A Novel Cell-Free Translation/Glycosylation System Prepared from Insect Cells

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概要

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• A cell-free translation/glycosylation system derived from lepidopteran(Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gap120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.

• 社団法人日本生物工学会の論文
• 2000-11-25

著者

• IMANISHI Shigeo

  Laboratory of Cell Engineering, National Institute of Sericaltunal and Entomological Science

• Hara T

  Microbiological Genetics Division Institute Of Genetic Resource Faculty Of Agriculture Kyushu Univer

• Tarui H

  Kyushu Univ. Fukuoka Jpn

• Imanishi Shigeo

  Laboratory Of Cell Engineering National Institute Of Sericaltunal And Entomological Science

• Imanishi Shigeo

  National Institute Of Agrobiological Sciences

• Imanishi Shigeo

  Laboratory Of Cell Engineering Department Of Insect Genetics And Breeding National Institute Of Seri

• TARUI HIROSHI

  Institute of Genetic Resources, Faculty of Agriculture, Kyushu University

• HARA TOSHIO

  Institute of Genetic Resources, Faculty of Agriculture, Kyushu University

• Imanishi Shigeo

  National Inst. Agrobiological Sci. Tsukuba Jpn

関連論文

• Differential Level in Co-Down-Modulation of CD4 and CXCR4 Primed by HIV-1 gp120 in Response to Phorbol Ester, PMA, among HIV-1 Isolates
• A Novel Cell-Free Translation/Glycosylation System Prepared from Insect Cells
• Insect Cell Extract Preparation by the Nitrogen Disruption Method for Cell-Free Translation
• Introduction of Kanamycin Resistant Gene to Pock-forming PlasmidpSA1.1 of Streptomyces azureus and Identification of spoIIIE-like Genein the Recombinant Plasmid
• Isolation and Characterization of a 41 kDa Sericin from the Wild Silkmoth Antheraea yamamai
• Factors Affecting Recombinant Protein Yields in Insect Cell/Baculovirus Expression System
• SUBCELLULAR LOCALIZATION OF INNEXIN, GAP JUNCTION RELATED PROTEIN IN INSECT CELLS(Cell Biology and Morphology,Abstracts of papers presented at the 75^ Annual Meeting of the Zoological Society of Japan)
• Calreticulin is Transiently Induced after Immunogen Treatment in the Fat Body of the Silkworm Bombyx mori
• Ecdysone Responsiveness of Several Cell Lines Derived from Bombyx mori
• Novel Bombyx mori cell lines cultivable at 37℃

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