• Title, Summary, Keyword: Biocon

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A New Signal Sequence for Recombinant Protein Secretion in Pichia pastoris

  • Govindappa, Nagaraj;Hanumanthappa, Manjunatha;Venkatarangaiah, Krishna;Periyasamy, Sankar;Sreenivas, Suma;Soni, Rajeev;Sastry, Kedarnath
    • 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.

Study on Reducing Methods of Natural Food-borne Pathogenic Microorganisms Originated from Saengshik (생식 중 자연환경유래 위해미생물 저감화 방법에 관한 연구)

  • Chang, Tae-Eun;Han, Jeong-Su;Song, Ok-Ja;Chung, Dong-Hwa;Shin, Il-Shik
    • Korean Journal of Food Science and Technology
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    • v.36 no.6
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    • pp.1020-1025
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    • 2004
  • In previous paper, contaminations of food-borne pathogenic bacteria of Saengshik was found to occur during processing, because detection rates of food-borne pathogenic bacteria in final products were higher than those of raw materials. In this study, methods to reduce food-borne pathogenic bacteria and improved manufacturing process were developed for microbial safety of Saengshik. Food-borne pathogenic bacteria in raw materials were reduced to about 0.5-2.0 log cfu/g when seven kinds of raw materials were washed with electrolyzed water and ozonated water, but food-borne pathogenic bacteria could not be removed completely. After improvement of manufacturing process, numbers of food-borne pathogenic bacteria were same or decreased to levels of raw materials. Gaseous ozone and Biocon could control air-borne bacteria under $1{\times}10^1$ cfu/1000 L of air in pulverization and mixing rooms.

Effect of Repetitive Magnetic Stimulation on Proliferation and Viability of Adipose Tissue-Derived Stromal Cells (반복자기자극이 지방유래 중간엽 줄기세포 증식과 활성에 미치는 영향)

  • Kim, Su-Jeong;Park, Hea-Woon;Cho, Yun-Woo;Lee, Joon-Ha;Seo, Jeong-Min;Shin, Hyoun-Jin;Kang, Jae-Hoon;Ahn, Sang-Ho
    • The Journal of Korean Physical Therapy
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    • v.21 no.3
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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.

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