Expression and characterization of the Ha-ras p21 protein produced at high levels in the insect/baculovirus system.
Normal and mutated cDNAs of Ha-ras have each been cloned into a standard (pAc373) and a novel (p36C) baculovirus transfer vector and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus immediately downstream of the polyhedrin promoter. Spodoptera frugiperda cells infected with recombinant virus containing the normal Ha-ras gene express very high levels of ras p21 protein (approximately 20% of total cell protein), whereas the mutant protein was expressed at considerably lower levels. Molecular analysis showed that this was most likely due to a post-transcriptional event. The expression vector p36C produced considerably higher levels of recombinant p21 compared to the more commonly used pAc373. The majority of the normal ras p21 protein is soluble, cytoplasmic, and appears to be nonacylated. However, about 10% of the p21 associates with the membrane fraction of infected cells and migrates as a slightly faster band on gels. Furthermore, this band is sensitive to hydroxylamine treatment and shows specific incorporation of [3H]palmitate, strongly suggesting that it is the palmitoylated form of p21, which is the biologically active form of the protein. Both the soluble and membrane-associated p21 have been purified to homogeneity under nondenaturing conditions, the latter in the presence of detergents. The isolation of native palmitoylated p21 has not been reported previously. The difference in hydrophobicity between these two proteins has been demonstrated by Triton X-114 partitioning. The use of the insect/baculovirus expression system to express relatively high levels (20 mg/liter) of palmitoylated p21 should aid experiments to resolve the structural and functional properties of this molecule.
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M J
MJ
Department of Molecular Biology, Wellcome Foundation, Beckenham, Kent, United Kingdom.
Hall
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A
Rhodes
S
S
Skinner
R H
RH
Murphy
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V
Sydenham
M
M
Lowe
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PN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D011905:Genes, ras; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping
D010169:Palmitic Acids; D011994:Recombinant Proteins; D019308:Palmitic Acid; D004247:DNA; D015689:Oncogene Protein p21(ras)