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

为了提高亲水模型蛋白肿瘤坏死因子（TNF）与脂质体的关联效率，通过基因融合将一个酰化信号序列连接到TNF的n端。将编码拉希德白血病病毒gag蛋白、galphai1蛋白或gphapha1蛋白酰化信号的DNA序列融合到编码TNF成熟结构域的cDNA 5'端，分别得到+C_<14>-TNF cDNA、+Galphai1-TNF cDNA和+ gphapha1 -TNF cDNA。使用兔网织细胞裂解液对编码这些融合cdna的mrna进行体外翻译，产生分子质量为18kDa的融合tnf，这是通过[^3H]-亮氨酸的结合和抗tnf抗体的免疫沉淀测定的。对[^3H]-脂肪酸掺入这些融合tnf的分析表明，只有具有n -肉豆肉酰化信号的融合tnf才能观察到蛋白质的有效酰化。为了产生大量的酰化融合tnf，我们将+C_<14>-TNF cDNA亚克隆到杆状病毒表达系统的转移载体pAcYM1中，并用线性化的杆状病毒DNA AcVAPK6共转染到Sf细胞中。纯化重组表达病毒后，用重组病毒感染Sf细胞表达融合TNF。用抗TNF抗体对感染的Sf细胞的总细胞裂解物进行Western blotting分析，发现融合TNF的有效表达，分子量为18kDa。对[^3H]-肉豆蔻酸掺入融合TNF的分析显示，在该表达系统中，重组融合TNF发生了有效的肉豆蔻酰化。因此，体外和体内两种表达系统均成功生成了n -肉豆芽酰基化融合TNF。

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.