Gross, M., Sweet, R. W., Sathe, G., Yokoyama, S., Fasano, O., Goldfarb, M., Wigler, M. H., Rosenberg, M. (1985) Purification and characterization of human H-RAS proteins expressed in Escherichia coli. Molecular and Cellular Biology, 5 (5). pp. 1015-1024. ISSN 0270-7306
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Abstract
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.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > genes: types > RAS bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression biotechnology > chromatography > protein purification |
| CSHL Authors: | |
| Communities: | CSHL labs > Wigler lab |
| Depositing User: | CSHL Librarian |
| Date: | 1985 |
| Date Deposited: | 13 Apr 2012 20:38 |
| Last Modified: | 04 Nov 2016 20:48 |
| PMCID: | PMC366817 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/26188 |
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