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

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 p2lv, 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 GDPaS and GTPaS. It has low specificity for the Sp isomer of GTP,BS. Together with the data for GDP,BS and GTP-yS 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.

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

“…Methylanthraniloyl (mant) derivatives of GDP, GTP, and XTP were prepared as described (17). Nucleotides were separated analytically by HPLC as previously described (17,23).…”

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

“…C-terminally truncated Rap1A (residues 1 ± 167) was prepared from ptacRap1A and puri®ed by a two-column puri®cation procedure similar to that of Ras (Tucker et al, 1986) as described before (Herrmann et al, 1996). The S17N mutation was introduced into ptacRap1A (residues 1 ± 171) using the megaprimer PCR method (Picard et al, Under the same conditions, the interaction of wild-type H-Ras .…”

“…The volume was 1200 ml 1994) and the mutagenesis primer 5'-GTTGGGAG-GAATGCATTGACAGTTCAG-3' (codon for Asn17 underlined). Full-length Rap1B, starting from a cDNA clone kindly given to us by Dr Hans Bos, was expressed from the E. coli expressed vector ptac such that the translation initiation site is the same as that for Ras (Tucker et al, 1986) and Rap1A: GAATTCTATG, with the EcoRI restriction site and translation initiation codon underlined. It is puri®ed by the same procedure as for Rap1A.…”

“…ptac-Rap2A was puri®ed similarly to ptac-Rap1A. Full length H-ras, K-ras, N-ras and R-ras were puri®ed as described before (Tucker et al, 1986;Herrmann et al, 1996). The R-ras clone was kindly provided by Dr Alan Hall.…”

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

“…The cells were then lysed and rabS was purified using a QSepharose column, as described by Tucker et al (1986). The protein was further purified by running a 0-0.5 M NaCl gradient through an S-Sepharose column equilibrated with 25 mM HEPES pH 6.8, 10 mM MgCl2, 100 mM GDP.…”