Purification and characterization of human H-RAS proteins expressed in Escherichia coli

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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URL: https://www.ncbi.nlm.nih.gov/pubmed/3923330
DOI: 10.1128/MCB.5.5.1015

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: http://repository.cshl.edu/id/eprint/26188

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