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PMID: 3015600 Published · ppublish English Journal Article

Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site.

The EMBO journal ·Vol. 5 ·No. 6 ·1986-06-00 ·Pages 1351-8

Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS, Wittinghofer A

Abstract

v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

MeSH Terms
Cell Line Cloning, Molecular DNA Restriction Enzymes Escherichia coli/genetics Genetic Vectors Guanine Nucleotides/metabolism Mutation Neoplasm Proteins/genetics,metabolism Oncogene Protein p21(ras) Oncogenes Plasmids Protein Binding Proto-Oncogene Proteins/genetics,metabolism Proto-Oncogene Proteins p21(ras)
Chemicals
Guanine Nucleotides Neoplasm Proteins Proto-Oncogene Proteins DNA Restriction Enzymes Oncogene Protein p21(ras) Proto-Oncogene Proteins p21(ras)
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Tucker J
Sczakiel G
Feuerstein J
John J
Goody R S
Wittinghofer A
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1986-06-00
Pages
1351-8
Language
English
Region
England
NLM ID
8208664
PMCID
PMC1166947
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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