In vitro synthesis of an N-myristoylated fusion protein that binds to the liposomal surface.
In vitro synthesis of an N-myristoylated fusion protein that binds to the liposomal surface.
复制标题
体外合成与脂质体表面结合的 N-肉豆蔻酰化融合蛋白。
DOI：
10.1006/abbi.1996.0063
复制
发表时间：
1996
影响因子：
3.9
通讯作者：
J. Klostergaard
中科院分区：
文献类型：
--
作者：
T. Utsumi;J. Kuranami;E. Tou;A. Ide;K. Akimaru;M. Hung;J. Klostergaard
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n -肉豆蔻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n -肉豆蔻酰化信号的DNA序列与编码TNF成熟结构域的cDNA 5′端融合，得到n -肉豆蔻酰化融合的TNF cDNA。利用兔网织细胞裂解液对编码该融合cDNA的mRNA进行体外翻译，产生了n -肉豆蔻酰基化的融合TNF，其分子量为18 kDa，通过加入[3H]肉豆蔻酸和抗TNF抗体的免疫沉淀测定。用Ala取代肉豆蔻酰化信号中的Gly2完全抑制了[3H]-肉豆蔻酸进入融合蛋白。使用Ficoll密度梯度离心的脂质体结合试验显示，将n -肉豆蔻酰基化融合TNF与双棕榈酰磷脂酰胆碱脂质体孵育可导致蛋白质与脂质体完全结合，而非肉豆蔻酰基化的对应体结合则少得多。因此，通过体外翻译/转录系统合成了对脂质体具有高亲和力的n -肉豆蔻酰基化融合TNF。
To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an N-myristoylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to be 5'-end of the cDNA coding for the mature domain of TNF to give N-myristoylated fusion TNF cDNA. In vitro translation of the mRNA coding for this fusion cDNA using rabbit reticulocyte lysate gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly2 in the myristoylation signal with Ala entirely inhibited the incorporation of [3H]-myristic acid into the fusion protein. A liposome binding assay using Ficoll density gradient centrifugation revealed that incubating the N-myristoylated fusion TNF with dipalmitoyl phosphatidylcholine-liposomes caused the complete binding of the protein to the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for liposomes, was synthesized by the in vitro translation/transcription system.