Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems.
Kirsten-ras is the oncogene most frequently activated in human tumors. Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21. When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease. However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E. coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system. Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation. A C-terminally truncated form (residues 1-166) was also expressed at high levels in E. coli for x-ray crystallographic studies. The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP. Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein.
Lowe
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PN
Department of Molecular Sciences, Wellcome Research Laboratorie, Langley Court, Beckenham Kent United Kingdom.
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M J
MJ
Bradley
S
S
Rhodes
S
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Sydenham
M
M
Paterson
H
H
Skinner
R H
RH
D000818:Animals; D016367:Baculoviridae; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003461:Crystallography; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D007313:Insecta; D007526:Isoelectric Point; D051379:Mice; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras)