and NCI-H460 cell line, with lower level of HSP27, was measured by NaI(Tl) scintillation counter. The modification of substituting C-terminus residue of FP7 to tyrosine (FP6Y) did not affect its interaction with HSP27. Accumulation of [$^{125}I$]iodo-FP6Y in NCI-H1299 cells was 3 fold higher than in NCI-H460 cells. The novel radio-iodinated FP6Y would be used as a tracer for targeting HSP27 protein.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.807-812
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• 2002

In order to analyze the cellular proteins which interact with core protein of hepatitis C virus (HCV), a yeast two-hybrid screening technique was employed. A carboxyl terminus truncated core protein, which contained amino acid residues from the 1st to 120th, was used as a bait to screen cellular proteins. The expression library prepared from HeLa cell was screened and 400 positive clones were selected. The 75 clones from the positive clones were sequenced and analyzed by undergoing the Blast search. Interestingly, 7 out of the 75 clones encoded E7 antigen of human papilloma virus (HPV). We studied in detail the Interaction between the truncated version of HCV core and E7 antigen in vitro. The core$_{120}$ protein expressed in chimeric form with G57 was able to bring down the E7 protein of HPV type 18 expressed in bacteria. It is therefore suggested that the core of HCV might affect the interaction between E7 and a normal cellular tumor suppressor, known as Rb protein.