• Journal of Periodontal and Implant Science
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• v.38 no.1
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• pp.41-50
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• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.21 no.2
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• pp.110-119
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• 1999

The principle of guided tissue regeneration (GTR), as applied to bone healing, is based on the prevention of connective tissue from entering the bony defect during the healing phase. This allows the slower bone producing cells to migrate into and reproduce bone within the defect. The principle of guided tissue regeneration has demonstrated a level of success in regenerating bone defect. Several types of membrane barrier, each one with distinct properties, have been utilized to apply this principle in bone regeneration. The purpose of this study is to introduce and discuss the attributes of rubber dam as a barrier membrane and evaluate whether improved bone regeneration can be achieved by GTR using rubber dam. In the 15 New Zealand white rabbits, full-thickness bone defects on three sites of each rabbit calvaria were made. Non membrane group served as a control and experimental group 1 was covered with rubber dam and group 2 covered with Gore-Tex$^{TM}$ membrane. Macroscopic, radiographic, microscopic examinations were made serially on 1, 2, 3, 6, 12 weeks after operation. The results were as follows: 1. Macroscopically, the control site was collapsed and filled with connective tissue throughout the experimental period. But the defects of experimental groups 1 and 2 were filled with bone-like mass and showed the hard consistency on palpation. 2. Radiographically, the early new bone formation appeared similarly from the host bone in groups 1 and 2. 3. Microscopically, there were much connective tissue at the central part of control site but the defect of group 1 and 2 was filled with the mature bony trabeculae on the 12th week. This results suggest that rubber dam can be effectively used as a barrier membrane for guided bone regeneration.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• Journal of Microbiology
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• v.42 no.2
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• pp.111-116
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• 2004

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli $tRNA_{1}$$^{Gln}$ with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-$tRNA_{1}$$^{Gln}$ formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also amelio-rated growth inhibition, presumably by competitively preventing $tRNA_{1}$$^{Gln}$ misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of $tRNA_{1}$$^{Gln}$, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-$tRNA^{Gln}$ amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mis-charging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli Glu-$tRNA_{1}$$^{Gln}$, and converts it to the cognate Gln-$tRNA_{1}$$^{Gln}$ species. B. subtilis GluRS-dependent Glu-$tRNA_{1}$$^{Gln}$ formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.149-154
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• 2005

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures of Escherichia coli YK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically de-fined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of $0.25\;gg^{-1}h^{-1}$, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.289-295
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• 1982

Streptomyces bobili (YS-40) isolated from soil was tested that it had drug resistance against penicillin, cephalosporin series antibiotics and other antibiotics in the previous paper. The treatment of Streptomyces bobili, (YS-40) with ethidium bromide (EtBr), acriflavine and sodium dodecyl sulfate. (SDS) resulted in the elimination of R-plasmid from the host strain. Minimum growth inhibitory concentrations (MIC) of Hg, Ag, penicillin-G, ampicillin, chloramphenicol, oxytetracycline, streptomycin and kanamycin were found to be 15, 10, > 3, 000, > 100, > 1, 000, > 100, < 5 and < 5$\mu\textrm{g}$/$m\ell$ respectively. Among the curing agents, EtBr was proved to be the most powerful compound for the elimination of R-plasmid in the strain and the elimination rate with EtBr(10$\mu\textrm{g}$/$m\ell$) was about 98%. Optimal pH to. the elimination of R-plasmid was pH 7.0 and the R-plasmid in the cells incubated for 24 hrs was proved to be eliminated most effectively. Aerial mass color, soluble pigment formation and reverse side color were reported to be often the plasmid associated characteristics of the R-plasmid bearing bacteria. But these characteristics of the uncured and cured Streptomyces bobili, (YS-40) showed no changes in the most of the pigment formation media tested in this work.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1246-1256
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• 2015

