Controlled Expression of Lysis Genes Encoded in T4 Phage for the Gentle Disruption of Escherichia coli Cells
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概要
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Two lysis genes, gene-t and gene-e, encoded in bacteriophage T4 were cloned in vectors pUC118 and pET26b(+), respectively. Immediately after the induction of gene-t expression, growth of Escherichia coli. JM 109 cells was halted. Since the expression of gene-I was toxic to the cells, the expressed gene-t Product(gp-t)could not he detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). On the other hand, expression of gene-e cloned in vector plasmid pUC118 did not give rise to any detectable chage in E. coli JM 109 cells; normal cell growth was observed, and the gene-e product(gp-e)was identified by SDS-PAGE. Lysis genes cloned in vector pET26b(+)were expressed in BL21(DE3)host cells carrying plasmid pLysS, which encodes T7 Iysozyme. It is thought that the expressed protein is translocated into the periplasmic space driven by the signal peptide of the pelB outermembrane protein of E. coli fused in the frame at the N-terminal end of the lysis protein. Immediate cell disruption was observed when the gene-t cloned in pET26b(+)was expressed in the logarithmic growth phase. β-Galactosidase activity was observed in the centrifuged supernatant of the cell culture producing gp-t. Almost 100% of the activity of the β-galactosidase produced in the BL21(DE3)pLysS cells was identified in the supernatant 30 min after the start of the induction period of gene-t, indicating that complete cell disruption had occurred at that time. The production of gp-e with the N-terminal fusion of the signal peptide of pelB did not cause immediate cell lysis, but it did result in morphological change in the BL21(DE3)pLysS cells, from rod-shaped to elliptical. Resuspension of the gp-e-producing BL21(DE3)pLysS cell pellet with pure water caused cell lysis followed by the release of β-galactosidase into the medium. Almost look of the β-galactosidase activity was identified in the resispended supernatant.
- 社団法人日本生物工学会の論文
- 1998-01-25
著者
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丹治 保典
Graduate School Of Bioscience And Biotechnology Tokyo Institute Of Technology
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TANJI YASUNORI
Department of Bioengineering, Tokyo Institute of Technology
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UNNO HAJIME
Department of Bioengineering, Tokyo Institute of Technology
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XING Xin-hui
Division of Materials Science and Chemical Engineering Yokohama National University
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XING XIN-HUI
Department of Chemical System Engineering, Yokohama National University
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ASAMI KAZUHIRO
Department of Bioengineering, Faculty of Biotechnology, Tokyo Institute of Technology
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海野 肇
東京工大 生命理工
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Unno Hajime
Dep. Of Biotechnology Tokyo Inst. Of Technol. Yokohama 226-8501 Jpn
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Xing X‐h
Yokohama National Univ. Yokohama Jpn
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Tanji Yasunori
Dep. Of Bioengineering Graduate School Of Bioscience And Biotechnology Tokyo Inst. Of Technol.
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Tanji Yasunori
Research Institute For Iron Steel And Other Metals Tohoku University
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Asami Kazuhiro
Department Of Bioengineering Faculty Of Biotechnology Tokyo Institute Of Technology
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Xing Xin-hui
Department Of Bioengineering Tokyo Institute Of Technology
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Unno Hajime
Department Of Bioengineering Faculty Of Bioscience And Biotechnology Tokyo Institute Of Technology
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Tanji Yasunori
Department Of Bioengineering Faculty Of Bioscience And Biotechnology Tokyo Institute Of Technology
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Asami Kouji
Department Of Bioengineering Faculty Of Biotechnology Tokyo Institute Of Technology
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Xing X‐h
中国清華大
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