IN-VITRO SYNTHESIS OF AN N-MYRISTOYLATED FUSION PROTEIN THAT BINDS TOTHE LIPOSOMAL SURFACE

To increase the efficiency of association of tumor necrosis factor (TN F), a hydrophilic model protein, with liposomes, an N-myristoylation s ignal sequence was linked to the N-terminus of TNF by gene fusion. A D NA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to the 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 reti culocyte lysate gave rise to an N-myristoylated fusion TNF with a mole cular mass of 18 kDa as determined by the incorporation of [H-3]myrist ic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly(2) in the myristoylation signal with Ala entirely inhibited th e incorporation of [H-3]myristic acid into the fusion protein. A lipos ome binding assay using Ficoll density gradient centrifugation reveale d that incubating the N-myristoylated fusion TNF with dipalmitoyl phos phatidylcholine-liposomes caused the complete binding of the protein t o the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for lipos omes, was synthesized by the in vitro transcription/translation system . (C) 1996 Academic Press, Inc.