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Applied Biochemistry and Biotechnology

, Volume 53, Issue 1, pp 29–35 | Cite as

Improvement ofescherichia coli cellfree system by utilization of cell extract having additional property

Problems and countermeasures
  • Norihiro Nishimura
  • Yoshihisa Kitaoka
  • Mitsuru Niwano
Article

Abstract

In theEscherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E.coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.

Index entries

Cell-free system copper ion ribonuclease Escherichia coli protein synthesis 

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Copyright information

© Humana Press Inc 1995

Authors and Affiliations

  • Norihiro Nishimura
    • 1
  • Yoshihisa Kitaoka
    • 1
  • Mitsuru Niwano
    • 1
  1. 1.Biotechnology Research LaboratoryKobe Steel Ltd.TsukubaJapan

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