• Journal of Microbiology
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• v.34 no.1
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• pp.74-81
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• 1996

Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the .alpha.-amylase gene from an .alpha.-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the .alpha.-amylase gene for easy replacement of various foregn structural genes. To evaluate this secretion vectors, the .betha.-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active .betha.-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each .betha.-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed .betha.- lactamases were located idn the culture medium. The amount of the secreted .betha.-lactamase was about 80% of the total secreted proteins in the culture medium.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.40-45
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• 2010

The gene encoding $\beta$-agarase (agaB) which hydrolyzes $\beta$-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal ($MF{\alpha}1$), in which the transcription of $MF{\alpha}1$-AgaB was under the control of AOX1 (alcohol oxidase 1, methanol inducible) promoter. The constructed plasmid pPIC-AgaB (9 kb) was integrated into HIS4 gene locus of Pichia pastoris genome. Successful integration was confirmed by performing colony PCR. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the active expression of agaB in P.pastoris. By SDS-PAGE and zymographic analysis, the molecular weight of $\beta$-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular $\beta$-agarase reached 1.34, 1.42 and 1.53 units/mL by inducing 0.1, 0.5, and 1% methanol, respectively, at baffled flask culture of P.pastoris GS115/pPIC-AgaB for 48 hr. Most of the enzyme activity was found in the extacellular fraction and the secretion efficiency showed 98%. Thermostability of recombinant $\beta$-agarase was also increased by glycosylation.

• Tuberculosis and Respiratory Diseases
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• v.45 no.1
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• pp.128-139
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• 1998

Background: It is well known that various cytokines and growth factors secreted mainly from alveolar macrophages do the key role in the pathogenesis of IPF. But recently it has been known that structural cells like fibroblast can also release cytokines. So the phenotypic changes in fibroblasts of IPF may do a role in continuous progression of fibrosis. The aim of this study is to find out whether there is a change in the biologic properties of the lung fibroblasts of IPF. Subjects and Method: The study was done on 13 patients with IPF diagnosed by open or thoracoscopic lung biopsy and 7 control patients who underwent resectional surgery for lung cancer. Lung fibroblast cell lines (FB) were established by explant culture technique from the biopsy or resected specimen Result: Basal proliferation of the fibroblast of IPF(IFB) measured by BrdU uptake tended to be highter than control fibroblast(NFB) (0.212 0.107 vs $0.319{\pm}0.143$, p=0.0922), also there was no significant difference in proliferation after the stimulation with PDGF or 10% serum. On the contrary, the degree of inhibition in proliferation by PGE2 was significantly lower($33.0{\pm}13.1%$) in IFB than control($46.7{\pm}10.0%$, p=0.0429). The IFB secreted significantly higher amount of MCP-l($l574{\pm}1283$ pg/ml) spontaneously than NFB($243{\pm}100$ pg/ml) and also after the stimulation with TGF-$\beta$($3.23{\pm}1.31$ ng/ml vs $0.552{\pm}0.236$ ng/ml, p=0.0012). Similarly IL-8 and IL-6 seretion of IFB was significantly higher than NFB at basal state and with TGF-$beta$ stimulation. But after the maximal stimulation with IL-1,8, no significant difference in cytokine secretion was found between IFB and NFB. Conclusion : Above data suggest that the fibroblasts of IPF were phenotypically changed and these change may do a role in the pathogenesis of IPF.