Purification and characterization of human H-ras proteins expressed in Escherichia coli.
The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.
D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006150:Guanine Nucleotides / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D009857:Oncogenes; D001749:Urinary Bladder Neoplasms / Q000235:genetics
D004274:DNA, Recombinant; D006150:Guanine Nucleotides; D009363:Neoplasm Proteins; D019204:GTP-Binding Proteins