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.

为了提高亲水模型蛋白肿瘤坏死因子 (TNF) 与脂质体的结合效率，通过基因融合将酰化信号序列连接到 TNF 的 N 末端。将编码拉希德白血病病毒-gag蛋白、Galphai1-蛋白或Gsα-蛋白的酰化信号的DNA序列融合至编码TNF成熟结构域的cDNA的5'-末端，得到+C_ 14 -TNF分别为 cDNA、+Galphai1-TNF cDNA 和 +Gsalpha-TNF cDNA。使用兔网织红细胞体外翻译编码这些融合 cDNA 的 mRNA裂解物产生分子量为18kDa的融合TNF，通过[^3H]-亮氨酸的掺入和抗TNF抗体的免疫沉淀来测定。对这些融合TNFs中掺入[^3H]-脂肪酸的分析表明，只有具有N-肉豆蔻酰化信号的融合TNFs才能观察到蛋白质的有效酰化。为了产生大量酰化融合TNFs，+C_<14 >-TNF cDNA亚克隆至杆状病毒表达系统（pAcYM1）转移载体中，并与线性化杆状病毒DNA共转染至Sf细胞（AcVAPK6）。纯化重组表达病毒后，用重组病毒感染Sf细胞，表达融合TNF。使用抗TNF抗体对感染的Sf细胞的总细胞裂解物进行蛋白质印迹分析，结果显示融合TNF的有效表达，分子量为18kDa。对[^3H]-肉豆蔻酸掺入融合TNF的分析表明，重组融合TNF在该表达系统中发生了有效的肉豆蔻酰化。因此，体外和体内表达系统均成功产生了N-肉豆蔻酰化融合TNF 。