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 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.
Utsumi
T
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Kuranami
J
J
Tou
E
E
Ide
A
A
Akimaru
K
K
Hung
M C
MC
Klostergaard
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004337:Drug Carriers; D015683:Gene Products, gag / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes; D008969:Molecular Sequence Data; D009154:Mutation; D009227:Myristic Acids / Q000737:chemistry; D014176:Protein Biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D004337:Drug Carriers; D015683:Gene Products, gag; D008081:Liposomes; D009227:Myristic Acids; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha