Preparation and characterization of an N-myristoylated fusion protein that binds to the membrane surface

与膜表面结合的 N-肉豆蔻酰化融合蛋白的制备和表征

基本信息

批准号：
06660112
负责人：
UTSUMI Toshihiko
金额：
$ 1.34万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

为了提高肿瘤坏死因子（TNF）的效率（TNF），一种与脂质体的亲水性模型蛋白相关的效率，通过基因融合将酰化信号序列与TNF的N末端联系起来。 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使用兔网状细胞裂解物为这些融合cDNA编码，从而导致融合型TNF，分子质量为18KDA，这是通过掺入[^3H] - 灰年和抗TNF抗体的免疫沉淀来确定的。 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使用线性化杆状病毒DNA（ACVAPK6）到SF细胞。纯化重组表达病毒后，通过用重组病毒感染SF细胞来表达融合TNF。使用抗TNF抗体对感染SF细胞的总细胞裂解物的蛋白质印迹分析表明，融合TNF的有效表达与分子质量为18KDA。分析[^3H] - 狂热酸到融合TNF中的分析表明，重组融合TNF的有效肉豆蔻酰化发生在该表达系统中。