Chlamydophila psittaci is an important intracellular pathogen. Persistent infection is an important state of the host-parasite interaction in this chlamydial infection, which plays a significant role in spreading the organism within animal populations and in causing chronic chlamydiosis and serious sequelae. In this study, a C. psittaci persistent infection cell model was induced by penicillin G, and real-time quantitative PCR was used to study the transcriptional levels of 10 C. psittaci genes (dnaA, dnaK, ftsW, ftsY, grpE, rpsD, incC, omcB, CPSIT_0846, and CPSIT_0042) in acute and penicillin-G-induced persistent infection cultures. Compared with the acute cultures, the penicillin-G-treated cultures showed a reduced chlamydial inclusion size and a significantly decreased number of elementary body particles. Additionally, some enlarged aberrant reticulate body particles were present in the penicillin-G-treated cultures but not the acute ones. The expression levels of genes encoding products for cell division (FtsW, FtsY) and outer membrane protein E encoding gene (CPSIT_0042) were downregulated (p < 0.05) from 6 h post-infection onward in the persistent infection cultures. Also from 6 h post-infection, the expression levels of DnaA, DnaK, IncC, RpsD, GrpE, and CPSIT_0846 were upregulated (p < 0.05); however, the expression level of OmcB in the persistent infection was< almost the same as that in the acute infection (p > 0.05). These results provide new insight regarding molecular activities that accompany persistence of C. psittaci, which may play important roles in the pathogenesis of C. psittaci infection.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.679-685
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• 2011

Xanthomonas oryzae pv. oryzae (Xoo) produces a putative effector, XoAvrBs2. We expressed XoAvrBs2 homologously in Xoo with a TAP-tag at the C-terminus to enable quantitative analysis of protein expression and secretion. Addition of rice leaf extracts from both Xoo-sensitive and Xoo-resistant rice cultivars to the Xoo cells induced expression of the XoAvrBs2 gene at the transcriptional and translational levels, and also stimulated a remarkable amount of XoAvrBs2 secretion into the medium. In a T3SS-defective Xoo mutant strain, secretion of the TAPtagged XoAvrBs2 was blocked. Thus, we elucidated the transcriptional and translational expressions of the XoAvrBs2 gene in Xoo was induced in vitro by the interaction with rice and the induced secretion of XoAvrBs2 was T3SSdependent. It is the first report to measure the homologous expression and secretion of XoAvrBs2 in vitro by rice leaf extract. Our system for the quantitative analysis of effector protein expression and secretion could be generally used for the study of host-pathogen interactions.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.349-354
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• 2005

A virus isolate collected from infected paprika (Capsicum annuum var. grossum) was characterized as Potato virus Y (PVY) based on biological, serological, cytopathological, and molecular properties. In host range studies, the paprika isolate produced the mosaic symptom on some tobacco, tomato and pepper (Capsicum annuum). A new paprika isolate also infected potato cultivars which is different biological characteristic compared to the other popular potyvirus infecting paprika, Pepper mottle virus (PepMoV). Previously reported PVY strains, $PVY^o$ and $PVY^N$ did not infect pepper and typical PepMoV isolates did not infect potato. Distinctive inclusion patterns of the scroll, pinwheel, long laminated inclusions, and helper components in the cytoplasm of infected cells were also different to those observed by the typical PVY isolate infections. However, the paprika isolate reacted to the monoclonal antibody of $PVY^N$ strain with high absorbance readings. RT-PCR amplification, cloning, and sequencing of the 3' untranslated region and a part of coat protein gene also added additional evidence of the paprika isolate as the $PVY^N$-related isolate. Multiple alignments as well as cluster dendrograms of PVY-paprika isolate revealed close phylogenetic relationship to the $PVY^N$ subgroup. Altogether, these results suggest that a new PVY isolate infecting paprika contained distinct characteristics compared to the other previously described PVY strains with closer relationship to the $PVY^N$ strain.

• International Journal of Industrial Entomology and Biomaterials
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• v.9 no.2
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• pp.217-223
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• 2004

A cathepsin L-like cysteine protease, v-cath, encoded by the baculovirus has been shown to playa role in host liquefaction. We have identified a v-cath gene in the silkworm virus, Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. The 969 bp v-cath has an open reading frame of 323 amino acids. A putative cleavage site and catalytic sites were conserved in BmNPV-K1 v-cath. The predicted three-dimensional structure of BmNPV-K1 v-cath revealed that the overall fold of BmNPV-K1 v-cath is similar to that of other proteases of the papain family. The deduced amino acid sequence of BmNPV-K1 v-cath showed 98% and 97% protein sequence identity to BmNPV T3 strain and to Autographa californica nuclear polyhedrosis virus, respectively. The BmNPV-K1 v-cath differed at 4 amino acid positions from BmNPV T3. The v-cath gene in BmNPV-K1 genome is located on the EcoRV 6 kb and XhoI 9 kb fragments. Northern hybridization analysis of BmNPV K1 v-cath gene revealed that it is expressed late in infection.