• Journal of Microbiology and Biotechnology
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• 제24권3호
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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.

• 한국식품과학회지
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• 제36권6호
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• pp.1020-1025
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• 2004

생식제품 및 제조 공정 중 위해 미생물의 제어 및 저감화를 위하여 제조공정을 개선하고 전해수, 오존수 등 살균효과에 대하여 조사하였다. 7가지의 위해미생물 오염도가 높은 생식원료에 대한 전해수의 살균효과가 차아염소산소다수 또는 오존수에 비하여 전체적으로 강한 것으로 나타났으며 오존수의 살균효과는 차아염소산소다수와 비슷하거나 다소 강한 것으로 나타났다. 그러나 전해수나 오존수를 이용하여 생식원료를 세정, 살균하여도 원료의 종류에 따라 살균효과에 차이가 있으며, 비 가열가공이라는 생식의 특성상 원료에서 검출되는 위해미생물은 최종제품까지 생잔할 가능성이 높기 때문에, 각 원료에 따른 살균처리지침을 위한 database 축적이 요구된다. 한편 모델공장을 선택하여 공장위생, 종업원 개인위생 및 제조공정을 개선한 후 공정개선의 미생물학적 효과를 조사한 결과 공정개선 전에는 원료에서 보다 최종제품에서 위해미생물의 수가 증가하는 경향을 나타내었으나, 공정개선 후에는 원료의 균수를 그대로 유지하거나, 감소하였으며 외주 원료의 혼합으로 인하여 위해미생물의 균수가 증가하였다. 따라서 생식의 제조공정에서 위해미생물을 저감화를 위해서는 외주 원료의 철저한 위생관리가 요구되며, 전해수 오존수 등을 이용한 원료의 비가열 세정살균과 지속적이고 위생적인 공정관리가 이루어진다면 위해 미생물의 균수를 저감화할 수 있을 것으로 사료된다.

• The Journal of Korean Physical Therapy
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• 제21권3호
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• pp.87-93
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• 2009

Purpose: TThis study examined the effect of repetitive magnetic stimulation (RMS) on the viability and proliferative response of human adipose tissue-derived stromal cells (hATSCs) in vitro. Methods: The hATSCs were cultured primarily from human adipose tissue harvested by liposuction and incubated in a $37^{\circ}C$ plastic chamber. The cells were exposed to a repetitive magnetic field using a customized magnetic stimulator (Biocon-5000, Mcube Technology). The RMS parameters were set as follows: repetition rate=10Hz, 25Hz (stimulus intensity 100%= 0.1 Tesla, at 4cm from the coil), stimulated time= 1, 5, and 20 minutes. Twenty four hours after one application of RMS, the hATSCs were compared with the sham stimulation, which were kept under the same conditions without the application of RMS. The cells were observed by optical microscopy to determine the morphology and assessed by trypan blue staining for cell proliferation. The apoptosis and viability of the hATSCs were also analyzed by fluorescence-activated cell sorting (FACS) analysis of Annexin V and MTT assay. Results: After RMS, the morphology of the hATSCs was not changed and the apoptosis of hATSCs were not increased compared to the sham stimulation. The viability of the cells was similar to the cells given the sham stimulation. Interestingly, the level of hATSC proliferation was significantly higher in all RMS groups. Conclusion: The application of RMS may not cause a change in morphology and viability of hATSCs but can increase the level of cell proliferation in vitro. RMS might be useful as an adjuvant tool in combination with stem cell therapy without adverse effects.