| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an acylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the acylation signal of Rasheed leukemia virus-gag protein, Galphai1-protein or Gsalpha-protein was fused to the 5'-end of the cDNA coding for the mature domain of TNF to give +C_<14>-TNF cDNA,+Galphai1-TNF cDNA and +Gsalpha-TNF cDNA,respectively.
In vitro translation of mRNAs coding for these fusion cDNAs using rabbit reticulocyte lysate gave rise to fusion TNFs with a molecular mass of 18kDa as determined by the incorporation of [^3H]-leucine and by immunoprecipitation with anti-TNF antibody. Analysis of incorporation of [^3H]-fatty acids into these fusion TNFs revealed that the effective acylation of protein was observed exclusively with fusion TNFs having N-myristoylation signal.
In order to produce a large amount of acylated fusion TNFs, +C_<14>-TNF cDNA was subcloned into transfer vector of baculovirus expression system (pAcYM1) and cotransfected to Sf cells with linearized baculovirus DNA (AcVAPK6). After purifying recombinant expression virus, fusion TNF was expressed by infecting Sf cells with the recombinant virus. Western blotting analysis of total cell lysates of the infected Sf cells using anti-TNF antibody revealed the efficient expression of fusion TNF with a molecular mass of 18kDa. Analysis of the incorporation of [^3H]-myristic acid into the fusion TNF revealed that the effective myristoylation of recombinant fusion TNF occurred in this expression system.
Thus, N-myristoylated fusion TNF was successfully generated by both in vitro and in vivo expression system.
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