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

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.

journal_name

Mol Cell Biol

authors

Gross M,Sweet RW,Sathe G,Yokoyama S,Fasano O,Goldfarb M,Wigler M,Rosenberg M

doi

10.1128/mcb.5.5.1015

subject

Has Abstract

pub_date

1985-05-01 00:00:00

pages

1015-24

issue

5

eissn

0270-7306

issn

1098-5549

journal_volume

5

pub_type

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