Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems

摘要: Kirsten-ras is the oncogene most frequently activated in human tumors. Studies of its biological function have been limited by nonavailability significant amounts major protein product, (4B) p21. When expressed Escherichia coli K12, recombinant was rapidly cleaved upon cell lysis lysine-rich C terminus region, probably ompT protease. However, soluble full-length obtained when gene an E. strain lacking gene, and also a baculovirus/insect expression system. Additionally, system produced about half membrane-associated form, which post-translationally modified polyisoprenylation carboxyl-methylation. A C-terminally truncated form (residues 1-166) at high levels for x-ray crystallographic studies. The kinetics GDP release GTP hydrolysis purified proteins are similar to those corresponding Harvey-ras proteins, though there small differences relative affinities GTP. Biological activity Kirsten Val-12 p21 demonstrated microinjection into Swiss 3T3 cells, resulting morphological transformation, with lower potency than that Harvey protein.