In vitro synthesis of an N-myristoylated fusion protein that binds to the liposomal surface

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 w...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1996-02, Vol.326 (2), p.179-184
Main Authors: Utsumi, T, Kuranami, J, Tou, E, Ide, A, Akimaru, K, Hung, M C, Klostergaard, J
Format: Article
Language:English
Subjects:
AIDS/HIV
Amino Acid Sequence
Animals
Base Sequence
Cloning, Molecular
DNA Primers - genetics
DNA, Complementary - genetics
Drug Carriers
Gene Products, gag - biosynthesis
Gene Products, gag - chemistry
Gene Products, gag - genetics
Humans
In Vitro Techniques
Liposomes
Molecular Sequence Data
Mutation
Myristic Acids - chemistry
Protein Biosynthesis
Rabbits
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Reticulocytes - metabolism
Tumor Necrosis Factor-alpha - biosynthesis
Tumor Necrosis Factor-alpha - chemistry
Tumor Necrosis Factor-alpha - genetics
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Summary: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.
ISSN:0003-9861
DOI:10.1006/abbi.1996.0063