Improvement of GFPuv-β3GnT2 Fusion Protein Production by Suppressing Protease in Baculovirus Expression System

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  • KATO Tatsuya
    <i>Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University</i>
  • MURATA Takeomi
    <i>Laboratory of Biochemistry, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University</i>
  • USUI Taichi
    <i>Laboratory of Biochemistry, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University</i>
  • PARK Enoch Y
    <i>Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University</i>

書誌事項

タイトル別名
  • Improvement of GFPuv-.BETA.3GnT2 Fusion Protein Production by Suppressing Protease in Baculovirus Expression System
  • Improvement of GFPuv ベータ 3GnT2 Fusion Protein Production by Suppressing Protease in Baculovirus Expression System
  • Improvement of GFPuv-β3GnT2 protein production by suppressin protease in baculovirus expression system

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  The effects of protease inhibitors on the production of recombinant protein were investigated using a recombinant baculovirus containing GFPuv-human β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) connected to the prepromelittin signal sequence. The addition of leupeptin as a cysteine protease inhibitor at 2.5 μg/ml improved intra- and extracellular β3GnT activities 5- and 3-fold, respectively, compared to those without addition, which was due to a suppression of protease activity. With the leupeptin addition only four degraded molecular bands lower than 32 kDa appeared, but 9 degraded molecular bands between 29 kDa and 41 kDa existed without addition. In contrast, pepstatin A as a carboxyl protease inhibitor had no influence on the improvement of β3GnT production, judging from SDS-PAGE. Moreover, when 50 μM carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), known as a proteasome inhibitor, was used in combination with the leupeptin, a ladder of low molecular mass bands of fusion protein was diminished. The intracellular β3GnT activity increased 9-fold, to as high as that without addition of two kinds of protease, but the extracellular activity was not different from that with the addition of only leupeptin. These findings indicate that the decrease in cell viability causes the decrease in the secretion rate of intracellular fusion protein, resulting the accumulation of the full-length of fusion protein.<br>

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