• Molecules and Cells
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• v.19 no.2
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• pp.205-211
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• 2005

The Arg-Gly-Asp (RGD) sequence serves as the primary recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the biological activities of matrix proteins. We have initiated structure-function studies of two RGD containing peptides, RGD-5(AGGDD) and cyclic RGD-6(CARGDDC). Assays have shown that cyclic RGD-peptides inhibit platelet aggregation more efficiently than linear ones. NMR data revealed that RGD-5 and RGD-6 have entirely different conformation. RGD-5 has a linear extended structure and RGD-6 has a stable loop conformation. In RGD-5 the guanidinium group of Arg2 and the carboxyl group of Asp4 lie in parallel, whereas the side-chains of Arg3 and Asp5 of RGD-6 are located in different planes, supporting the idea that the stability of the cyclic form derives from the packing of the side chain of the Arg and Asp residues. The structural features of these peptides could provide a basis for designing new drugs against diseases related to platelet aggregation and as cancer antagonists.

• Journal of Microbiology
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• v.38 no.1
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• pp.1-7
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• 2000

Eight strains of Aeromonas hydrophila isolated from diseased trout in Korea were characterized and compared with an American type strain by various methods including biochemical and physiological tests, PCR, randomly amplified polymorphic DNA (RAPD), plasmid profiling, and gel electrophoresis of total, membrane, and extracellular proteins. Virulence factors such as surface array proteins, cytotoxin, hemolysin, haemagglutinin, and protease were also investigated. The Korean strains showed heterogeneity in Iysine decarboxylase production, utilization of various carbon sources, and production of acetoin. Five strains had the same profiles of total and membrane proteins. Six strains haemagglutinated with trout red blood cells (RBCs) which was inhibited by fucose, galactose, and mannose, except for No. 1 where haemagglutination was inhibited by only galactose and mannose, but not by fucose. Four isolates haemagglutinated with human RBCs which was inhibited by fucose and mannose yet not by galactose. The type strain haemagglutinated only with trout RBCs which was inhibited by fucose, galactose, and mannose. Every isolate secreted protease, hemolysin, cytotoxin, and siderophore, but no enterotoxin. Results showed that the Korean isolates, except for No.7, had very different biochemical and molecular characteristics from those of the American type strain.

• Microbiology and Biotechnology Letters
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• v.51 no.2
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• pp.167-173
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• 2023

Proteolytic enzymes secreted by Aspergillus, as pathogenicity factors, affect blood coagulation and fibrinolysis, and therefore the target proteins of their action in the bloodstream are of significant interest. In the present study, the action of the isolated protease of A. terreus 2 on different human plasma proteins was shown. The protease of A. terreus 2 exhibited the highest proteolytic activity against hemoglobin, which was 2.5 times higher than the albuminolytic activity shown in both of the protein substrates used. In addition, the protease has significant ability to hydrolyze both fibrin and fibrinogen. However, the inability of the A. terreus 2 protease to coagulate rabbit blood plasma and coagulate human and bovine fibrinogen indicates the severity of the enzyme's action on human blood coagulation factors. It should be considered as a potential indicator of this isolated protease's participation in fungal pathogenesis. The protease shows no hemolytic activity. Furthermore, its activity is insignificantly inhibited by thrombin inhibitors, and is not inhibited by plasmin inhibitors.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.11
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• pp.1507-1514
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• 2008

Cholesterol oxidase catalyses the conversion of cholesterol to 4-cholesten-3-one. This enzyme has been used for clinical assay of human serum cholesterol and for reduction of cholesterol level in foods and feeds. In order to search the microorganism which has a high extracellular and stable activity of cholesterol oxidase, soil microorganisms were screened. As a result, the one with the highest extracellular cholesterol oxidase activity was obtained and named as the BEN 115. The BEN 115 strain was identified as one of the Nocardia species based on our taxonomic studies. The cholesterol oxidase from this strain was shown to have two bands of extracellular proteins on SDS-PAGE and Western blot. Their molecular masses were estimated to be about 55 and 57 kDa, respectively. In addition, this cholesterol oxidase was considerably stable at the broad range of pH $3.5{\sim}9.5$ and at the temperature of $25{\sim}55^{\circ}C$. The optimum pH and temperature of this cholesterol oxidase were pH 5.5 and $35^{\circ}C$, respectively. The activity of extracellular cholesterol oxidase could be enhanced 1.6 to 2.0 folds by the addition of nonionic detergent such as Triton X-114, Triton X-100, or Tween-80 into the culturing broth. The substrate specificities against campesterol, sitosterol and stigmasterol were measured to be 50%, 50%, and 27%, respectively, compared to the cholesterol. These results suggest that Nocardia sp. BEN 115 may be useful as a microbial source of cholesterol oxidase production.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.337-345
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• 2014

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro $MAT{\alpha}$ sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.3
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• pp.250-265
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• 2008

Distraction osteogenesis is a well-established clinical treatment for limb length discrepancy and skeletal deformities. Appropriate mechanical tension-stress is believed not to break the callus but rather to stimulate osteogenesis. In contrast to fracture healing, the mode of bone formation in distraction osteogenesis is primarily intramembranous ossification. Although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the basic biology of the process is still not well known. Moreover, the molecular mechanisms in distraction osteogenesis remain largely unclear. Recent studies have implicated the growth factor cascade is likely to play an important role in distraction. And current reserch suggested that mechanical tension-stress modulates cell shape and phenotype, and stimulates the expression of the mRNA for bone matrix proteins. The purpose of this study is to examine the pattern of expression of growth factors($TGF-{\beta}1$, IGF-I, bFGF) and extracellular matrix proteins(osteoclacin, osteonectin) related to osteogenesis by osteodistraction of the mandible in rabbits. 24 rabbits is used for this experiment. Experimental group are gradual distraction(0.7mm, twice/day), acute distraction(1.4mm, twice/day) and control group is only osteotomized. After 5 days latency, osteotomic site is distracted for each 7 days and 3.5 days. Consolidation period is 28 days. The animal is sacrificed at the 3th, 7th, 14th, 28th. The distracted bone is examined by immunohistochemical analysis and RT-PCR analysis. The results obtained from this study were as follow : No significant difference was found on clinical examination according to distraction rate, but gradual distraction was shown to improve regenerate bone formation on radiographic and histologic examination. Growth factors and extracelluar matrix proteins expression increased in distraction group than control group. From these results, it could be stated that graudal distraction is shown to improve and accelerate bone formation and mechanical stress like distraction has considerable effects on osteogenesis related factors. And rabbit is the most appropriate animal model for further reseach on the molecular mechanisms that mediate osteodistraction. It is believed that understanding the biomolecular mechanisms that mediate distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone healing.

• BMB Reports
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• v.45 no.4
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• pp.253-258
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• 2012

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically-validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.

• KSBB Journal
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• v.30 no.3
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.218-218
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• 2002

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.